A001.doc

9th Meeting of The Society for Natural ImmunityNovember 4-8, 2005

NK Hawaii -- Poipu Beach, Kauai

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A001Lectin-Like Transcript 1 (LLT1) is a ligand for CD161 receptor. Hatice Aldemir1, Virginie Prod’homme1, Marie-Jeanne Dumaurier1, Christelle Retiere2, Julie Cazareth1, Franck Bihl1, and Veronique M. Braud 1 . 1CNRS UMR6097, University of Nice-Sophia Antipolis, 660 Route des Lucioles, 06560 Valbonne, France, 2Etablissement Francais du Sang, 44011 Nantes cedex 1, France.

Human NK cells and subsets of T cells or NKT cells express the orphan C-type lectin receptor CD161 (NKR-P1A) of unknown function. In contrast to rodents that possess several NKR-P1 genes coding for either activating or inhibitory receptors, the nature of signals delivered by the single human NKR-P1A receptor is still to be clarified. We report that the Lectin-Like Transcript 1 (LLT1) molecule is a ligand for CD161 receptor. Engagement of CD161 on NK cells with LLT1 expressed on target cells inhibited NK cell-mediated cytotoxicity and IFN- secretion. Conversely, LLT1/CD161 interaction in the presence of a TCR signal enhanced IFN- production by T cells. These findings identify a novel ligand/receptor pair that differentially regulates NK and T cell functions.





A002Synapses and nanotubes in innate immunity. Catarina R. Almeida, Bjorn Önfelt, Shlomo Nedvetzki, Stefanie Sowinski, Bebhinn Treanor and Daniel M Davis (with numerous collaborators). Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

Micrometer-scale segregation of proteins across an intercellular contact is the hallmark characteristic of an immunological synapse (IS) and here, we report that the extent of segregation of ICAM-1 from MHC class I protein at the NK cell IS varies with the level of target cell expression of MHC class I protein. At NK cell synapses with target cells expressing low levels of MHC protein (i.e. 104/cell surface), ICAM-1 and MHC protein are largely co-localized, whereas at synapses involving target cells expressing high levels of MHC protein (105/cell surface), clusters of ICAM-1 and MHC protein are clearly segregated. In addition, with target cells expressing a low amount of HLA-C, a multi-focal patterning of MHC protein occurs at the NK cell IS whereas for higher levels of target cell expression, patterning of MHC protein was homogeneous, ring-shaped, or containing multiple exclusions. Thus, supramolecular patterning and segregation of proteins at an intercellular contact reflects protein expression levels and therefore, could be used generally to report such information between cells.

After disassembly of the IS, membrane tethers or nanotubes leave cells connected for some time. Here, we demonstrate the transport of vesicles and other cargo within membrane nanotubes that connect various immune cells. Some of these nanotubular connections contain both f-actin and tubulin while others contain only f-actin, with relative frequencies that depend on cell type. Lipid vesicles were observed to move within a subset of tubes in a step-wise and ATP-dependent manner. Thus, nanotubular connections between cells are more complex than simple ubiquitous membrane tethers and can traffic different cargoes in a controllable manner.

Finally, we report that after seven days of co-culture with autologous mature macrophages, NK cells proliferate, secrete IFN-, and increase expression of activating receptors NKG2D and NKp44. After co-culture, NK cells show increased cytotoxicity to susceptible target cells. This activation of NK cells is dependent on the intercellular contact with macrophages where ICAM-1, 2B4 and macrophage f-actin accumulate. Activation of macrophages by LPS induces transcription of ULBP1, 2 and 3, and renders macrophages directly susceptible to NK cell cytotoxicity via NKG2D recognition. At this cytolytic NK cell/macrophage





immunological synapse, NK cell f-actin and macrophage ICAM-1 often accumulate within a peripheral ring at the synapse around a central cluster of NKG2D and activating adaptors DAP10 and CD3. Thus, macrophages can augment NK cell effector functions whereas LPS-activated macrophages are directly lysed; the different outcomes correlating with distinct NK cell/macrophage immune synapses.





A003Dramatic alterations of NK cell subsets and function starting in Acute HIV-1 infection. Galit Alter, Nickolas Teigen, and Marcus Altfeld. Massachusetts General Hospital, 149 13th Street, Room 6613, Charlestown MA 02129 USA.

NK cells are critical in the first line defense against viral infections. Here we characterized for the first time the phenotypic and functional evolution of the NK cell compartment by multiparameter flow-cytometry starting in acute HIV-1 infection. Acute HIV-1 infection was associated with elevated NK cell numbers, an expansion of CD3negCD56dimCD16pos NK cells and an early depletion of CD3negCD56brightCD16neg NK cells. Ongoing viral replication resulted in a secondary depletion of CD3negCD56dimCD16pos NK cells with a paralleled increase in functionally anergic CD3negCD56negCD16pos NK cells. While NK cell activity was elevated in acute HIV-1 infection following stimulation with MHC devoid target cells, maximal NK cell response to mitogens was already significantly reduced in acute infection. The increased NK cell activity to MHC devoid target cells early in infection was associated with increased expression of KIR on NK cells. During the first year of infection, NK cell numbers, function, perforin expression, and cytolytic activity following stimulation with MHC devoid target cells decreased to levels observed in chronically HIV-1 infected subjects. This was associated with the accumulation of functionally anergic CD3negCD56negCD16pos NK cells, expressing high levels of SHIP-1 and reduced levels of perforin. Taken together, these data demonstrate significant perturbations in NK cell distribution and function as early as acute HIV-1 infection, which may have important consequences on the subsequent immune control of opportunistic infections, as well as HIV-1 replication itself.





A004Phosphorylation of a KIR by protein kinase C impacts on receptor expression on the surface of NK cells. Diana A. Alvarez Arias and Kerry S. Campbell, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA

The KIR3DL1 (3DL1) inhibitory receptor negatively regulates human NK cell activation by recruiting the SHP1 and SHP2 protein tyrosine phosphatases following phosphorylation of two cytoplasmic tyrosine residues. Remarkably, phosphoamino acid analysis of 3DL1 from 32P-orthophosphate-labelled primary NK and NK-92 cells revealed strong constitutive phosphorylation on serine (S) and weak phosphorylation on threonine (T). Truncation mapping in NK-92 cells and in vitro phosphorylation analysis identified S364 and S367 as casein kinase II phosphorylation sites and S394 and T386 as protein kinase C (PKC) sites. PKC stimulation with phorbol ester did not increase 3DL1 phosphorylation, indicating that the S/T phosphorylation is constitutive and stable. To determine whether serine phosphorylation plays a role in the receptor function or expression/turnover, we created NK cell lines containing S/T to alanine (A) mutants of 3DL1. None of these mutations affected the inhibitory capacity of 3DL1 in cytotoxicity assays. However, the 3DL1-S394A mutant exhibited an increased level of surface expression in NK-92 cells and an increased turnover rate relative to the wild type receptor. In contrast, mutation of S394 to a phosphomimetic residue, aspartic acid, decreased 3DL1 expression and turnover. Our results indicate that serine phosphorylation of 3DL1 by PKC regulates surface expression in NK cells. Supported by NIH grants R01-CA083859 and CA-09035.





A005MHC class I transfer from surrounding cells is restricted by endogenous expression of the corresponding MHC class I ligands on the NK cell itself. Katja Andersson1, Geoffrey S. Williams2, Marjet Elemans1, Ramit Mehr3, Daniel M. Davis2 and Petter Höglund1. 1Strategic Research Center for studies of Integrative Recognition in the Immune System (IRIS),Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, Nobels Väg 16, Stockholm, Sweden. 2Division of Cell and Molecular Biology, Imperial College London, London, UK. 3 Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel.

Upon in vitro co-culture, Ly49A+ NK cells acquire exogenous MHC class I ligands (H-2Dd) from surrounding target cells. Intriguingly, endogenous expression of H-2Dd, e.g. H-2Dd displayed on the surface of the Ly49A+ NK cell itself, dramatically reduced the capacity of the Ly49A+ NK cells to acquire exogenous H-2Dd ligands. The low expression level of Ly49A on H-2Dd+Ly49A+ NK cells can not alone explain the reduced extent of acquisition. Instead, a cis-interaction between endogenously expressed H-2Dd molecules and Ly49A receptors on the NK cell may reduce the ability of the NK cell to acquire MHC proteins from other cells. Supporting this hypothesis, acid treatment of the NK cell, that ruptures endogenous cis-interactions, resulted in an instant augmentation of Ly49A expression levels (as measured by the YE1/48 antibody but not with the JR9 antibody) followed by an enhanced capability of acid-treated NK cells to acquire H-2Dd from surrounding cells. Our data suggest that endogenous expression of MHC class I impairs the ability also of human NK cells to acquire MHC class I from surrounding cells. In parallel with MHC class I acquisition, both murine and human target cells acquire inhibitory receptors from NK cells, suggesting the existence of bi-directional transfer of proteins between NK cells and target cells. Our ex vivo models represent useful tools in future studies of the mechanisms and functions of protein transfer between NK cells and target cells.





A006Beyond plasmacytoid dendritic cells: interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective anti-viral immunity. Christopher E. Andoniou1,2, Serani L.H. van Dommelen1,2, Valentina Voigt1,2, Daniel M. Andrews1,2, Geraldine Brizard2, Carine Asselin-Paturel3, Thomas Delale3, Katryn J. Stacey4, Giorgio Trinchieri3, Mariapia A. Degli-Esposti1,2. 1Immunology and Virology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Australia; 2Centre for Experimental Immunology, Lions Eye Institute, Perth, Australia; 3Schering-Plough Laboratory for Immunological Research, Dardilly, France; 4Institute for Molecular Bioscience, Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Australia

Dendritic cells (DCs) are critical for the initiation of adaptive immunity. Recently, the ability of DCs to regulate aspects of innate immunity, in particular natural killer (NK) cell function, has been recognized. NK cells play a critical role in controlling viral infections, including those with the herpesvirus cytomegalovirus. The anti-viral activities of NK cells involve lysis of infected targets and the release of cytokines, such as IFN-. Recently, we have investigated the molecular mechanisms that participate in DC-NK interactions during cytomegalovirus infection. Importantly, we have analysed the role of DCs other than plasmacytoid DCs (pDCs), to determine whether the ability to activate innate immunity is a unique feature of PDCs, or whether other DC subsets can participate in such activities. The important role of conventional CD11b+DCs in regulating NK cell activities in response to cytomegalovirus infection, and the mechanisms that participate in DC-NK cell interactions during infection will be discussed. Importantly, the therapeutic value of activating NK cell responses using conventional DCs for the control of viral infection has been investigated and will be reviewed.





A007Characterization of human NK cell subsets that are activated by HLAclass I-

tumor targets in HLA class I-typed donors. Nicolas Anfossi, Pascale André, Violette Breso, François Romagné, Charles A. Stewart, Sophie Roetynck, Sophie Guia, Sophie Ugolini and Eric Vivier. Innate Pharma®, 119-121 ancien chemin de Cassis, 13009 Marseille and Centre d’Immunologie INSERM-CNRS de Marseille Luminy, Parc scientifique et technologique de Marseille Luminy, Case 906, 13288 Marseille Cedex 09, France.

Recent studies have shown that cytotoxic cell function could be assessed by flow cytometric analysis of CD107/LAMP cell surface mobilization. We confirmed that cytotoxic activity of human NK cells, measured by Chromium 51 release assay (natural cytotoxicity and ADCC) is correlated with CD107/LAMP mobilization. Based on this assay, we show here that NK cells activated by the prototypical MHC class I- NK cell target K562 include three subsets: IFN-+CD107-, IFN--

CD107+ and IFN-+CD107+ NK cells, indicating that cytotoxicity is not correlated with cytokine production. We also further determined the phenotype of the NK cells that are activated by K562 targets, and found that the majority of IFN- producing NK cells are CD56dim. Moreover, only NK cells that express low level of the Natural Cytotoxicity Receptor NKp46 were activated by K562 cells. Finally, the response to K562 was dependent upon the HLA class I genotype of the donors and the pattern of HLA-class I-specific inhibitory receptors expressed by NK cells (Killer cell Ig-like receptors and CD94/NKG2A). These results suggest a mechanism of NK cell education to self tolerance, which involves the interaction between MHC class I molecules and their inhibitory NK receptors.





A008Inhibition of NKp30 activating receptor by the pp65 protein from human cytomegalovirusTal I Arnon and Ofer Mandelboim. The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem, 91120, Israel.

Natural killer (NK) cells are bone marrow derived lymphocytes that constitute a key frontline defense against a range of hazardous conditions, including viral infection and tumor transformation. In humans, NK cell deficiency is associated with recurrent systemic infections, even in the presence of functional T and B cells. One of the most frequent complications associated with NK cell deficiency involves increased sensitivity to viruses belonging to the herpes family, such as the human cytomegalovirus (HCMV). The importance of NK cells in CMV immunity has been directly demonstrated in mice, where multiple viral escape mechanisms have been developed to evade NK attack and where depletion or functional impairment of NK cells leads to an increased susceptibility to CMV.

Here we demonstrate a direct interaction between two major proteins; the HCMV tegument protein pp65 and the NK activating receptor, NKp30. We show that the binding of pp65 to NKp30 is specific and functional and thus represents the first NKp30 ligand to be identified. Surprisingly, the recognition of pp65 by NKp30 does not lead to NK activation, but rather to a general inhibition of NK ability to kill normal, tumor and virally infected cells. Finally, we show that the inhibition of NK activity by pp65 is mediated via the dissociation of the zeta chain from NKp30, which consequently diminishes the activating signals.





A009Nod2 regulation of NK cell cytotoxicity. Verónica Athié-Morales 1 and Clair M. Gardiner2. 1Xoma Ireland Limited and 2School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland

NK cells have long been recognised for their ability to kill virally infected cells. However, NK cell deficient patients suffer from recurrent bacterial infections suggesting that NK cells might also play an important role in the innate immune response against bacteria. Cells of the innate immune system sense pathogen associated molecular patterns (PAMPs) through pathogen recognition receptors (PRRs). It is now recognised that Toll-like receptors (TLR) along with Nuclear Oligomerization Domain (Nod) proteins are the two main families of mammalian PRRs. Recent data demonstrating a functional role of TLR2 and TLR5 in the direct recognition of bacterial PAMPs confirmed the involvement of NK cells against bacterial infections. Nod2 is a cytoplasmic PRR with expression reported on intestinal epithelial cells, macrophages and neutrophils. Nod2 plays an essential role in the detection of invasive bacteria in the gut and mutations of Nod2 are associated with the development of Chron`s disease. Muramyl dipeptide (MDP), the specific ligand for Nod2, is a naturally occurring peptide contained in peptidoglycan (PGN) from both Gram + and Gram – bacteria. Here we demonstrate that Nod2 is present and functional in primary NK cells and the NKL cell line. MDP/ Nod2 signals in NK cells as demonstrated by IB degradation and consequent NFB activation. MDP is naturally internalised by NK cells and differently from other cell types does not require transfection in NK cells. Reports showing localisation of Nod2 in cytoplasmic vesicles suggested a possible requirement of lysosomal acification for MDP/ Nod2 signalling as previously shown for TLR7, TLR8 and TLR9. Using the inhibitor chloroquine, we demonstrate that MDP/ Nod2 signalling is independent of lysosomal acidification in NK cells. Moreover, MDP/ Nod2 induces CD69 up-regulation and cytotoxicity against the Daudi cell line. The present data demonstrate a functional role for MDP/ Nod2 in NK cell biology and further highlights the importance of NK cells in the innate immune response against bacterial infections.





A010Genomics and Evolution of the NKC in a New World Monkey and a Prosimian Species. Anne Averdam1, Mario Sontag2, Richard Reinhardt2, Lutz Walter1

1 Department of Primate Genetics, German Primate Center, Göttingen, Germany2 Max Planck Institute for Molecular Genetics, Berlin, Germany

The order Primates is divided into Catarrhini (humans, apes, Old World monkeys), Platyrrhini (New World monkeys), and Strepsirrhini (prosimians and tarsius). We have analysed the genomics of the natural killer gene complex (NKC) extending between CD94 and LY49L in a platyrrhine species, the common marmoset (Callithrix jacchus), and a strepsirrhine species, the grey mouse lemur (Microcebus murinus), by mapping and sequencing of respective BAC clonal contigs. The marmoset NKC region between CD94 and LY49L encompasses 180 kb and contains additionally the NKG2A, NKG2C, NKG2D, and NKG2F genes. LY49L and NKG2F are pseudogenes due to absence of exons 4 to 7 and a frame shift mutation in exon 1, respectively. The other identified loci appear functional and expression could be verified by RT-PCR using cDNA from blood PBMC. Interestingly, for the marmoset CD94 gene, which is rather monomorphic in human, several alleles could be identified. Two CD94 alleles differ in the presence of an additional codon (Thr) in exon 3 that codes for the stalk region. Currently, we are sequencing the coding regions of CD94, NKG2A, NKG2C, and NKG2D in several marmoset individuals to determine the degree of genetic variability.The corresponding grey mouse lemur NKC region encompasses about 550 kb, which is about twice as large as in human and is due to duplications of loci. Besides single NKG2D and LY49L loci, we could identify three CD94 and eight NKG2 genes. Preliminary sequence analysis suggests that two NKG2 genes are pseudogenes. Interestingly, the LY49L gene in the mouse lemur appears to be functional. Phylogenetic analysis of primate NKG2A, NKG2C, NKG2E, and NKG2F sequences indicates orthologous relationship of the marmoset loci and paralogous relationship of the lemur loci.





A011Longitudinal analysis and modeling identifies changes in innate immunity parameters as predictive correlates of CD4 reconstitution in ART-treated HIV-1 infection. L. Azzoni, J. Chehimi, R. June, M. Farabaugh, B. Thiel, L. Zhou and L. J. Montaner. The Wistar Institute, 3601 Spruce St., Philadelphia, PA USA

Background: Innate immunity effectors with anti viral function (e.g. NK and Dendritic cells) are impaired in the course of HIV infection. Cross-sectional studies suggest incomplete recovery upon viral suppression. Here we report a longitudinal (> 18 month) study of the correlates of ART-mediated viral suppression in chronic HIV-1 infection. Methods: Cryopreserved PBMC were obtained from 76 patients enrolled in the WIHS and MACS cohorts; a viremic (pre-ART) and 2 or 3 subsequent non-viremic (on ART) time points were studied for each patient. Frequency and phenotype of NK, T and dendritic (DC) cells was studied by flow cytometry. In a subset of samples we also investigated activation-induced IL-12 and IFN- production and APC function (by mixed lymphocyte cultures, MLR).Statistical analysis: 78 variables were analyzed, and log transformed as needed to approximate a linear distribution. Within-group changes were tested with an ANOVA model. Variables that best correlated with changes in CD4 count over time were identified using a GEE-based covariance test.Results: CD4 counts significantly increased over time, whereas CD38 expression on CD4+ and CD8+ T cells decreased upon viral suppression. Based on our covariance analysis, a unit change in WBC counts or MLR Stimulation Index was predictive of changes in CD4 counts () of 46.8 and 29.7 units, respectively.A decrease in the expression of activation markers [CD38 MFI on CD8+ (= -337.7) and CD4+ T cells (= -234.33); % HLA-DR+ NK cells (=-0.555)] was significantly predictive of amount of CD4 counts change over time. Over the observation time, univariate analysis indicated that CD161+/56+/16+ NK cells increased significantly, whereas CD161+/CD56-/CD16+ NK cells decreased. Plasmacytoid DC frequency and IFN- production in response to Influenza or CpG-2216, as well as Myeloid DC frequency and production of IL-12 in response to LPS, was also significantly increased. The effect of gender was minimal and required no adjustment of main findings.Conclusions: We identify significant time-dependent changes in innate immune parameters and immune activation following long-term viral suppression, indicating that CD4 recovery is associated with suppression of innate and





adaptive immune activation, and with recovery of APC/MLR function. Our results also support the hypothesis that expansion of NK cells subsets during suppressive ART does not depend on their sustained activation.





A012Disconnect between the recovery of NK cell-mediated cytotoxicity to HIV-infected targets and restoration of NK subsets in ART-suppressed HIV-1-infected individuals. L. Azzoni1, J. Chehimi1, M. Farabaugh1, S. Creer1, K. Mounzer2, J. Kostman2, C. Gallo2, J. Ondercin2, J. Shull2 and L. J. Montaner1.

1 The Wistar Institute, 3601 Spruce St., Philadelphia, PA USA2 Philadelphia FIGHT, 1233 Locust St. Philadelphia, PA USA

Background: Mature Natural Killer (NK) frequency and cytotoxicity is profoundly impaired in viremic HIV-infected individuals. Only incomplete functional recovery of mature NK cells has been reported in ART-suppressed patients. We report the effects of 6 months of suppressive ART (with 8-week viral suppression) on frequency and function of NK, plasmacytoid (PDC) and myeloid dendritic cells (MDC).Methods: We followed 27 viremic HIV-1-infected patients monthly after initiating ART inclusive for up to 8 weeks after suppression to <50 HIV RNA c/ml. 18 HIV- donors were used as controls. Frequency and phenotype of NK, T and dendritic (DC) cell subsets was studied by flow cytometry on whole blood. The function of NK cells [IL-12+IL-15-induced IFN- production, spontaneous and induced (IL-12/IL-15, IFN-, CpG-2216) cytotoxicity to HIV-1-infected targets], APC and myeloid DC (MLR, LPS-induced IL-12 production, dextran uptake) and PDC (IFN- production induced by Influenza virus PR-8 strain or TLR-9-specific CpG-2216) were also studied. Statistical analysis: within-group changes over time were tested with the Friedman ANOVA or Wilcoxon signed ranks test. Differences between HIV+ and control individuals were tested with the Mann-Whitney test. Correlations between variables at specific time points were tested with the Spearman test.Results. In addition to the expected raise of CD4 counts upon treatment (median baseline= 229.5; endpoint= 359 cells/mm3), we observed a significant increase in CD56+ NK cells (baseline 35.52, endpoint 47.31 cells/mm3), which remained significantly lower than in control individuals (median= 180.8). In contrast, both spontaneous and cytokine or PDC-induced (CpG) cytotoxicity quickly increased to levels similar to control individuals upon complete viral suppression. PDC and MDC frequencies were normalized within 8-weeks of viral suppression despite a lack of restoration of the PDC-mediated IFN- production in response to CpG or Influenza virus at levels observed in HIV- subjects.





Conclusions: The rapid reconstitution of cytotoxic activity by NK cells via direct or cytokine and CpG-mediated pathways indicates a quick reversal of a functional block (e.g. by soluble viral proteins tat or gp120) in the absence of full restoration of mature NK subsets upon viral suppression, suggesting that a partial NK cell subset recovery may be sufficient to quickly restore the full cytotoxic potential once viral suppression is achieved.





A013

NK cell behavior in lymph nodes revealed by static and real time imaging. Marc Bajenoff1, Béatrice Breart2, Alex Y. C. Huang3, Hai Qi3, Julie Cazareth1,

Veronique Braud 2 , Ronald N. Germain3, and Nicolas Glaichenhaus1. 1INSERM E03-44 and 2CNRS UMR6097, Université de Nice-Sophia Antipolis, 660 Route des Lucioles, 06560 Valbonne, France. 3 Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 10 Center Dr. MSC-1892, Bethesda, MD 20892-1892, USA

Natural Killer (NK) cells promote dendritic cell (DC) maturation and influence T cell differentiation in vitro. To better understand the nature of the putative interactions among these cells in vivo, we have used immunohistochemical analysis and dynamic intravital imaging to study NK cell localization and behavior in lymph nodes (LNs) in the steady state and upon infection with Leishmania major. In naive mice, NK cells reside in the LN outer paracortex and interact with DCs in the same region previously shown to be the site of early antigen-dependent T cell activation. In contrast to T cells, intravital microscopy revealed that NK cells were slowly motile. L. major induced NK cells to accumulate in the outer paracortex and to secrete interferon- but did not modify NK cell movement. NK cells thus form a relatively fixed network in a strategic area of the LN where they can receive inflammatory signals, interact with DC, and regulate T cell responses.





A014Human T cell leukemia virus (HTLV)-infected primary CD4+ T-cells are refractory to killing by autologous natural killer cells despite down modulation of major histocompatibility complex class I molecules. Prabal Banerjee, Gerold Feuer and Edward Barker. Department of Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA.

Human T-cell leukemia virus (HTLV) is the etiologic agent of adult T cell leukemia (ATL) and establishes a chronic infection in the host despite vigorous virus specific immune responses. The major histocompatibility complex class I (MHC-I) molecules which are the key for antigen presentation to host cytotoxic T lymphocytes (CTLs) are the targets for many viruses. Interference with assembly or expression of MHC-I complex can contribute to evasion of CTLs although natural killer (NK) cells can recognize and destroy cells lacking them. Previous studies have shown that PBMCs insolated from patients with adult T cell leukemia (ATL) and HTLV-1 infected cell lines have altered MHC-I expression . HTLV-2 is related to HTLV-1 and they share a high degree of sequence homology particularly in the Tax region although HTLV-2 does not lead to disease in infected individuals. To investigate whether HTLV-1 and -2 infections of primary CD4+ T cells can modulate MHC-I molecule expression, we infected primary CD4+ T cells with HTLV-1 and –2 in vitro and assessed the level of MHC I molecules expressed on infected cells and uninfected controls during peak virus production. Analysis of MHC-I expression on HTLV-1 and -2 infected CD4+ T-cells revealed that infection leads to a decrease in MHC class I molecules. Because decreased expression of MHC-I on CD+ T-cells can lead to destruction of these lymphocytes by NK cells we wanted to determine whether NK cells are capable of killing HTLV-1 and -2 infected autologous primary T-cells. Our data show that despite the decreased MHC class I expression, autologous NK cells were incapable of killing HTLV-1 and -2 infected CD4+ T-cells, yet were effective in the destruction of the NK cell sensitive cell line, K562. Future studies will focus on determining the mechanisms underlying the ability of HTLV-1 and -2 infected T cells to evade NK cell killing.





A015The Cdc42-interacting protein 4 is a potential link between the actin and microtubular networks at the NK cell cytolytic immunological synapse. Pinaki B. Banerjee 1 , Rena Zheng1, Megan Suhoski2, Linda Monaco-Shawver1, Jordan S. Orange1,2 1Joseph Stokes Research Institute of the Children’s Hospital of Philadelphia, 2University of Pennsylvania School of Medicine Immunology Graduate Group. 3615 Civic Center Blvd, Philadelphia PA 19104.The temporospatial receptor-ligand interactions between an NK cell and a susceptible target cell forms an interface known as the NK cell activating immunological synapse (NKIS). The formation of the NKIS requires the cytoskeleton and our earlier work established that it is formed in distinct cytoskeleton-dependent stages. In particular, Wiskott-Aldrich Syndrome Protein (WASp)-dependent actin polymerization precedes microtubular function. We have shown that WASp function is required for filamentous actin (F-actin) reorganization, cell surface receptor clustering at the NKIS, and lytic granule polarization to the NKIS, whereas microtubular function is only required for the latter. The Cdc42-interacting protein 4 (CIP4) is a potential link between the F-actin and microtubule-dependent events as it interacts with WASp through its SH3 domain and tubulin through its FCH domain. Thus, to investigate the role of CIP4 in NKIS formation, we first determined the presence of CIP4 mRNA and protein in various human NK cell lines, as well as its localization by laser scanning confocal microscopy. We found that CIP4 was expressed and accumulated at the cytolytic NKIS with the microtubule organizing center (MTOC). In contrast, CIP4 was not found at noncytolytic synapses. The function of CIP4 was evaluated in human NK cells through stable overexpression of retrovirally transduced CIP4 or CIP4 devoid of its SH3 and FCH domains. Resulting cell lines were sorted for their level of transgene expression using a green fluorescent protein reporter driven by an internal ribosomal entry site. All transduced cells with high-level expression of CIP4 or CIP4 mutants were inhibited in their cytotoxic activity, whereas those with low transgene expression were only intermediately affected. These cells all accumulated F-actin normally at the NKIS, but failed to polarize their MTOC. Furthermore, the localization of CIP4 in cells overexpressing CIP4 was found not only with the MTOC but also throughout the F-actin cortex. Co-immunoprecipitation of CIP4 from lysate of cells overexpressing CIP4 identified its association with both tubulin and WASp, whereas it was primarily associated with only tubulin in lysates from cells with





endogenous CIP4 expression. Thus, the appropriate expression of CIP4 is essential for MTOC polarization to the F-actin cortex of the NKIS as well as for NK cell function. In addition, studies of WASp negative NK cells, demonstrated that CIP4 failed to accumulate at the NKIS. These data suggest that CIP4 can link F-actin and tubulin to facilitate formation and function of the NK cell activating IS.





A016Natural Killer cell and Macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum. Myriam Baratin*, Sophie Roetynck*, Catherine Lépolard†, Christine Falk‡, Jürg Gysin†, Eric Vivier*, Sophie Ugolini*. *

Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Université de la Méditerranée, Marseille, France. † Unité de Parasitologie Expérimentale, URA Institut Pasteur, Université de la Méditerranée, Marseille, France. ‡ Institute for Molecular Immunology, National Research Center for Environment and Health, Münich, Germany.

Interferon- (IFN-) secretion by Natural Killer (NK) cells is pivotal to several tumor and viral immune responses, during which NK and dendritic cells cooperation is required. We show here that macrophages are mandatory for NK cell IFN- secretion in response to erythrocytes infected with Plasmodium falciparum, a causative agent of human malaria. In addition, direct sensing of Plasmodium falciparum infection by NK cells induces their production of the proinflammatory chemokine CXCL8, without triggering their granule-mediated cytolytic programs. Despite their reported role in Plasmodium falciparum recognition, Toll-like receptors (TLR)2, TLR9, TLR11 are individually dispensable for NK cell activation induced by Plasmodium falciparum-infected erythrocytes. However, IL-18R expression on NK cells, IL-18 production by macrophages and MyD88 on both cell types are essential components of this previously undescribed pathway of NK cell activation in response to a parasite infection.





A017Plasmacytoid and conventional dendritic cells mediate rapid activation of NK cells to herpes simplex virus type 1. Daniel P. Barr 1 , Patrick C. Reading1, Magdalena Wojtasiak1, Paul G. Whitney1, Gabrielle T. Belz2 and Andrew G. Brooks1

1Department of Microbiology and Immunology, University of Melbourne, Parkville, 3010 Australia2Division of Immunology, Walter and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, 3050 Australia.

Natural killer (NK) cells play a crucial role in the initial response to viral infections but the mechanisms controlling their activation are unclear. We demonstrate that activation of NK cells leading to the production of interferon- immediately following infection with herpes simplex virus type 1 is not mediated directly by the virus. Instead NK cell activation requires the presence of dendritic cells (DC). We further identify a synergistic role for DC derived IL-12 and IL-18 as key mediators in the early activation of NK cells. Moreover the rapid activation of NK cells by this mechanism was mediated by both conventional and plasmacytoid DC indicating versatility in the induction of innate immune responses by specific DC populations.





A018Innate CD8 T rather than NK cells rapidly co-localize with lesions induced by Listeria monocytogenes infection and are more effective than NK cells in providing IFN- mediated innate protection. Rance E. Berg, Emily Crossley, Sean Murray and James Forman. University of Texas Southwestern Medical Center, Center for Immunology, 6000 Harry Hines, Dallas, TX 75390-9093.

During the innate immune response to Listeria monocytogenes (LM), the secretion of IFN- is crucial in controlling bacterial numbers. We have recently shown that CD8 T cells have the ability to rapidly secrete IFN- independent of antigen, in response to IL-12 and IL-18, during a LM infection. In the current study, we compared the relative abilities of NK and CD8 T cells to provide innate immune protection. Upon transfer of either NK or memory OT-I T cells (specific for the ovalbumin protein) into IFN- deficient hosts that were subsequently infected with wild-type LM, both cell types were found in the spleen and had the ability to secrete IFN-. However, the OT-I T cells were more effective at providing innate immune protection as determined by spleen and liver LM burdens. We used immunocytochemistry to demonstrate that upon infection with LM, marginal zone macrophages were localized to the T cell area of the splenic follicle. Transferred memory OT-I T cells were also found in the T cell area of the spleen, co-localizing with the LM and macrophages. In sharp contrast, NK cells were found predominantly in the red pulp region of the spleen.

Memory OT-I T cells were found to be randomly distributed in the liver parenchyma of naïve mice. However, both at 1 and 3 days after infection, these memory cells were preferentially found surrounding LM lesions in the liver. Additional data will be presented on the kinetics of the innate response and the in vivo contribution of CD8, NK, and NKT cell populations in both spleen and liver. These results highlight the importance of CD8 T cells in innate immune responses to LM and suggest that their protective ability is the result of their co-localization with LM infected cells.





A019Analysis of the expression and cell binding properties of the IgV and IgC isoforms of NKp30. Jayaram Bettadapura1, Kimberly A. Hewitt1, Joseph G. Altin2, Craig Freeman1, Christopher R. Parish1, and Hilary S. Warren.1 1Division of Immunology and Genetics, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia. and 2 School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra, ACT, Australia. [email protected]

The natural cytotoxicity receptor NKp30 (NCR3, CD337) is an NK cell specific activation receptor involved in the interaction of NK cells with tumour cells and dendritic cells. Alternative splicing and differential use of polyadenylation signals results in nine different transcripts for NKp30, of which six could potentially encode functional proteins. Three transcripts encoding an extracellular IgV domain, and three transcripts encoding an extracellular IgC domain each combine with transcripts encoding three different cytoplasmic domains to give the six transcripts. Although NKp30 transcripts are detected in many cell lines and tissues, cell surface expression detected with the available NKp30 mAb appears restricted to NK cells. In order to understand the relevance of the different NKp30 isoforms to NK cell function, we have carried out quantitative real-time PCR analysis of the different NKp30 transcripts in NK cells and other cell lines and have examined the effects of various cytokines on expression of the different NKp30 isoforms in NK cells. We have expressed the IgV and IgC extracellular domains of NKp30 as 6-His tagged proteins using a Baculovirus expression system. Using soluble recombinant NKp30(IgV) and NKp30(IgC) proteins bound to fluorochrome-labelled liposomes, we have compared the ability of the multimeric complexes of these two forms of NKp30 to bind to different cell types and have evaluated the role of heparan sulphate proteoglycans in this process. Our studies reveal that the IgV and IgC forms of NKp30 bind to different cellular ligands.





A020Deregulation of Endogenous IL-15 Promotes NK Cell Expansion and Graft Rejection in a Murine Model of Allogeneic Bone Marrow Transplantation. Bradley W. Blaser, Sameek Roychowdhury, Daniel J. Kim, Noah R. Schwind, Donna F. Kusewitt, Martin Guimond, Bruce R. Blazar and Michael A. Caligiuri

The Integrated Biomedical Science Graduate Program and the Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.The University of Minnesota Cancer Center and Department of Pediatrics, Division of Bone Marrow Transplantation, Minneapolis, Minnesota, 55455.

Hybrid resistance is the rejection of parental (P) type donor hematopoietic cells by F1 hybrid recipient NK cells in allogeneic bone marrow transplantation (BMT). Because IL-15 is a critical survival and activation factor for NK cells, we hypothesized that deregulation of endogenous IL-15 expression would increase graft rejection in a PF1 murine model of allogeneic BMT. Bone marrow (BM) cells (5 x 106) from wild type C57Bl/6 mice (wt B6) or B6 mice that overexpress IL-15 (IL-15 tg B6) were infused with wt B6 splenocytes (2 x 106) into lethally irradiated allogeneic (B6D2F1) or syngeneic (B6) recipient mice. Our results demonstrated that allogeneic recipients of wt B6 BM cells did not experience bone marrow graft rejection or acute GVHD (100% survival) while graft rejection was invariably lethal in allogeneic recipients of IL-15 tg B6 BM cells (median survival time 25 days, P=0.002). Mortality due to bone marrow graft rejection was confirmed by blinded analysis of histopathology which showed diffuse marked hypoplasia of sternal and femoral bone marrow in allogeneic recipients of IL-15 tg B6 BM cells. Histologic evidence of acute graft versus host disease was minimal to absent. IL-15 did not directly inhibit BM cell engraftment because syngeneic IL-15 tg B6 BM cell recipients did not show evidence of graft rejection (100% survival). Finally, allogeneic and syngeneic recipients of IL-15 tg B6 BM cells displayed a significant expansion of splenic NK1.1+CD4-CD8- NK cells compared to allogeneic and syngeneic recipients of wt B6 BM cells, respectively (allogeneic: 0.640.04% versus 0.160.02%, P=0.0005; syngeneic: 8.60.2% versus 3.61.1%, P=0.01). These data demonstrating enhanced graft rejection due to deregulated expression of IL-15 are consistent with the phenomenon of hybrid resistance and the “missing self” hypothesis for NK cell activation. While lymphocyte subset depletion studies will be required to formally establish the role of NK cells in this process, these early results argue against the administration of





IL-15 in allogeneic BMT donor-recipient pairs where NK cell alloreactivity in the host-versus-graft direction is predicted.





A021Activation of natural killer Cells by the intracellular bacterium Francisella tularensis. Sirosh M. Bokhari, Jing Yu, Nicholas McWilliams, Connie Chen and Michael J. Parmely. Department if Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.

Natural killer (NK) cells are triggered to produce interferon- (IFN-) during infection and this cytokine response is important in evoking an early T cell-independent host defense against microbes. IL-12 and TNF- act as co-activators during this process. Francisella tularensis, the causative agent of tularemia, is a facultative intracellular bacterium potentially lethal in human beings. Systemic infection results in liver colonization and hepatic damage and is highly lethal. The live vaccine strain (LVS) of F. tularensis, which is attenuated for humans, is pathogenic in mice. The production of IFN- is thought to be a key protective immune response in naïve mice. The primary objective of this study was to determine the phenotype and tissue distribution of the IFN--producing cells in naïve mice infected with F. tularensis LVS and to elucidate the requirements for cell activation. Intraperitoneal (i.p.) challenge of naïve B6 and CF1 mice with LVS induced an IFN- response within 16 hours. Serum IFN- concentrations reached maximum levels several hours after the bacteremic phase. Hepatic and splenic lymphocytes were the primary source of IFN- during this response. No significant response was detected in lymphocytes from the blood, thymus or lymph nodes. The liver and spleen were also major targets of systemic infection. In vitro challenge of B6 hepatic and splenic lymphocytes with LVS also elicited IFN- production, demonstrating that the IFN- producing cells were resident within these organs and not recruited from other sites following infection. Two-color flow cytometry identified the IFN- producing lymphocytes to be predominantly CD3-negative. More than 80% of the IFN- producing lymphocytes were NK1.1-positive. By contrast, heat-killed LVS failed to induce a response in vivo or in vitro, suggesting that heat labile bacterial components or viable organisms are essential for eliciting the response. These results indicate that NK cells are activated early after i.p. challenge with F. tularensis LVS and that the mechanism probably involves heat labile bacterial components triggering the IFN- production. F. tularensis LVS may provide a useful model for the study of NK cell activation by microbial ligands. These





findings also suggest that NK cells mediate host defense against F. tularensis during systemic infections.





A022The tumor suppressor TSLC1/NECL-2 triggers NK cell and CD8+ T cell responses through the cell surface receptor CRTAM. Kent S. Boles, Gaëlle Le Friec, Winfried Barchet, Tom Diacovo, Marina Cella, Marco Colonna. Washington University School of Medicine, 660 S. Euclid, St Louis, MO, 63110 USAThe tumor suppressor in lung cancer-1 (TSLC1) gene is frequently silenced in human lung carcinomas and its expression suppresses tumorigenesis in nude mice. TSLC1 encodes a cell surface protein called Necl-2 that belongs to the Nectin and Nectin-like (Necl) family of molecules. Necl-2 mediates epithelial cell junctions by homotypic contacts and/or heterotypic interactions with other Nectins and Necls. Thus, it may inhibit tumorigenesis by ensuring that epithelial cells grow in organized layers. Here we demonstrate that NK cells and CD8+ T cells recognize Necl-2 through a receptor known as class I-restricted T cell-associated molecule (CRTAM), which is expressed only on activated cells. CRTAM-Necl-2 interactions promote cytotoxicity of NK cells and IFN- secretion of CD8+ T cells in vitro as well as NK cell-mediated rejection of tumors expressing Necl-2 in vivo. These results provide evidence for a novel mechanism of tumor suppression mediated by TSLC1 that involves cytotoxic lymphocytes. Furthermore, they reveal Necl-2 as one of the molecular targets that allows the immunosurveillance network to distinguish tumor cells from normal cells.





A023The HIV down modulates ligands for natural killer cell activation receptors on primary CD4+ T-lymphocytes. Matthew I. Bonaparte and Edward Barker, SUNY Upstate Medical University, 750 E. Adams Street, 2204 WH, Syracuse, NY 13210, USA

We have demonstrated that natural killer (NK) cells lacking inhibitory receptors to HLA-C and –E can destroy, to a limited degree, human immunodeficiency virus (HIV)-infected cells. The ability of NK cells to kill virus-infected cells is dependent not only on the loss or decreased expression of ligands for NK cell inhibitory receptors (i.e., major histocompatibility complex (MHC) class I molecules), but the presence of activation ligands on the target cells as well. More importantly, HIV may modulate the ligands for NK cell activation receptors and decrease the ability to trigger NK cells. This in turn reduces the ability to kill the HIV-infected cells. In this study we determined which NK activation ligands are present on primary CD4+ T-cells and whether HIV modulates the surface expression of these ligands. For this study we stained the surface of uninfected and HIV-infected primary CD4+ T-cells with antibodies directed to MICA and MICB that are ligands for the activation receptor NKG2D, CD155 that are ligands for the activation receptor DNAM-1, and CD48 that are ligands for the NK cell activation receptor CD244. In addition we used soluble NK cell receptors (NCRS) NKp30 and NKp46 to identify ligands for NK cell activation receptors on uninfected and HIV-infected CD4+ T-cells. From our study we demonstrated that ligands for NKp30, NKp46, NKG2D, and DNAM-1 were not present on either uninfected or infected primary CD4+ T-lymphocytes even though they were present on cell lines. However, we did observe the presence of CD48 and found that HIV decreased its surface expression by 60%. Recently it has been demonstrated that ICAM-1, -2 and -3 are capable of activating NK cells by triggering LFA-1 molecules (independent of their role in adhesion). We demonstrated that HIV increases ICAM-1 and -3 on CD4+ T-cells, but decreases ICAM-2 expression by 55%. This finding is important given that binding of LFA-1 by ICAM-2, but not ICAM-1 and -3, leads to target cell killing by NK cells. Future studies will determine if selective down-modulation of CD48 and ICAM-2 by HIV prevents NK cells from killing HIV-infected cells despite the decreased expression of MHC class I molecules.





A024CD56bright Natural Killer cells switch to type 2 immunity in first trimester human pregnancy. Angela M. Borzychowski1,2, B. Anne Croy 3 , Chris W.G. Redman1 and Ian L. Sargent1. 1Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford, United Kingdom OX3 9DU. 2Department of Biomedical Sciences, University of Guelph, Guelph Ontario, Canada N1G 2W1. 3Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6.

In normal human pregnancy, type 1 (cell-mediated) immunity is thought to be suppressed to protect the fetus from immune rejection, while type 2 (humoral) immunity is enhanced or unchanged. Type 1 lymphocyte-derived cytokines are also thought to have roles in the pathogenesis of disorders of pregnancy, such as pre-eclampsia. However, it is now apparent that the Th1/Th2 paradigm of pregnancy is too simplistic, as in normal human pregnancy there is activation of the innate immune system resulting in a systemic inflammatory response, which is exaggerated in pre-eclampsia. Furthermore, in pre-eclampsia and healthy third trimester pregnancy, greater changes in type1 and type 2 immunity are found in natural killer (NK) cells rather than in T cell subsets. In this study, our aim was to quantify type 1 and type 2 T and NK cell subsets throughout healthy pregnancy in order to determine at which stage of gestation the shift to type 2 immunity occurs. We have used type 1 (IL-18 receptor) and type 2 (ST2L) lymphocyte function markers in four-colour flow cytometry to characterise peripheral blood lymphocyte populations (Th, Tc, NK CD65bright, NK CD56dim and NKT) from pregnant women followed throughout each trimester of healthy pregnancy and non-pregnant control women (n=11 per group). Overall, there was a decrease in total type 1 (IL-18R+) lymphocytes in third trimester pregnancy relative to controls and earlier gestational timepoints. However, when lymphocyte subpopulations were analysed separately, the type 1:type 2 ratio of CD56bright NK cells was found to be significantly decreased in all trimesters of healthy pregnancy relative to non-pregnant control women. In contrast, type 1:type 2 ratios of CD56dim NK cells and Th cells were significantly lower only in the third trimester, relative to controls. The data confirm that normal human pregnancy is dominated by an elevated type 2 immune response, which involves both T cells and NK cells. However, as the increase in type 2 cells in the first trimester of pregnancy was only apparent in the CD56bright NK population, this suggests that





these cytokine producing NK cells, and not Th cells, may be the trigger for the type 2 lymphocyte shift during pregnancy. Funded by NSERC and Wellbeing of Women, UK





A025Receptor/ligand interactions that are involved in NK-mediated killing of tumor cells. Cristina Bottino, Roberta Castriconi, Alessandra Dondero, Francesca Bellora, Andrea Petretto, Alessandro Moretta and Lorenzo Moretta. Istituto Giannina Gaslini, L.go G. Gaslini 5, 16148 Genoa, Italy and Dipartimento di Medicina Sperimentale, University of Genoa, Via Leon Battista Alberti, 2, 16132 Genoa Italy

Natural Killer (NK) cells are equipped with a series of surface receptors that recognize different cellular ligands on potential target cells. Some of these ligands (e.g. HLA class I) prevent the NK-mediated attack by interacting with inhibitory NK receptors (e.g. Killer Ig-like Receptors). Other ligands are specifically recognized by activating NK receptors that once engaged induce both cytotoxicity and lymphokine release. The general concept is that tumor transformation results in downregulation of surface HLA class I molecules together with upregulation or de novo expression of ligands for triggering NK receptors. Thus, transformed cells become highly susceptible to NK-mediated lysis. We analyzed the expression of a large panel of surface molecules on different tumor cells. Moreover, we investigated the susceptibility of tumors to NK-mediated cytolytic activity and we dissected which receptor/ligand interactions participate in this process. We show that a significant heterogeneity in susceptibility to lysis exists among tumors of different as well as of similar histotype. Moreover, the receptor/ligand interactions involved in NK-mediated recognition of target cells varies among the various tumors analyzed and their susceptibility to lysis directly correlates with the number of ligands for NK triggering receptor expressed at the cell surface. Altogether our data show that tumor cells set up various strategies to escape the NK-mediated attack. These include downregulation or lack of expression of ligands for activating NK receptors.





A026Activated T cells kill autologous and allogenic tumor cells. Francoise Bouet1, Olivier Toutirais1, Laurent Sulpice2, Nathalie Rioux-Leclercq1, Cécile Thomas de la Pintière2, Noelle Genetet1 and Véronique Catros 1 . 1UPRES 3891, Faculté de Médecine de Rennes, 2 avenue du Pr Leon Bernard, Rennes, 35043, France. 2CHU de Rennes, 2 rue Henri Le Guilloux, Rennes, 35033, France.Although generally portrayed as a minor subset of human T cells, peripheral blood T cells rapidly proliferate following activation by natural or synthetic phosphoantigens. We have investigated the ability of the main subset of peripheral lymphocytes, the 92 T cells to kill tumor cells in vitro and the question of the relevance of their potential interest for immunotherapy in cancer patients is adressed.In 9/9 healthy donors and 4/4 cancer patients (3 colorectal cancer: CRC; 1 sarcoma) selective amplification (70-97 %) of 92 T cells could be achieved from freshly prepared peripheral blood lymphocytes (PBL) with a single BrHPP (Phosphostim™, Innate Pharma) ex vivo treatment. Cytotoxic activity of 92 T cells was demonstrated on tumor cell cultures derived from tumor biopsies (2 CRC; 3 renal cell carcinoma : RCC) but not on normal cells. Both NKG2D and TCR were involved in recognition of target cells. In assays done with 92T cells from patients, strong reactivity against tumor cells could be observed either in autologous or in allogenic situation. Tumor cells were commercial (4CRC) or laboratory established (3 CRC, 5 RCC, 2 sarcomas, 1 pancreatic tumor, 2 glioblastomas) cell lines. Expression of classical and non classical MHC molecules, NKG2D ligand, F1ATPase and HSP were investigated in sensitive tumor cells. Preliminary assays using blocking monoclonal antibodies suggest that several molecules expression by tumor cells contribute to their targeting by 92 T cells.





A027The inhibitory receptor NKG2A determines lysis of vaccinia virus infected autologous targets by natural killer cells. Collin R Brooks*, Tim Elliott* Peter Parham† and Salim I Khakoo**Southampton University School of Medicine, Southampton General Hospital, Tremona Road, Southampton, UK and †Departments of Structural Biology, Microbiology and Immunology, Stanford University, 299 Campus Drive WestStanford CA 94305 USA.

The mechanisms by which Natural Killer (NK) cells recognize virally infected cells are poorly understood. In order for NK cells to be activated upon contact with an infected cell the balance between the activating and inhibitory signals that regulate NK cell function must be altered in favor of activation. To address this we have studied human liver-derived NK cells and vaccinia infection using the pathogenic WR strain. In cytotoxicity assays six out of seven NK cell clones expressing high levels of NKG2A, as determined by flow cytometry and quantitative RT-PCR, lysed the autologous vaccinia-infected, but not the mock-infected, autologous BLCL. No correlation was found between lysis of infected targets and activating receptor expression. None of the five clones expressing low levels of NKG2A lysed the vaccinia infected autologous cell line. Similarly vaccinia infection permitted lysis of 721.221 transfectants expressing HLA-A*0101, and hence HLA-E, but not of transfectants expressing HLA-C allotypes. Quantitative RT-PCR demonstrated an inverse correlation between the level of inhibitory KIR expression and that of NKG2A (r2=0.62, p<0.01), but no correlation was found amongst that of the activating KIR and NKG2 genes. Consistent with NKG2A as a key determinant in the ability to lyse vaccinia infected targets, vaccinia infection selectively down-regulated HLA-E, but left total levels of MHC class I intact. These data demonstrate that release from an inhibitory receptor:ligand interaction is one mechanism that permits NK cell recognition of a virally infected target, and that the variegated expression of inhibitory receptors for MHC class I in humans generates a repertoire of NK cells with different anti-viral potentials.





A028The SH2-domain-containing inositol 5'-phosphatase (SHIP-1) is the main mediator of the inhibitory action of the KLRG1 molecule. Laurent Brossay , Céline Fugère, and Hao Jun Jonathan Chong. Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island 02912, USA

The killer cell lectin-like receptor G1 (KLRG1) is the mouse homolog of the rat mast cell function-associated antigen (MAFA) and is expressed on natural killer (NK) cells and a subset of T cells. KLRG1 is a C-type lectin inhibitory receptor (CLIR) that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. In order to investigate the molecular mechanisms underlying KLRG1 function, the T cell hybridoma DO11 was stably transfected with wild-type and mutant KLRG1. Using these cell lines, we show that KLRG1 undergoes phosphorylation upon pervanadate treatment and that phosphorylation occurs at the tyrosine residue. Various signaling molecules were examined for co-precipitation with KLRG1, and we found that KLRG1 associates preferentially with SHIP-1, to a lesser extent with SHP-2 but not with SHP-1. Using site-directed mutagenesis, the Y+3 leucine residue was identified as critical for optimal association of KLRG1 with SHIP-1 whereas the Y+1 serine residue antagonizes KLRG1 association with SHP-2. Partial inhibition of TCR signaling was obtained upon engagement of sorted high KLRG1 expressor, which is dependent on the presence of the ITIM tyrosine residue, highlighting the physiological role of the KLRG1 molecule on T cells. Supported by NIH grant AI58181





A029

Role of NK Cells in Dominant H2k-linked Innate Murine Cytomegalovirus Resistance in MA/My mice. Michael G. Brown, Abhijit Dighe, Xuefang Xie, Marisela Rodriguez and Pearl Sabastian. Division of Rheumatology and Immunology, Departments of Internal Medicine and Microbiology, University of Virginia, Charlottesville, VA 22908.

Innate resistance in acute murine (M) CMV-infected C57BL/6 mice requires Ly49H+ NK cells. Interestingly, we recently found that MCMV resistance in NZW mice involves Ly49H-independent NK cell-mediated immunity even though NZW NK cells display Ly49H-like receptors. Herein, we studied MCMV resistance in MA/My mice since their NK cell receptors do not bind anti-Ly49H mAb or soluble m157 MHC class I-related viral ligands. Because NKC-Ly49 haplotypes in MA/My and C57L are related, but their innate MCMV defenses are quite different, we studied MCMV immunity in their hybrid offspring using classical genetics strategies. We used quantitative real-time PCR to assess MCMV control traits in (C57L x MA/My) x C57L backcross and (C57L x MA/My)F2 intercross offspring. Individual offspring genomes were subsequently genotyped using a genome-wide panel of simple sequence length polymorphism (SSLP) markers. Quantitative trait loci (QTL) were subsequently mapped using SSLP marker regression and permutation analyses. We report that H2k-linked QTL determine ~35% of the genetic variance associated with MCMV control in this genetic system, including MHC and non-MHC locations. The H2b haplotype most frequently, but not absolutely, correlated with MCMV susceptibility, thus confirming a role for non-MHC genes in MCMV control. To extend our findings, we confirmed a dominant role of H2k-linked genes in MCMV immunity in our novel C57L.M-H2k-NKCmamy interval specific congenic strains. Moreover, we found that H2k-linked resistance is contributed in part through NK cells since their removal from C57L.M-H2k mice prior to MCMV infection diminished MCMV resistance. Taken together, effective NK cell-mediated MCMV control in this genetic system required polymorphic H2k genes without need of Ly49H-m157 interactions. Our data implicate a broad antiviral role for NK cells in MCMV control using multiple independent defense mechanisms distinguished by genetic diversity.





A030Separate signals for granule polarization and degranulation control target cell lysis by NK cells. Yenan T. Bryceson1,2, Michael E. March1, Hans-Gustaf Ljunggren2, and Eric O. Long1

1Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA. 2Center for Infectious Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden.

Natural killer (NK) cells express multiple receptors that bind ligands on target cells. Due to the multiplicity of receptor–ligand interactions during NK–target cell contact, the relative contribution of each receptor to NK cell activation has been difficult to assess. By using insect cells that express ligands of human NK cell receptors, we have shown that cytotoxicity by freshly isolated, resting NK cells is controlled by separate signals for granule polarization and degranulation. ICAM-1 on insect cells was sufficient to induce LFA-1–dependent granule polarization, but not degranulation. Conversely, binding of the Fc receptor CD16 to IgG–coated insect cells was sufficient to induce degranulation, but not polarization. Combined, these two signals resulted in efficient cytotoxicity by resting NK cells. Furthermore, ligands of natural cytotoxicity activation receptors (e.g. NKG2D, 2B4, DNAM-1) induced degranulation only when combined in specific synergistic pairs. Therefore, functional activation of resting NK cells is tightly regulated and occurs by mutual co-stimulation of non-activating receptors. The results reveal complex hierarchy, synergy, and redundancy among activation receptors on NK cells.





A031IL-21 downregulates NKG2D/DAP10 expression and function in primary human NK cells. Steven J. Burgess, Alina Marusina, Ishani Pathmanathan, Francisco Borrego and John E. Coligan.Receptor Cell Biology Section, laboratory of Allergic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD USA, 20852.

Natural Killer (NK) cells function to eliminate tumorigenic and virally infected cells without prior sensitization. To prevent inappropriate NK cell activation and cellular cytotoxicity, NK cells express several types of inhibitory receptors that function to suppress activation receptor signals. Upon cellular stress, such as viral infection or malignant transformation, host cells can orchestrate their own destruction, in part, by upregulating ligands for NK activation receptors such as NKG2D/DAP10 complex. NK cells require cytokines, namely IL-2 and IL-15, among others, for proliferative and effector functions. IL-21 is a recently described cytokine produced by activated CD4+ T cells and is proposed to modulate T, B and NK cell function. Thus, the fact that the in vivo source of IL-21 is from activated CD4+ T cells may represent a novel regulatory link between adaptive and innate immunity. IL-21 signals not only through IL-21R, but also interacts with a common gamma cytokine receptor chain, also shared by IL-2, IL-7, IL-9 and IL-15 receptors. We are analyzing the mechanisms of how IL-21 functions to modulate human NK cell activity with particular emphasis on NKG2D/DAP10 activation receptor.We report that in vitro culturing of human primary NK and CD8 T cells with IL-21 in combination with IL-2 results in significant reduction of the cell surface expression of NKG2D/DAP10, compared to cells treated with IL-2 alone. Moreover, we observed a 20% reduction in cytolytic activity of IL-21 treated human NK cells towards P815 target cells in an NKG2D redirected lysis assay. IL-21 was also able to significantly reduce NKG2D-mediated degranulation compared to IL-2 treated control cells. The mechanisms of IL-21-mediated NKG2D/DAP10 down regulation occurred at the level of transcription, as IL-21 treatment of cells was able to dramatically reduce the reporter activity of a DAP10-luciferase construct and DAP10 transcripts as assessed by real time RT-PCR compared with IL-2 treated cells. Interestingly, IL-21 was able to upregulate expression of other NK activation receptors NKP-30 and 2B4 as well as the





inhibitory receptor CD94/NKG2A. These data suggests that IL-21 can, in part, exert its regulatory effect on NK cells by modulating the expression of recognition receptors which, intriguingly, involves the down regulation of NKG2D/DAP10 activation receptor.





A032NK cell Interaction with Freshly Isolated Ovarian Carcinomas Trigger Degranulation and Cytotoxicity. Mattias Carlsten1,2, Niklas Björkström2, Yenan Bryceson2, Håkan Norell1, Kjell Schedvins3, Rolf Kiessling1, Hans-Gustaf Ljunggren2, Karl-Johan Malmberg2

1Immune and Gene Therapy Laboratory, Cancer Center Karolinska, Department of Oncology and Pathology, Karolinska Institutet, S-171 76 Stockholm, Sweden, 2

Center for Infectious Medicine, Karolinska University Hospital Huddinge, S-14186 Huddinge, Sweden, 3Department of Obstetrics and Gynecology, Karolinska University Hospital Solna, S-171 76 Stockholm, Sweden.

KIR-HLA mismatched allogeneic stem-cell transplantation (SCT) is associated with NK cell mediated allo-reactivity against hematopoietic tumors. However, the role of NK cells in allogeneic SCT against solid malignancies remains elusive. Here, we demonstrate that resting and short-term IL-2 activated, allogeneic NK cells specifically recognize and kill ovarian carcinoma cells obtained from peritoneal effusions during primary surgery. All ovarian cancers expressed low levels of HLA class I and induced degranulation in resting and IL-2 activated NK cells independent of KIR-HLA donor-recipient matching. Ovarian carcinomas displayed heterogeneous expression of ligands for NKG2D, DNAM-1 and NKG2A/C. Although NK cell degranulation and tumor cell lysis were influenced by specific ligand-receptor interactions, the overall capacity of NK cells to recognize ovarian carcinomas could not be predicted by the donor and recipient HLA class I genotypes or by their respective NK receptor and ligand repertoires. In summary, we identify ovarian carcinoma as a novel candidate for allogeneic NK cell therapy and demonstrate that donor selection may require individualized functional analysis.





A033Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells. James R. Carlyle 1 , Aruz Mesci1, Belma Ljutic1, Simon Belanger2, Etienne Rousselle2, Marie-France Proteau2, & Andrew P. Makrigiannis2

1Sunnybrook & Women’s Research Institute, University of Toronto, ON, Canada2Laboratory of Molecular Immunology, Institut de Recherches Cliniques de Montréal, QC, Canada

NK cells in selected mouse strains have long been defined using NK-1 alloantigen-specific antisera or the anti-NK1.1 mAb, PK136. Strain-dependent NK1.1 reactivity of mouse NK cells was thought to be due to selective NKR-P1 expression in B6 but not in BALB/c mice. However, closely-related NKR-P1 family members also possess differential NK1.1 reactivity: NKR-P1B shares the NK1.1 epitope with NKR-P1C; other NKR-P1’s do not react with PK136. Thus, the BALB/c defect could be due to NKR-P1 allelic differences, defects in NKR-P1 expression, or both. To address this, we have undertaken NK1.1 epitope mapping and genomic mapping of the BALB/c Nkrp1 region.

The BALB/c Nkrp1-Ocil/Clr locus gene organization was deduced using a BAC library. Positive genomic clones were identified by PCR and sequencing. Like the Ly49 gene repertoire, the BALB/c Nkrp1/Clr repertoire has significantly diverged compared to that of the B6 haplotype. Strikingly, the B6 Nkrp1d gene appears to represent a divergent allele of the Nkrp1b gene in BALB/c and other mouse strains. Notably, BALB/c NK cells express abundant and functional NKR-P1B and NKR-P1C transcripts, and comparison to known NK1.1 alloantigen sequences reveals only a few differences. Epitope mapping studies demonstrate that PK136 mAb recognizes, in part, a distal C-terminal epitope shared by NKR-P1BSw/SJL/P1CB6 but absent in NKR-P1A/D/FB6 and NKR-P1B/CBALB. Mutation of a single amino acid in the NKR-P1BBALB cDNA confers NK1.1 reactivity. In addition, NKR-P1BBALB, like NKR-P1BSw/SJL, was found to be a functional receptor for Clrb. Thus, allelic divergence of the Nkrp1b/c gene products and divergence of the BALB/c Nkrp1/Clr region explains a longstanding confusion regarding the strain-dependent NK1.1 alloantigen reactivity of mouse NK cells.

Supported by a Career Development Award from the HFSP (JRC), a New Investigator Award from the CIHR (APM), and grants from the CIHR (JRC, APM).





A034Epigenetic Control of Highly Homologous Killer Immunoglobulin-like

Receptor Gene Alleles. Huei-Wei Chan 1 , Jeffrey S. Miller2, Mikel B. Moore1,

Charles T. Lutz1*Departments of Pathology and Laboratory Medicine, and Microbiology, Immunology and Molecular Genetics, Markey Cancer Center, University of

Kentucky, Lexington, Kentucky1; Department of Medicine, University of

Minnesota, MMC 806, 420 Delaware St. SE, Minneapolis, MN 554552

Mature human natural killer (NK) lymphocytes express the highly homologous killer immunoglobulin-like receptor (KIR) genes in a stochastic fashion and KIR transcription precisely correlates with allele-specific DNA methylation. Here we demonstrate that CpG methylation of a minimal KIR promoter inhibited transcription. In peripheral blood NK cells and long-term cell lines, expressed KIR genes were associated with a moderate level of acetylated histone H3 and H4 and trimethylated histone H3 lysine 4. Histone modifications were preferentially associated with the transcribed allele in NK cell lines with monoallelic KIR expression. Although reduced, a substantial amount of histone acetylation and H3 lysine 4 trimethylation also was associated with nonexpressed KIR genes. DNA hypomethylation correlated with increased chromatin accessibility, both in vitro and in vivo. Treatment of NK cell lines and developing NK cells with the DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine, caused a dramatic increase in KIR RNA and protein expression, but little change in histone modification. Our findings suggest that KIR transcription is primarily controlled by DNA methylation.





A035Anti-HIV activity of the MHC non-restricted human cytotoxic T cell line TALL-104.J. Chehimi, L. Azzoni, L. Shawver, M. Farabaugh, SA. Creer, and L. J. MontanerThe Wistar Institute, 3601 Spruce Street, Room 480, Philadelphia PA 19104 USA

Background: While actively investigated, immune-based therapy approaches exploiting MHC-restricted effectors such as CD8+T cell face limitations of immune escape, Little is known about the therapeutic potential of anti-HIV, non-MHC-restricted effectors. This study analyzes the antiviral activity of TALL-104, a MHC non-restricted human cytotoxic T cell line, (CD3/TCRab+ CD8+ CD56+ CD16-) that has been extensively characterized as an adoptive immune therapy tool for the treatment of cancer patients in pre-clinical studies. Methods: In vitro HIV (HIV-IIIB and Ba-L) replication in TALL-104 was assessed measuring p24 production by ELISA. TALL-104 cytotoxic function to ACH2 and U1 cells, and cell lines infected in vitro with Influenza virus, CMV and HIV was evaluated in standard 51Cr release assays (E:T ratios from 1:1 to 25:1). The effect of TALL-104 cells on HIV replication in chronically infected SUP-T1 (HIV-IIIB), PHA-PBMC (HIV-NL4-3) and monocytes-derived macrophages (MDM, HIV-89.6) was assessed measuring p24 production in cell-free supernatants and cell lysates.by ELISA. Expression of Fas-L, TRAIL and activating cytotoxic receptors NKp46 and NKG2D was determined by flow cytometry.Results: TALL-104 cells are not permissive to either X4 or R5 viruses. TALL-104 lysed latently infected U1 and ACH2 cells with a higher (3-4 fold) magnitude when HIV-1 replication was induced by TPA. TALL-104 cytotoxic function extends to other cell types infected in vitro with Influenza (PR-8), CMV or HIV-1 (5-10 fold over uninfected control cells). TALL-104 cells potently inhibited HIV-1 replication in chronically infected SUP-T1 (strain IIIB, 30-90% inhibition), PBMC (strain NL4-3, 70-80% inhibition) and MDM (strain 89.6, 75%-95% inhibition) without exerting direct cytotoxicity toward uninfected PHA-PBMC and MDM. Our results demonstrate that TALL-104 cells express Fas-L, TRAIL and activating cytotoxic receptors NKp46 and NKG2D which may represent potential mechanisms for the observed TALL-104-mediated anti-HIV activity.Conclusions: We present evidence documenting the ability of TALL-104 cells to inhibit in vitro viral replication in acutely and chronically HIV-infected cells. TALL-104s anti-HIV activity identifies a potential new non-MHC-restricted approach to





inhibit HIV-1 replication.





A036Dendritic Cell Requirement to Activate NK Cytotoxic Activity Against Viral infected but not against Tumor targets in HIV-1 Infected Individuals. J. Chehimi*1, L. Azzoni1, C. Tomescu1, M. Farabough1, S.A. Creer1, J. Ondercin2, J. Shull2, K. Mounzer2, J. Kostman3, L. J. Montaner1

1The Wistar Institute, 3601 Spruce Street, Room 480, Philadelphia PA 19104 USA; 2Philadelphia Field Initiating Group for HIV-1 Trials, Philadelphia, PA, USA; 3Philadelphia Field Initiating Group for HIV-1 Trials and the Div. of Infectious Diseases, Univ. of Pennsylvania, Philadelphia, PA, USA

Background. Dendritic cells (DC) and natural killer (NK) cells frequencies and function are impaired in HIV infection. This study analyzes the role of plasmacytoid (PDC) and myeloid (MDC) DC in regulating NK cytotoxic activity to virally infected and tumor targets in HIV-1-infected individualsMethods. PBMC from ART-suppressed (<50 RNA c/ml) and viremic (>100,000 c/ml) HIV-1 infected individuals and uninfected controls were used as sources of DC and NK cell subsets. DC subsets and NK were enriched using magnetic beads or erythrocyte rosetting. HLA-DR+ cells were depleted from PBMC by complement-mediated lysis. NK cytotoxicity was tested in a 51Cr release assay using HSV-infected fibroblasts or K562 target cells. Direct cell contact requirements between NK and DC subsets were assessed using trans-well cultures: NK cell activation was evaluated measuring CD69 surface expression by flow cytometry after co-culture with DC primed with or without CpG-2216, IFN-a or Influenza-PR8.Results. HLA-DR+ cells were required for NK cell-mediated lysis of Herpes-infected targets (35.45% vs. 6.75% for HLA-DR-); no requirement for HLA-DR+ cells was observed with K562 target cells (48.75% vs. 41.8% for HLA-DR-. The addition of HLA-DR+ cells from suppressed HIV+ donors (<50 c/ml), but not from viremic donors (> 100,000 c/ml) to HLA-DR- from HIV- controls restored NK cytotoxicity against infected targets (38.6% compared to 48.5% for total PBMC). Determination of the cell subset regulating NK responses was addressed by testing the effects of highly enriched PDC and MDC cells derived from suppressed individuals as compared to controls. Our results indicate that PDC had a greater permissive role than MDC for NK-mediated killing of infected targets (PDC= 29.5%, MDC= 13.9%, total PBMC= 35.9 % for HIV+ donors; PDC=33.31%, MDC=9.47%; total PBMC=44.64% for controls). The study of CD69 expression on NK cells in permeable membrane (trans-well) based culture experiments supported a major role for soluble factors (IFN-a) as the mediator of PDC accessory function in NK cell activation and cytotoxicity.





Conclusions. Our data indicate that in HIV infection, NK responses against Herpes-infected targets depend on the presence of both mature NK and functional DC subsets with greater activity from PDC subsets. Taken together, our results support a critical role for HLA-DR+ cells/PDC in regulating NK cell cytotoxic responses against virally infected cells, but not tumor targets, where no evidence of HLA-DR+ cell-mediated help was observed.





A037Suppression of Tumor Formation in Lymph Nodes by L-Selectin-Mediated Natural Killer Cell Recruitment. Shihao Chen 1 , Hiroto Kawashima1, John B. Lowe2, Lewis L. Lanier3, 4, and Minoru Fukuda1, 4. 1Glycobiology Program, The Burnham Institute, La Jolla, CA 92037. 2Howard Hughes Medical Institute, Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan 48109. 3Department of Microbiology and Immunology and the Cancer Research Institute, University of California San Francisco, 513 Parnassus Ave., HSE 1001G, Box 0414, San Francisco, CA 94143-0414. 4Contributed equally to this work.

Natural killer (NK) cells are known to reject certain tumors in vivo; however, the ability of NK cells to prevent metastasis of tumors into secondary lymphoid organs has not been addressed. Here, we report that in tumor-bearing hosts, NK cells are recruited to regional lymph nodes in wild-type mice, but not in mice deficient for L-selectin or L-selectin ligands. By adoptive transfer and complete Freund’s adjuvant stimulation experiments, we demonstrated that L-selectin on NK cells and L-selectin ligands in endothelial cells are essential for NK cell recruitment to lymph nodes. Furthermore, freshly isolated resident lymph node NK cells lysed tumors efficiently, and metastasis of B16 melanoma cells to draining lymph nodes was suppressed in wild-type or RAG-1-deficient mice, but not when NK cells were depleted. Although L-selectin deficient NK cells lysed tumor cells in vitro as efficient as wilt-type NK cells, NK cell-dependent suppression of tumor metastasis was diminished in mice deficient for L-selectin or L-selectin ligands, presumably as a result of insufficient NK cell recruitment to the lymph nodes. These findings indicate that L-selectin-mediated NK cell recruitment plays a crucial role in the control of tumor metastasis into secondary lymphoid organs.





A038KIR2DS1 is an activating NK Receptor with Ligand specificity for HLA-Cwlys80 Group C2 molecules in Individuals that lack HLA-CwLys80 alleles. Joseph H. Chewning, Annamalai Selvakumar, Xiao-Rong Liu, Charlotte N. Gudme, Bo Dupont. Immunology Program and Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY USA.

HLA class I ligand specificity for inhibitory KIRs is well established for the HLA-Cw3 (C1-group=CwAsn80) with KIR2DL2/3, HLA-Cw4 (C2-group=CwLys80) with KIR2DL1 and HLA-B-Bw4 with KIR3DL1. C2 molecules also demonstrate weak binding to 2DS1, while no interaction between the C1-group and 2DS2 has been detected. We have investigated a possible relationship between the HLA-Cw and KIR2DS1 genotypes and 2DS1+ NK cells that have cognate ligand specificity for C2 group molecules. We demonstrate that individuals positive for 2DL1,2DS1 and homozygous for C1 group HLA-Cw alleles consistently contain NK cells with functional 2DS1 receptors. These NK cells are activated by ligand binding to C2 group positive target cells. NK activation is induced by all tested C2 group alleles: Cw*0202,*0401,*0501,*0602,*1602 and *1701. C2 molecules induced cytotoxicity by IL2-cultured bulk NK cell lines against BLCLs homozygous for Bw4,C2 group (Mean(M)=50%; Range(R)=44-59%) and Bw6,C2 group (M=25%; R=22-30%). 2DS1 positive NK clones (N=24) were activated by C2 group molecules and cytotoxic against Bw4,C2 positive BLCL target cells: M=51%; (R)= 30-59% and Bw6,C2 positive targets:M=49%;R=26-54%. Cytotoxicity was inhibited by anti-KIR2DL/S1(EB6): M=13% against Bw4,C2 and M=12% against Bw6,C2. mAb4E(anti HLA-B/Cw,Fab2) also inhibited the C2 ligand induced cytotoxicity to M=7% on Bw4,C2 targets and M=20% on Bw6,C2 targets. 19 additional NK clones were 2DS1 and 2DL2/3. Similar to the 2DS1 single positive NK clones, they displayed cytotoxicity against C2 group positive target cells: Bw4,C2:M=54%; Bw6,C2:M=48%. EB6 and 4E effectively blocked the cytotoxicity. In parallel experiments, the same NK clones were inhibited when tested in cytotoxicity against 721.221+Cw*0304 positive target cells while cytotoxic against 721.221. Interferon-gamma production was increased on recognition of Cw4-expressing target cell lines by both freshly isolated, uncultured NK cells and by IL-2 propagated EB6 positive NK clones. EB6/KIR2DS1 was identified within the cSMAC of the activated NK immune synapse formed by EB6 positive NK clones with C2-expressing cell lines, but 2DS1 was not located in the synapse of 721.221. This study demonstrates that





KIR2DS1 is a functionally active C2 group specific, activating receptor on NK cells when present in donors that are lacking the cognate HLA-Cw C2 group of HLA alleles. Upon ligand binding, NK cell mediate cytotoxicity and interferon-gamma production in both IL-2 cultured and freshly isolated NK cells. These findings may have important implications for matching of donors and recipients for allogeneic hematopoietic stem cell transplantation.





A039Multiplicity and plasticity of Natural Killer cell signaling pathways. Sabrina Chiesa1, Michael Mingueneau1, Nicolas Fuseri1, Bernard Malissen1, David H. Raulet2, Marie Malissen1, Eric Vivier1 and Elena Tomasello1

1Centre d’Immunologie de Marseille-Luminy, INSERM – CNRS – Université de la Méditerranée, Marseille Cedex 09, France; 2Department of Molecular and Cell Biology, University of California, Berkeley CA 94720-3200

Natural Killer (NK) cells express an array of activating receptors that associate with DAP12 (KARAP), CD3 and/or FcR ITAM (Immunoreceptor Tyrosine-based Activation Motif)-bearing signaling subunits. In T and mast cells, ITAM-dependent signals are integrated by critical scaffolding elements such as LAT (linker for activation of T cells) and NTAL (non-T cell activation linker). Using mice that are deficient for ITAM-bearing molecules, LAT or NTAL, we show that NK cell cytotoxicity and interferon- secretion are initiated by ITAM-dependent and –independent as well as LAT/NTAL-dependent and -independent pathways. The role of these various signaling circuits depends on the target cell as well as on the activation status of the NK cell. The multiplicity and the plasticity of the pathways that initiate NK cell effector functions provide a rationale for their robustness to multiple pharmacological agents and genetic mutations in both human and mouse.





A040Effective NK cell and KIR reconstitution after hematopoietic cell transplantation (HCT) is affected by stem cell source and graft type and correlates with clinical outcomes. Sarah Cooley, Valarie McCullar, Rosanna Wangen, Tracy L. Bergemann, Stephen Spellman, Daniel J. Weisdorf, and Jeffrey S. Miller. University of Minnesota, MMC 806, 420 Delaware St. SE, Minneapolis, MN 55455 USA.KIR ligand-mismatched T-cell depleted (TCD) HCT results in improved clinical outcomes but unrelated unmanipulated bone marrow (UBM) transplants using the same strategy do not. We hypothesized that NK cell and KIR reconsitiution may be affected either by the T-cell content of the graft or by the stem cell source. To evaluate the effect of the T-cell depletion status we analyzed PBL collected by the National Marrow Donor Program after 77 unrelated transplants (40 TCD, 37 UBM). Although recipients of both transplant types had an approximate 4-fold increase in NK cells compared to their donors, the KIR expression was suppressed, significantly more so after UBM transplants (Donor: 48.42 ± 2.35% versus Recipient: 26.74 ± 1.94, n = 36; P < .001) than after TCD transplants (Donor: 53.34 ± 3.25% versus Recipient: 42.68 ± 3.32, n = 38; P = .017). The percentage of recipient NK cells producing IFN- (measured by intracellular cytokine staining) was significantly higher after UBM than after TCD transplants (UBM: 53.96 ± 4.47%; TCD 34.86 ± 5.75%, P = .007). Multivariate Cox proportional hazards models showed a correlation between increased NK cell IFN- production and the development of acute graft-versus-host-disease, and between decreased KIR expression and inferior survival. Because of interest in umbilical cord blood as an alternative stem cell source allowing more HLA disparity we compared NK and KIR reconstitution after cord blood, autologous (Auto), allogeneic sibling (AlloSib) and unrelated (TCD or UBM) transplants. While the KIR expression on recovering NK cells after all transplant types was significantly less than that on normal donor cells (all with p < .0006), the UBM recipients had significantly less KIR recovery when compared to the other transplant types (which were all similar). For example, the KIR reconstitution is more normal after umbilical cord blood transplants than after allogeneic UBM transplants (Cord: 37.99 ± 2.54%; UBM: 27.31 ± 2.06%, P = .0027). These results show that in adult unrelated donor transplants T-cells in the graft affect in vivo NK and KIR reconstitution with clinically significant consequences. Graft T-cells may directly compete for cytokines and growth factors, or be a surrogate marker for other transplant factors such as the degree





of post-transplant immunosuppression. In unmanipulated grafts derived from other stem cell sources, however, KIR expression is similar to that seen in adult unrelated TCD grafts, suggesting that the stem cell source also affects the degree of KIR reconstitution. Further investigation of the underlying mechanisms will allow for selection of the optimal combination of transplant characteristics to improve clinical outcomes.





A041Altered NK cell functions induced by chronic exposure to NKG2D-ligand expressing cells. Jérôme D. Coudert*, Marco Colonna‡, Eric Vivier† and Werner Held*.* Ludwig Institute for Cancer Research, Lausanne Branch; Epalinges SWITZERLAND.‡ Department of Pathology and Immunology, Washington University School of Medicine; 660 S. Euclid; St. Louis, MO USA.† Centre d’Immunologie INSERM/CNRS de Marseille-Luminy; Marseille FRANCE.

NKG2D is an activation receptor, which allows NK cells to detect diseased host cells. The engagement of NKG2D with corresponding ligand results in surface modulation of the NKG2D receptor and reduced function upon subsequent receptor engagement. However, it is not clear, whether the NKG2D receptor complex and/or its signaling capacity is preserved.We show that the prolonged encounter with tumor cell-bound, but not soluble, ligand can completely uncouple the NKG2D receptor from the intracellular mobilization of calcium and the exertion of NKG2D-mediated cytolysis. However, cytolytic effector function is intact since NKG2D ligand-exposed NK cells can kill via the Ly49D receptor. While NKG2D-dependent cytotoxicity is impaired, prolonged ligand exposure results in constitutive IFN production. The functional changes are associated with a reduced presence of the relevant signal transducing adaptors DAP-10 and KARAP/DAP-12. This is likely the consequence of constitutive NKG2D engagement and signaling since NKG2D function and adaptor expression is restored to normal when the stimulating tumor cells are removed. Since the chronic exposure to tumor cells expressing NKG2D-ligand alters NKG2D signaling, it may facilitate the evasion of tumor cells from NK cell reactions.We are currently determining whether chronic NKG2D triggering selectively affects NKG2D-dependent effector functions or whether other NK cell activation pathways, e.g. missing self recognition, ADCC or virus-derived protein recognition are also impaired. In addition we are also addressing whether chronic triggering of non-NKG2D NK cell activation pathways negatively regulate NK cell functions. These experiments may allow us to determine whether there is cross-talk between distinct NK cell activation receptors and their signaling pathways.





This work may contribute to a better understanding of NK cell tolerance mechanisms.





A042Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer. Erika Cretney 1 , Yoshihiro Hayakawa1, Dale I. Godfrey2 and Mark J. Smyth1. 1Cancer Immunology Program, Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3001, Australia, 2Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, 3010, Australia.

The CD1d reactive glycolipid, -galactosylceramide (-GalCer), potently activates T cell receptor- type I invariant NKT cells that then secondarily stimulate the proliferation and activation of other leukocytes, including NK cells. Here we report a rational approach to improving the anti-tumor activity of -GalCer by using delayed IL-21 treatment to mature the -GalCer-expanded pool of NK cells into highly cytotoxic effector cells. In a series of experimental and spontaneous metastases models in mice, we demonstrate far superior anti-tumor activity of the -GalCer/IL-21 combination above either agent alone. Superior anti-tumor activity was critically dependent upon the increased perforin-mediated cytolytic activity of NK cells. Transfer of -GalCer pulsed dendritic cells followed by systemic IL-21 caused an even more significant reduction in established (day 8) metastatic burden and prolonged survival. In addition, this combination more effectively prevented chemical carcinogenesis. Combinations of IL-21 with other NK cell activating cytokines such as IL-2 and IL-12 were much less effective in the same experimental metastases models, and these cytokines did not effectively substitute for IL-21 in combination with -GalCer. Overall, the data suggest that NK cell anti-tumor function can be greatly enhanced by strategies designed to expand and differentiate NK cells via DC activation of NKT cells.





A043Differential transcription of Eomes and T-bet during maturation of mouse uterine natural killer cells. 1 B. Anne Croy, 2Chandrakant Tayade, 2Yuan Fang, 2Gordon P Black, VA Paffaro, Junior3, 4Adrian Erlebacher. 1 Department of Anatomy and Cell Biology, Queen’s University, Kingston, ON, Canada K7L 3N6. 2Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph N1G 2W1, Canada. 3 Biological Sciences, Efoa/Ceufe, Alfenas, MG, Brazil. 4Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA 02115.

During endometrial decidualization in humans and rodents, transient but abundant numbers of uterine (u)NK cells appear, proliferate and differentiate. UNK cells share features with peripheral NK cells but are specialized to promote pregnancy-associated structural changes in maternal spiral arteries mediated by production of IFN-. In peripheral NK cells and CD8+ T cells, the transcription factors T-bet and eomesodermin (Eomes) regulate maturation and effector functions, including IFN- production. No studies are reported for uNK cells. Implantation sites in peripheral NK cell-deficient T-bet null mice had uNK cells normal in morphology and number and appropriately modified spiral arteries. Since Eomes null mice are not viable, we used quantitative Real-time PCR for comparisons between C57Bl/6J (B6) and alymphoid (Rag20/0c0/0) mice to assess uNK cell expression of Eomes, T-bet, and the target genes IFN-, granzyme A and perforin. Transcription was compared between gestation dated (gd) endometrial samples (mixed cell composition) and 200 morphologically homogeneous, laser capture micro-dissected uNK cells of different maturation stages. In endometrium, Eomes transcripts greatly outnumbered those of T-bet, whether donors were non-pregnant or pregnant, and increased to gd10. T-bet expression continued to increase to gd12 in B6 and gd18 in alymphoid mice. In uNK cells, transcripts for T-bet, Eomes and IFN- were most abundant in mature cells while transcripts for Granzyme A and perforin were more abundant in immature cells. Thus, Eomes dominance to T-bet discriminates regulation of the uNK cell subset from that observed for peripheral NK cells.Supported by NSERC and the Canada Research Chair’s Program.





A044Studies of structurally related Ly49 receptors in rats. 1Ke-Zheng Dai, 1Christian Naper, 2Mike Daws, 2Lise Kveberg, 2Erik Dissen, 2Bent Rolstad, 3James C. Ryan, and 1John T. Vaage. 1Institute of Immunology (IMMI), Rikshospitalet University Hospital, Songsvannasu 20, Oslo 0027 Norway;

2Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway; and the 3Veterans Affairs Medical Center, the Northern California Institute for Research and Education and the University of California, San Francisco, CA

Rat NK cells that possess certain NKC alloresponder genes mediate the prompt rejection of allogeneic recirculating lymphocytes, a rejection phenomenon termed allogeneic lymphocyte cytotoxicity (ALC). Using mAb DAR13 we previously identified Ly49s3 (Ly49 stimulatory receptor 3) as a candidate ALC alloresponder gene. The Ly49s3 receptor binds nonclassical class Ib-encoded MHC allodeterminants on lymphoblast target cells and triggers natural killing. Here we report that mAb DAR13 not only reacts with Ly49s3, but also with three other structurally related Ly49 molecules with opposing functions, Ly49s4, Ly49i3 (Ly49 inhibitory receptor 3) and Ly49i4. Using DAR13 and a novel mAb STOK6, which is specific for Ly49s3, we show that expression of these three Ly49 family members varies markedly among different strains in a pattern related to their NKC genotypes. STOK6 staining of NK cells from MHC congenic rat strains confirmed that Ly49s3 functions as an MHC receptor, since it is down-regulated on the cell surface in the presence of its putative class Ib ligands. We are currently searching for the molecular ligand for Ly49s3 and its three close relatives, as well as for a separate set of structurally related Ly49 receptors, Ly49s5 and Ly49i5.





A045The number of circulating B cells directs NK cells towards rituximab-mediated ADCC and IFN production. Sébastien Dall’Ozzo, Nicolas Congy-Jolivet, Shehrazade Kantari, Guillaume Cartron, Chantal Le Guellec, Gilles Paintaud, Pierre Bardos, Hervé Watier, Gilles Thibault. EA3853 « Immuno-Pharmaco-Genetics of therapeutic Antibodies », Laboratoire d’Immunologie, Faculté de Médecine, Université François Rabelais, 10 boulevard Tonnellé 37032 Tours, Cedex, France. ADCC and antibody-dependent cytokine production (ADCP) by NK cells are not coordinately regulated although both are FcRIIIa-dependent. Previous results support ADCC as a major mechanism for the therapeutic effect of Rituximab (RTX)(a chimeric anti-CD20 mAb) in non-Hodgkin lymphoma (NHL) and suggest that it may be a less important mechanism in Chronic Lymphocytic Leukemia (CLL). A cytokine-release syndrome (CRS) has been observed during RTX infusion in patients with high number of circulating tumor B cells (as observed in CLL) and it has been assumed that FcRIIIa engagement on NK cells may be responsible for CRS. The fact that NK cells may be preferentially directed to ADCC in NHL and to ADCP in CLL suggests that the number of circulating B cells may influence these RTX-mediated responses. To test this hypothesis, blood samples with NK:B ratios ranging from 1.1:1 to 2.8:1, were firstly incubated with 0.1g/ml to 1000g/ml of RTX. In these conditions, a B-cell depletion was detected at concentrations≥1g/ml. It was similar in NK cell-depleted blood with ratios ranging from 0.03:1 to 0.45:1, whereas it was dramatically enhanced at concentrations≤10g/ml in autologous NK cell-enriched blood with ratios ranging from 12.4:1 to 28.2:1. A Depletion of B and NK cells was observed 3h after the onset of RTX infusion in NHL patients. Then, NK cell counts recover rapidly resulting in high NK:B ratio (≥30:1) 24h after infusion. On the other hand, IFN+ NK cells were detected in blood samples from 3 out of 4 CLL patients with ratios ranging from 0.005:1 to 0.05:1 but not in those from healthy donors at RTX concentration≥1µg/ml. Using Daudi cells, we observed that the % of IFN+ NK cells increased when the E:T ratio decreased from 10:1 to 0.5:1, conversely to ADCC. In addition the concentation-effect relationship of RTX-mediated ADCC and ADCP were different. Thus, weak FcRIIIa engagement by low number of B cells (high NK:B ratios) and low RTX concentrations is sufficient to sustain ADCC, whereas substantial ADCP requires high FcγRIIIa engagement by high number of B cells (low NK:B ratios) and high RTX concentrations. The NK:B ratio





is thus essential to direct the RTX-mediated NK cell functions. This conclusion is consistent with the observed effects of RTX in vivo.





A046Potential role of NK cells in AIDS-associated primary effusion lymphoma cell growth and infiltration: Implication of immune therapy. Md. Zahidunnabi Dewan,1,2 Hiroshi Terunuma,3,4 Xuewen Deng,3 Hiroyuki Abe,4 Yuetsu Tanaka,5 Mitsuo Honda2and Naoki Yamamoto1,2

1Department of Molecular Virology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,Tokyo 113-8519, Japan; 2AIDS Research Center, National institute of Infectious Disease, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; 3Biotherapy Institute of Japan, 2-4-8 Edagawa, Koutou-ku, Tokyo 135-0051, Japan; 4Kudan Clinic, 1-9-5 Kudankita, Chiyoda-ku, Tokyo 102-0073, Japan; 5Department of Infectious Disease and Immunology, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan

NK cells are an important component of the innate immune response against viral infections. This study demonstrates a direct role of NK cell in PEL cell growth and infiltration in vivo. PEL cell line BCBL-1 was inoculated either in NOD/SCID or NOD/SCID/cnull mice. BCBL-1 cells were able to produce tumor at inoculated site in NOD/SCID (T and B cell knock-out) mice with NK cells while completely failed to infiltrate into various organs. Immunosupression of NOD/SCID by treatment with an anti-murine antibody TM-1, which transiently abrogates NK cell activity in vivo, resulted in successful engraftment of tumor growth and infiltration in comparison with non-treated NOD/SCID mice. In contrast, NOG (T, B and NK cell knock-out) mice inoculated with BCBL-1 cells were most efficient in the formation of a large tumor and metastasis within 3 weeks. Adoptive transfer of activated human NK cells inhibited tumor growth and infiltration in NOG mice. Our results suggest that NK cells play an important role in primary growth and infiltration of PEL cells in SCID mice. This new xenotransplant model is relevant and can be recommended for use in clarifying the mechanism of growth of cancer cells as well as for developing new therapeutic strategies against cancer.





A047Functional analysis of NK cells in breast cancer patients and their role in primary and metastatic tumor growth. Md. Zahidunnabi Dewan,1,2 Hiroshi Terunuma,3,4 Masahiro Takada,5 Daisuke Nakano,5 Xuewen Deng,3 Hiroyuki Abe,4 Yuetsu Tanaka,6 Masakazu Toi5 and Naoki Yamamoto1,2

1Department of Molecular Virology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,Tokyo 113-8519, Japan; 2AIDS Research Center, National institute of Infectious Disease, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; 3Biotherapy Institute of Japan, 2-4-8 Edagawa, Koutou-ku, Tokyo 135-0051, Japan; 4Kudan Clinic, 1-9-5 Kudankita, Chiyoda-ku, Tokyo 102-0073, Japan; 5Division of Clinical Trials and Research, Breast Cancer Research and Treatment Program, Tokyo Metropolitan Komagome Hospital, Tokyo Medical Center for Cancer and Infectious Disease, 3-18-22 Honkomagome, Bunkyu-ku, Tokyo 113-8677, Japan; 6Department of Infectious Disease and Immunology, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan

NK cells play a central role in host defense against tumor and virus-infected cells. Functional defects of NK cells in breast cancer and their direct role in tumor growth and metastasis have not yet been clearly demonstrated. In the present study, we examined the phenotypic and functional characteristics of NK cells in breast cancer patients. Non-activated NK cells isolated from breast cancer patients were exhibited significantly lower cytolytic activity in vitro. Cytolytic activity of NK cells after in vitro stimulation was dramatically increased as compared with freshly isolated NK cells. In addition, we investigated the role of NK cells to induce primary and metastatic tumor of breast cancer cells in SCID mice. Breast cancer cell line MDA-231 was able to produce tumor at inoculated site in NOD/SCID (T and B cell knock-out) mice with NK cells while completely failed to metastasize into various organs. Immunosupression of NOD/SCID by treatment with an anti-murine antibody TM-1, which transiently abrogates NK cell activity in vivo, resulted in successful engraftment of tumor growth and metastasis in comparison with non-treated NOD/SCID mice. In contrast, NOD/SCID/cnull (T, B and NK cell knock-out) mice inoculated with MDA-231 cells were most efficient in the formation of a large tumor and metastasis within 3 weeks. Adoptive transfer of activated human NK cells inhibited tumor growth and metastasis in vivo. Our results suggest that NK cells play an important role in





tumor growth and metastasis of breast cancer cells. Thus, activated NK cells could be a promising immunotherapeutic strategy against breast cancer either combination with conventional therapy or single.





A048Involvement of natural killer T cells in the impaired liver regeneration in HBV transgenic mice. Zhongjun Donga, Jian Zhang b , Haiming Weia, Yongyan Chena, Rui Sunabc, Zhigang Tianab. aInstitute of Immunology, University of Science and Technology of China, Hefei 230027, China; bSchool of Pharmacy, Shandong University, Wenhua Western Road, Jinan, Shandong 250012, China;

Cytotoxic T lymphocytes (CTL) have always been considered as one of critical effector cells in the pathogenesis of hepatitis B virus (HBV) infection. In HBV transgenic (HBV-tg) mice, hepatitis B surface antigen (HBsAg)-specific CTL play a major role in the spread of liver necrosis under antigen non-specific amplification mechanisms. It is speculated that continuous liver necrosis may induce impaired liver regeneration during HBV persistent infection. Apart from these reports, there are few studies focusing on the effect of HBV infection on liver cells proliferation in human and mice. NKT cells are heterogeneous population of innate lymphocytes, in contrast to their relatively low presence in peripheral lymphatic system, NKT cells are quite abundant in the liver, implying an important role of this cell type in liver biology. Using a murine model of HBV infection, we demonstrate in this report that the impaired liver regeneration of HBV-tg mice was related with elevated levels of NKT and their IFN- production. Blockade of CD1d-NKT interaction via systemic administration of anti-CD1d antibody prevented the impaired liver regeneration caused by NKT cells.

Two-thirds partial hepatectomy (PHx) as liver regeneration model was performed in HBV transgenic (HBV-tg) mice and wild mice. The ratio of liver weight to body weight, proliferating cell nuclear antigen (PCNA) labeling and BrdU incorporation were applied to evaluate liver regeneration. The number and activation of hepatic NKT cells were analyzed with flow cytometry. After PHx, the ratio of liver weight to body weight in HBV-tg mice was less than that in wild mice, accompanied with decreased PCNA labeling and BrdU incorporation. NKT cells mainly including CD4+ NKT and double negative NKT cells significantly accumulated in hepatectomized-liver of HBV-tg mice, simultaneously with enhanced interferon gamma (IFN-) production and CD69 expression on hepatic NKT cells. The impaired liver regeneration of HBV-tg mice was ameliorated by NKT cells depletion with specific anti-NK1.1 antibody. Either blockade of CD1d-NKT cell interaction or neutralization of endogenous IFN- enhanced BrdU incorporation





in HBV-tg mice after PHx. NKT cells and their IFN- production may contribute to the impaired liver regeneration of HBV-tg mice.





A049Infection of Dendritic Cells with Influenza Virus Provides General Anti-viral and Flu-specific Signals for Natural Killer Cell Activation: Roles of NKG2D, ULBPs, NKp46 and IFN-. Monia Draghi*, Achal Pashine†, Barathi, Sanjanwala*, Ketevan Gendzekhadze*, David Cosman‡, Alessandro Moretta§, Nicholas M. Valiante† and Peter Parham* *Departments of Structural Biology and Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305; †Vaccines Research, Chiron Corporation, Emeryville, CA 94608; ‡Department of Molecular Biology, Amgen, Seattle, WA 98101; §Department of Experimental Medicine, University of Genova, Genova, Italy 16132

The molecular mechanisms underlying dendritic cell (DC) activation of NK cells in the context of viral infections have not been investigated in detail. Here we used influenza virus infection of human DC in vitro to model the coordinated responses of DC and NK cells to acute viral infection. Activation of resting NK cells after co-culture (18-24 h) with infected or uninfected DC was determined by measuring NK cell IFN- production, lytic activity and CD69 expression. Whereas, enhanced NK cell IFN- production and CD69 expression in response to virally infected DC was contact-dependent and governed by the NKG2D and NKp46 triggering receptors, increased NK lytic activity following co-culture with infected DC was largely dependent on DC IFN- production. Influenza infection increased ULBP1-3 surface expression by DC but did not induce the expression of the other family of human NKG2D ligands, MICA/B. Moreover, neutralizing antibodies to ULBPs or NKG2D, but not MICA/B, blocked NK IFN- production and up-regulation of CD69 in response to infected DC. No other stimulatory NK receptor studied (NKp30, NKp44 nor 2B4) appeared to be involved in the NK-DC response to influenza. Using synthetic double stranded RNA (Poly I:C) to stimulate DC, we were able to reconstitute some but not all of the NK cell stimulatory mechanisms identified for flu-infected DC. Specifically, Poly I:C treated DC stimulated NK activation events (e.g. IFN- production and CD69 expression) that were independent of NKp46 engagement and although NKG2D-ULBP interactions were again central, Poly I:C induced ULBP1 and 2 expression by DC but not ULBP3. Overall, these results indicate that rapid NK activation by DC during acute viral infection is governed by multiple factors, some of which appear specific to the anti-influenza response (e.g. NKp46-HA and NKG2D-





ULBP interactions). This suggests a previously unrecognized level of selectivity exists in the coordinated responses of NK cells and DC to different pathogens.





A050Natural killer cells mediate adaptive immune responses in the absence of T and B lymphocytes. Danielle L. Drayton, Jacqueline G. O’Leary, Mahmoud Goodarzi and Ulrich H. von Andrian. CBR Institute for Biomedical Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115

Immunity can be characterized as either “innate” or “adaptive”. Innate immunity is mediated, in part, by natural killer (NK) cells and macrophages expressing germline encoded receptors that recognize a finite set of pathogen-associated patterns. In contrast, adaptive immunity is thought to be mediated exclusively by T and B cells expressing recombination dependent antigen receptors that can recognize countless antigenic determinants. Moreover, adaptive immunity generates immunological memory such that antigen re-exposure provokes a more rapid and potent immune response. Contact hypersensitivity (CHS) is an adaptive immune response in which hapten application to the skin elicits a local sensitization-dependent inflammatory reaction. Studies in mice have revealed that CHS can be mediated by hapten-specific memory T cells and it is therefore believed that CHS is exclusively T cell dependent. However, we discovered recently that several T and B cell deficient mice, including mice deficient in recombination activating gene-2 (Rag2-/-), exhibit normal CHS responses to the haptens 2,4-dinitroflurobenzene (DNFB) and oxazolone (OXA). In these mice, NK cells mediated long-lived, hapten-specific CHS responses. This conclusion is supported by several observations – 1) Rag2-/- mice depleted of NK cells and Rag2-/- x C-/- mice that lack T, B and NK cells, fail to mount a CHS response to DNFB and OXA. 2) Adoptive transfer of purified NK cells from hapten sensitized Rag2-/- mice to Rag2-/- x C-/- recipients restored hapten-specific CHS responses 3) DNFB sensitized Rag2-/- mice only mounted a CHS response to DNFB, but not OXA and vice versa, and 4) Hapten-specific CHS responses in Rag2-/- mice persisted for at least four weeks following sensitization. Taken together, these data highlight a novel and unexpected role for NK cells in adaptive immunity, namely antigen-specificity and memory, and identify DCs as putative hapten-presenting cells in NK cell mediated CHS.





A051KIR-HLA combination analysis demonstrates the minimal essential KIR receptor in humans. Zeying Du, David W. Gjertson, Elaine F. Reed, and Rajalingam Raja. UCLA Immunogenetics Center, Dept. of Pathology and Lab. Medicine, University of California at Los Angeles, Los Angeles, CA 90095.

Interactions between KIR receptors and HLA class I molecules regulate NK cell function. KIR and HLA are encoded by unlinked polymorphic gene families, and their independent segregation results in the inheritance of a variable number and type of KIR-HLA pairs in individuals. The aim of the present study was to identify the simplest KIR-HLA combination in humans that could enable NK cells to perform the minimal essential function. We have characterized KIR genes in a panel of 800 unrelated individuals representing major ethnic groups. One third of the panel carried the simplest KIR genotype (3DL3-2DL3-2DP1-2DL1-3DP1-2DL4-3DL1-2DS4-3DL2) carrying three non-functional genes --- 2DP1 and 3DP1 are psudogenes, and 3DL3 is poorly transcribed. KIR2DL4 unlikely contributes to peripheral tolerance since it recognizes trophoblast-specific HLA-G. Subtyping of 2DS4 revealed that majority of the population carry only 2DS4*003 allele, which is not expressed due to a deletion in exon-5. Therefore, 12% of our study panel (n=800) does not carry any activating KIR genes, and posses only 4 inhibitory KIRs: 2DL3, 2DL1, 3DL1, and 3DL2. The inhibitory function of these receptors however depends on the presence of their cognate HLA class I ligand HLA-CN80, HLA-CK80, HLA-Bw4, and HLA-A3/11 respectively. HLA typing of these individuals revealed that only 3 Asian origins carried HLA ligands for all 4 inhibitory KIRs. No individual was found lacking HLA class I ligand for all four inhibitory KIR receptors. The majority had only one HLA-C ligand carrying asparagine at amino acid position 80 (N80), which is recognized by the weakly interacting 2DL3 receptor. We determined that 4% of our study population has only one KIR2DL3 receptor:HLA-CN80 ligand combination, which could provide the essential inhibitory signal to NK cells to mediate tolerance to self. Environmental challenges particularly those affecting HLA-C expression in these individuals might breakdown the self-tolerance and trigger autoimmunity.





A052Novel MACS Methods for Separation of Natural Killer Cell Subsets used for subsequent Gene Expression Profiling. Julia Dzionek, Anna Maria Turkiewicz, Stefan Reininghaus, Jürgen Schmitz, Andreas Bosio, Volker Huppert. Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, D-51429 Bergisch Gladbach, Germany

Natural Killer cells, initially regarded as “disturbing noise“ in in vitro experiments, are now regarded as an important (Hokland, Mol Immunol 2005) but heterogenous (Cooper, Blood 2001) lymphocyte subset. Different Natural Killer cell subsets have different functions (Cooper, Blood 2001; Mavilio, PNAS 2005; Trotta, Blood 2005) and further research will be required to identify the role of those NK cell subsets in immune responses.We have developed novel methods, based on magnetic cell sorting, for purification of several NK cell subsets and have evaluated the separation performance of these kits using peripheral blood mononuclear cells from 10 donors. More than 1x105 CD56brightCD16- cells from 1x108 PBMC can be isolated with >70% purity (>85% CD56+CD16-), and more than 1x106 CD56dimCD16+ cells with >94% purity. Additional subsets have been identified based on CD8 expression: >2x106 CD8+ NK cells can be purified with >90% purity.Only limited data has previously been published on gene expression profiling of NK cell subsets using DNA microarrays (Koopman, J Exp Med 2003). We thus have used these MACS sorted NK cell subsets from 3 donors for an immune system specific microarray analysis (“PIQOR Immunology”). Usually gene expression profiling experiments are combined with flow cytometry characterization and functional tests. Therefore, we used a novel amplification method to perform microarray analysis from mRNA isolated from as little as 1000 cells. Genes for surface molecules known to be differently expressed on NK cell subsets and immune effector molecules produced preferentially by single subsets served as control for the combination of amplification and microarray analysis (CD56, CD16, CD8, CD62L, CD122, Perforin, Granzyme B). Results of the analysis will be shown.





A053Chromosome 8 linked to specific NK cells deficiency in a multiplex family. Céline Eidenschenk 1 , Jean Dunne2, Claire Fourlinnie1, Emmanuelle Jouanguy1, Laurent Abel1, Jean-Laurent Casanova1, Conleth Feighery2. 1 Laboratoire de Génétique Humaine des Maladies Infectieuses, Université de Paris René Descartes-INSERM U550, Faculté de Médecine Necker, 75015 Paris, France, EU. 2 Department of Immunology, St. James’s Hospital, Dublin, Ireland.

The role of murine NK cells in protective immunity to viruses is now well established, whereas that of human NK cells remains unclear, due to a lack of well-defined primary immunodeficiency associated with a selective absence of NK cells. We described here four children with a specific NK cells deficiency. These children suffered from recurrent infections, one has developed an Epstein-Barr virus (EBV)-driven lymphoma. Patients were borned to a consanguineous Irish family of gypsy origin, suggesting an autosomal recessive mode of inheritance. We report here on a genome-wide search for the gene controlling the NK cells deficiency in this family. By genotyping 400 highly informative microsatellite markers covering the entire genome at approximately 10-cM intervals, we identified 6 chromosome regions using homozygosis mapping. These regions showed suggestive linkage with NK cells deficiency. We saturated all chromosome regions carrying markers. A unique region showed significant evidence for linkage, on chromosome 8. This region is actually 20Mbp length. A gene candidate strategy is now undertaken to find the molecular bases of NK cells deficiency in human.





A054A novel primary immunodeficiency associated with impaired lymphocyte survival and secondary neutropenia. Céline Eidenschenk1, Emmanuelle Jouanguy1, Jean-Jacques Mention3, Benoît Pasquier2, Anne Puel1, Jean-Claude Carel4, Françoise Le Deist2,5 and Jean-Laurent Casanova1,6. 1 Laboratoire de Génétique Humaine des Maladies Infectieuses, Université de Paris René Descartes-INSERM U550, Faculté de Médecine Necker, 75015 Paris, France, EU. 2 Développement Normal et Pathologique du Système Immunitaire, INSERM U429, Hôpital Necker Enfants Malades, Pavillon Kirmisson, 75015 Paris, France, EU. 3 Interaction de l’épithélium intestinal avec le système immunitaire, INSERM EMI 0212 Faculté de Médecine Necker, 75015 Paris, France, EU. 4 Département d’Endocrinologie Pédiatrique et INSERM U561, Groupe Hospitalier Cochin-Saint Vincent de Paul et Faculté de Médecine Cochin, Université Paris V, Paris, France, EU. 5 Centre d’étude des Déficits Immunitaires, Hôpital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France, EU. 6 Unité d’Immunologie et d’Hématologie Pédiatriques, Hôpital Necker Enfants Malades, 75015 Paris, France, EU.We previously reported the clinical phenotype of two siblings with a novel inherited developmental and immunodeficiency syndrome, consisting of severe intra-uterine growth retardation and an impaired development of specific lymphoid lineages, including a transient CD8 / T lymphopenia and a persistent lack of NK cells, NK-T cells, and / T cells. The only detectable myeloid disorder is a persistent neutropenia. We herein report a cellular phenotype of impaired cell survival of /T and B lymphocytes. Peripheral blood B and T lymphocytes were excessively apoptotic and /CD4 and CD8 T cell blasts showed an impaired survival in response to both IL-2 and IL-15. Impaired /T cell survival in response to both IL-2 and IL-15 documented in vitro most likely accounts for the lack of / T, NK-T and NK cells, as well as the transient CD8 /T lymphopenia documented in vivo. Known components of the IL-2/IL-15 survival pathway were normal, implying that the two siblings suffered from an inborn error affecting a novel component of the pathway. The severe intra-uterine growth retardation of the two siblings suggests that the defect probably intersects with insulin-mediated survival signals in utero. In contrast, we further report that neutropenia is not associated with a primary defect of impaired survival. Rather, we show an impaired IL-17 production in the patients which leads to an impaired induction of G-CSF production, accounting for the neutropenia. The lymphoid defect of the patients, which is caused by an impaired cell survival, is thus the primary lesion,





whereas the myeloid defect is a mere consequence of the lack of IL-17 producing lymphoid cells. This is the first human inherited disorder associated with an impaired cell survival, responsible for developmental and immunological phenotypes. Moreover, this is the first immunodeficiency associated with primary lymphoid and secondary myeloid phenotypes.





A055Molecular analysis of NTB-A signaling - a role for SAP and EAT-2 in NTB-A-mediated activation of human NK cells. Philipp Eissmann and Carsten Watzl, Institute for Immunology, University Heidelberg, INF 305, 69120 Heidelberg, Germany

The CD2 family member NTB-A (NK-T-B antigen) is expressed on NK-, T- and B-cells. Engagement of NTB-A on human NK cells by homophilic interaction with NTB-A expressing target cells can trigger cytotoxicity, cytokine production and proliferation. To better understand how NTB-A can activate human NK cells, we analyzed the molecular mechanisms of NTB-A signaling. We show that NTB-A is already tyrosine phosphorylated in non-stimulated human NK cells and is associated with SAP (X-linked lymphoproliferative gene product/ SH2D1A) and EAT-2 (EWS/FLI1 activated transcript 2). This base-line phosphorylation of NTB-A is mediated by Src-family kinases and may be a result of the homophilic interaction of NTB-A among neighboring NK cells. Blocking NTB-A phosphorylation abolishes the association with SAP and EAT-2, demonstrating that the recruitment of both molecules to the receptor is dependent on tyrosine phosphorylation. Co-immunoprecipitation experiments show that SAP and EAT-2 are able to bind to the same NTB-A molecule at the same time. NTB-A is not phosphorylated in a B-cell line lacking SAP and EAT-2, suggesting that these molecules might be important to induce NTB-A receptor phosphorylation. Stimulation of NTB-A by mixing human NK cells with NTB-A positive targets results only in a slight increase of NTB-A phosphorylation. This might reflect the importance of NTB-A homophilic interaction within a NK cell population, which already keeps the receptor in a status of high activation. The cytoplasmic tail of the NTB-A receptor contains three tyrosines, two of which are embedded within an ITSM (immunoreceptor tyrosine based switch motif). To analyze the contribution of the different tyrosines to NTB-A-mediated NK cell activation, we generated a NTB-A negative NK cell line, in which we expressed different tyrosine to phenylalanine mutants of NTB-A. Functional studies of these transfectants showed that the second tyrosine is sufficient and essential for NTB-A function. The third tyrosine is not sufficient, but is necessary for full NTB-A function. The first tyrosine, which is not embedded in an ITSM sequence, is not important for NTB-A-mediated NK cell activation. Our current studies aim to identify the binding sites for SAP and EAT-2 within the cytoplasmic tail of NTB-A. In our future studies we will analyze how SAP and EAT-2 contribute to the





activation of NK cells and whether the two molecules turn on different signaling pathways downstream of NTB-A.





A056Production of NCR1 monoclonal antibodies in ncr1 deficient mice. Moran Elboim, Roi Gazit and Ofer Mandelboim. The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem, 91120, Israel

Natural killer (NK) cells are large granular lymphocytes characterized by a CD56+CD3- phenotype. As part of innate immune response, NK cells are designed to recognize and eliminate a broad spectrum of pathologic conditions, ranging from transformed cells to pathogens infections. The specific cytotoxic effect of NK cells is not based on the expression of a monospecific antigen receptor, like in B or T cells. Instead, their ability to distinguish between normal and stressed cells relays on an array of both activating as well as inhibitory receptors that together regulate NK activity. Despite the fact that NK cells are part of the innate immune system, they are derived from a common precursor of T cells, to which they show close resemblance in many common features including the expression of surface receptors. In fact, most of the receptors that are expressed on NK cells are also found on T cells as well as other lymphocytes and until today, the only NK specific markers that have been discovered are the natural cytotoxic receptors (NCRs).While in human three NCRs have been characterized (NKp46, NKp30 and NKp44), in mice the only NCR identified is NCR1, a NKp46 homologue. Importantly, NCR1 expression is ubiquitous on most NK clones, thus representing a common and specific protein that solely marks the NK population. Recently, we have generated knockin mice in which the ncr1 gene was depleted and replaced with the reporter protein GFP. In order to produce specific ncr1 monoclonal antibodies we immunized ncr1-deficinet mice with a recombinant ncr1 protein fused to human IgG1, prepared hybridomas and screed for specific ncr1 antibodies. The production of mouse ncr1 mAbs is of great importance, as it would enable efficient depletion of mouse NK cells in vivo and serve a powerful tool to study NK biology both in vitro and in vivo.





A057Modelling spleen NK cell population dynamics. Marjet Elemans, Eleftheria Rosmaraki, Maria H. Johansson, Petter Höglund, Klas Kärre and Ramit Mehr. Strategic Research Center for studies of Integrative Recognition in the Immune System (IRIS), Microbiology and Tumorbiology Center (MTC), Karolinska Institutet, Box 280, SE-17177 Stockholm, Sweden.

The number of NK cells in the periphery is assumed to be approximately stable; the system is assumed to be homeostatic. However, it is unclear what determines NK cell homeostasis in the periphery and what are the important factors in the return to equilibrium, e.g. after radiation. Is the daily flow of cells from the bone marrow into the spleen constant, or is it determined by the density of cells in the spleen? Is the proliferation rate constant or density dependent? Is cell death increased when the number of NK cells reaches a high level?To be able to answer these questions, a mathematical modelling approach is necessary. We developed a mathematical model to simulate spleen NK dynamics in the mouse. Different versions of this model assume different modes of regulation of the population rates of inflow, proliferation and death. By fitting the models to data of BrdU-labelling experiments, we can evaluate which of the models is the most likely to be correct, and at what rates the different processes take place. Results and conclusions will be presented.





A058Inhibitory receptors block NKG2D-mediated NK cell cytotoxicity by controlling its raft association. Johanna Endt1, Fiona E. McCann2, Daniel M. Davis2, Carsten Watzl 1 1Institute for Immunology, University Heidelberg, INF305, 69120 Heidelberg, Germany2Department of Biological Sciences, Imperial College London, London, UK

Natural Killer (NK) cells are regulated by activating and inhibitory receptors. We recently demonstrated that the activating receptor 2B4 (CD244) is recruited into lipid rafts upon ligand binding and that this raft recruitment is essential for 2B4 phosphorylation and function. Inhibitory receptors control 2B4-mediated NK cell activation by blocking the raft recruitment of the 2B4 receptor.

Blocking the raft recruitment of activating receptors could be a general mechanism how inhibitory receptors control NK cells activation. To test this hypothesis we focused on NKG2D, an activating receptor that has been described to overcome the regulation through inhibitory receptors under certain conditions.

Here we show that stimulation of NKG2D, either by antibody-mediated cross-linking or cell mixing with NKG2D-ligand expressing target cells, results in the translocation of NKG2D into lipid rafts. This raft-recruitment is essential for the function of NKG2D. These data suggest that by controlling raft recruitment, inhibitory receptors could also regulate the activity of NKG2D. Indeed, we could demonstrate that the inhibitory receptor CD94/NKG2A can control NKG2D-mediated NK cell activation and that this inhibition is mediated by blocking the raft recruitment of the NKG2D receptor.

It was recently shown that inhibitory receptors target the signaling molecule Vav-1 by SHP-1-mediated dephosphorylation. Vav-1 can regulate actin reorganization, which is an important step for the polarization of lipid rafts. We now show that blocking actin reorganization inhibits NKG2D-mediated NK cell activation. In addition, Vav-1 phosphorylation induced upon contact with target cells expressing NKG2D ligands, is sensitive to inhibitors of actin reorganization. Also, NKG2D-mediated Vav-1 phosphorylation can be blocked by the engagement of the inhibitory CD94 receptor. This suggests that inhibitory receptors may control NKG2D raft association and NKG2D-mediated NK cell activation by targeting Vav-1.









A059Characterization of the receptor encoded by the KIR2DL5 gene. 1Ernesto Estefanía, 2Raquel Flores, 1Natalia Gómez-Lozano, 2Helena Aguilar, 2Miguel López-Botet, 1Carlos Vilches.1Servicio de Inmunología, Hospital Universitario Puerta de Hierro, San Martín de Porres 4, 28035 Madrid; and 2Molecular Immunopathology Unit, Universitat Pompeu Fabra (DCEXS), Barcelona, Spain.

Human KIR were first identified as cell surface receptors capable of inhibiting the NK cell-mediated lysis of allogeneic targets according to rules predicted by the missing-self hypothesis. The isolation of the genes encoding those receptors was followed by the identification of additional homologous genes, all of which constitute the KIR-gene family, composed in humans by 16 members. The products and functions of several of the newer genes, identified in genomic studies, remain uncharacterized due in part to a lack of specific reagents. Among those newer genes, KIR2DL5 exhibits a unique combination of features. Its exon-intron organization and sequence are most similar to those of KIR2DL4 (89% nucleotide sequence identity), a proposed receptor for the MHC class Ib molecule HLA-G that shows dual inhibitory/activating functions, is conserved in humans and primates, and is transcribed ubiquitously in NK cells. In contrast, KIR2DL5 resembles other KIR genes in its variegated pattern of transcription and its variable presence in the human genome. Using cDNA constructs, we have obtained antibodies that recognize specifically the product of the KIR2DL5 gene. We will provide evidence of the expression and function of KIR2DL5 as an inhibitory receptor of NK cells.





A060Plasticity of NK cells is Reflected by Differences between Cytotoxicity and Cytokine Production upon NKG2D, 2B4, KIR and NCR Triggering. Christine S. Falk, Barbara Mosetter, Monika Braun, Marion von Geldern, and Dolores J. Schendel. Institute for Molecular Immunology, GSF Research Center for Environment and Health, Munich, Germany.Introduction: The balance between activation and inhibition of natural killer (NK) cells is regulated by the integration of all positive and negative signals that are delivered by many different receptors. HLA class I molecules represent one of the major regulators in this scenario because they serve as ligands for most inhibitory receptors. Upon encounter with HLA class I-negative target cells, several activating receptors are triggered simultaneously depending on the ligand repertoire expressed by the target cell. Interactions with HLA class I-positive target cells, however, counterbalance these positive signals by crosslinking inhibitory receptors. Initial studies revealed that various NK cells and lines display remarkable differences with respect to their cytokine secretion pattern indicating that some NK cells may play a regulatory role for the induction of specific immune responses.Materials and Methods: Since most human target cells express a variety of activating ligands, individual signals by NKG2D, 2B4 and NRC, for instance, can only be compared using monoclonal antibodies coupled to beads or FcR-expressing murine target cells. In order to dissect individual signals, the capacity of NKG2D, 2B4, CD94/NKG2C and p50.3 (CD158i/ 2DS4) to mediate secretion and cytotoxicity was analyzed for four NK lines and several alloreactive NK clines. The patterns of 11 cytokines were detected by the multiplex protein array system (Luminex) and cytotoxicity was analyzed by redirected lysis experiments. Results and Discussion: In alloreactive NK cells and one NK line, cytotoxicity was induced by NKG2D, 2B4, NCR and sometimes CD94/NKG2C. These activating signals were efficiently inhibited by KIR2DL2 or CD94/NKG2C-mediated signals, respectively. Impaired inhibitory capacity by LAIR-1 was observed in some combinations. In contrast, no induction of cytotoxicity by single receptors could be achieved for two other NK lines indicating that additional activating receptors may be in charge in these NK cells. Remarkably, induction of cytotoxicity was accompanied by cytokine secretion (IL-8, IFN- and MIP-1ß) only for some NK lines. Although cytotoxicity could be induced by NKG2D, 2B4 and NCR in other NK lines, neither IL-8, IFN- or MIP-1ß were induced but, in stead, constitutive levels of IL-5, IL-13 and MIP-1ß were secreted. These initial findings indicate that NKG2D, 2B4 and NRC triggering results in cytotoxicity and cytokine induction in NK cells. However, in some NK cells these receptor-mediated signals seemed to be restricted to cytotoxicity and dissected from cytokine secretion. Taken together, the plasticity of NK cells applies





not only to their individual receptor repertoire but also to differential functions such as cytokine secretion suggesting that immune responses, in general, may be influenced by “regulatory NK cells”.





A061Glycoconjugates are involved in NKT-mediated activation of NK cells. Anna Fiserova, Jan Svoboda, Marketa Kuldova, Katarina Hulikova, Karel Krenek, Luca Vannucci, Karel Bezouska, Miloslav Pospisil and Vladimir Kren. Institute of Microbiology, ASCR, Videnska 1083, Prague, Czech Republic.

NKR-P1 is a multipotent receptor present on various cell types (NK, NKT, monocytes, granulocytes). Nevertheless, the genuine nature of NKR-P1 receptor functioning upon ligand interaction is still not fully clarified, also on account of the fact that its physiological ligand remains to be identified. Our previous results using the glycoconjugates (GCs) with N-acetyl--D-glucosamine (GlcNAc) moiety, as synthetic mimics, showed high affinity to recombinant NKR-P1 and CD69 proteins. Considering these results, the current study focuses on the effect of GCs (GlcNAc8-PAMAM, GlcNAc4-calix(4)arene) on cytotoxic cells (NK, NKT, CTL) - their distribution in spleen, peripheral blood or tumor microenvironment, and functional endpoints in B16F10 mouse melanoma model. Multicolor FACS analysis and cell-mediated cytotoxicity assay of purified spleen CTLs and NK cells were performed.

Examine morphology (FSC/SSC - on the basis of size and granularity), phenotype (CD49b/CD8) and activation (NK1.1, CD69) of cells in different immune compartments a differentiation trends under the GCs influence were identified. In spleen and blood, both NK cells (CD49b+/CD8-) and CTLs (CD49b-/CD8+) transformed to monocyte and granulocyte morphology without changing the expression of either NK1.1 or CD69 markers. In tumor, apart from morphological changes, we observed increase in CD69 expression, while major fraction of these activated TILs consisted of NK cells on behalf of down modulated CLTs. Upon GCs stimulation, NKT cells (CD49b+/CD8+) grow their numbers in the spleen and subsequently undergo further differentiation in peripheral blood by increasing the content of granules (detected in granulo-gate), but strongly down-modulate the NK1.1 expression. This may be caused by the receptor internalization, following the GCs coupling. In contrast, NKT cells infiltrating the tumor up-regulate the CD69. In order to evaluate whether GCs modulate effector functions, cytotoxic assay was performed on sorted cell populations (NK, CTL). An increase of both NK and CTL mediated killing of B16F10 target cells appeared solely in case of tumor-bearing mice. However, only purified NK cells of both tumor-bearing and control mice, achieved significant enhancement of cytotoxicity against NKR-P1-dependent targets (IC-21). This suggests the involvement of NK1.1 antigen in GCs-induced NK cell differentiation and lytic activity.

Summarizing these results, NKT cells can be considered as primary targets for glycoconjugates, initiating a cascade of events, leading to the NK cell





differentiation, their migration into tumor microenvironment and subsequent functional activation.This work was supported by grants 524/05/0267, IAA5020403, IAA400200503, IAA500200509 and Institutional Research Concept AV0Z50200510.





A062Microarray analysis of gene expression from splenic NK cells during MCMV infection in Ly49H transgenic mice. Nassima Fodil-Cornu1, Kwangsin Kim2*, Seung-Hwan Lee2* and Silvia M. Vidal1, 1Department of Human Genetics, Department of Microbiology and Immunology McGill Duff Medical Building, 3775 University Street, MontrealH3A 2B4 QC Canada, 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada, *Department of Microbiology and Immunology, Division of Biology and Medicine, Brown University,Providence, 02912 USA

NK cells play a pivotal role in innate immunity against tumors and infection, as well as in regulating the acquired immune response. In mice, the Cmv1/Ly49h locus expressed on NK cells determines innate resistance to MCMV. Acquisition of MCMV-resistance in transgenic mouse expressing Ly49H, FVBLy49h-tg, demonstrated the critical role of Ly49H in clearance of the infection, and provided an ideal model to characterize the role of NK cells in host defense. Therefore, in order to dissect the response of NK cells during MCMV infection, we performed a comparative gene expression analysis of splenic NK cells using an Affymetrix microarray, from i) FVBLy49h-tg mice at various infection time points, ii) FVB wild type and FVBLy49h-tg mice at 36h post-infection.

During the clearance of the virus, 22690 genes were analyzed at different time points in the resistant mice. Of those, only 231 genes showed different patterns of expression between day 0, 3, 5, and 8. The largest functional category for up-regulated genes involved proliferation, protein and DNA metabolisms, transcription and cell organization, contrasting with 15% of genes implicated in NK cell response. Interestingly, at day 3 post-infection, gene up regulation coincided with acute stage of MCMV infection. At later time post infection (day 8) the gene expression returned to basal level, possibly as a result of decreasing viral load.

Early during MCMV infection, only 35 out of 16,000 genes analyzed showed greater than 2.5 fold expression difference between the transgenic and non-transgenic mice. In the FVBLy49h-tg mice, the up-regulated genes were involved in NK cell proliferation, cytotoxicity as well as cell mediated immunity, for example the early T lymphocyte activation-1 gene (osteopontin). Notably, the gene expression profile in susceptible mice exhibited a significant up-regulation of pro-inflammatory cytokines such as IFN-, and , TNF associated receptor, serum amyloid A3 as well as neutrophil chemoattractans as MIP2, IP-10. Strikingly, the high IFN- production by NK cells of wild type FVB mice is not correlated to viral infection control, emphasizing that the contact between NK cells and MCMV infected target cells is crucial for clearing the virus. Similarly, antiviral cytokines are not sufficient to





control viral replication in the absence of Ly49h, and that direct killing of virus infected cells by NK cells expressing Ly49H is required for resistance.

These results suggest that the differential pattern of expression between resistant and susceptible mice depends on the presence or absence of Ly49h as well as on signals emanating from productively infected cells. Finally, genes differentially expressed on MCMV-susceptible NK cells may serve as useful biomarkers for HCMV susceptibility in risk populations, graft recipients in particular.





A063NK cell phenotype and function in chronic HCV-infected patients: modified cytotoxicity and cytokine secretion without decreased NCR function/expression. 1Manuela Fogli, 1Stefania Mazza, 2,3Paola Costa, 1Monica Basso, 1Antonio Picciotto, 1,

4M.Cristina Mingari, 1,2,3Lorenzo Moretta, 1,3Andrea De Maria.1University of Genova, 2G.Gaslini Institute,3CEBR,4IST-GE, Largo R.Benzi 10, 16132 Genova,ITALYBackground: Hepatitis C virus (HCV) readily establishes high-level lifelong persistent infection in the vast majority of immunocompetent adults.. Failure of HCV-specific CD8+CTL to clear replicaton has been found to be associated a decreased CD8+CTL memory maturation as well as decreased perforin contents and IFN production. Virus-induced conditioning of innate immune responses is a possible mechanism that may contribute to the impairment of virus-specific CD8+CTL responses. Plasmacytoid and myeloid dendritic and NK cells have been shown to be variably affected during HCV infection as well as during HIV-1 infection. Aim: determine to what extent triggering NK cell receptor expression and function is affected during chronic viraemic HCV.Methods: Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation from 15 patients with chronic viraemic HCV infection, and from 15 healthy control donors and 20 HIV-infected patients. Fresh NK cells were obtained by mAb-coated microbead negative selection. NK cell phenotype was analyzed by two- and three color cytofluorometric analysis (FACSCalibur) using mAbs specific for CD3, CD56, CD16, NKp30, NKp46, NKp44, NKp80, NKG2D, NKG2C, NKG2A, CD81, HLA-DR, CD69, CD25, p58.1, p58.2, p70, LIR1/NKG2, CD94/NKG2A. NK cell cytotoxicity was assayed in a 4hr-51Cr release assay using as targets P815 cells in a redirected killing assay and FO-1, Hep G2 and Daudi cells. Cytokine production of freshly separated NK cells was evaluated in supernatants of cells after specific triggering.Results: Cytofluorometric analysis showed no evidence of NK cell activation in HCV infected patients, as determined by HLA-DR, CD69 CD25 and NKp44 expression, while HLA-DR and CD69 are consistently expressed on NK cells derived from HIV-1 infected patients. Analysis of Natural Cytotoxicity Receptors (NCR) showed that NK cells from HCV infected patients had a trend towards increased expression of NKp30 and NKp46, while HIV-1 infected patients have decreased NCR expression. The expression of NKG2D, NKG2A and KIRs on NK cells in HCV patients was similar to healthy controls. Cytotoxic activity of fresh NK cells from the patients showed a conserved redirected killing activity via CD16 and NKp30 and NKp46. Reduced killing activity against Fo1, Daudi, and Hep G2 targets was observed, with decreased involvement of NKG2D in the lysis. Cytokine production profiles of freshly separated NK cells showed increased production of proinflammatory cytokines (IL-8 and IL-1ß) and of IL-10 in HCV patients, while similar levels of IFN and TNF were recorded.





Conclusions: in HCV infected patients fresh NK cells do not show defective expression of NCR or of inhibitory NK cell receptors. The functional differences point towards an increased production of IL-10 and of proinflammatory cytokines (IL8, IL1ß) together with a decreased lysis of tumor cell targets. These observations, in association with the increased expression of NKp30 and NKp46 on NK could contribute, once NK cells localize in the liver, to the recruitment of inflammatory cells with a Th2-skewed editing of DC function associated with decreased immune surveillance against transformed cells.





A064Selective natural killer cell proliferation during murine cytomegalovirus infection. Anthony R. French§, Hannah Sjolin*, Sungjin Kim‡, Liping Yang‡, Klas Karre*, and Wayne M. Yokoyama‡, .

§Division of Pediatric Rheumatology, Department of Pediatrics, Washington University School of Medicine, St Louis MO; *Microbiology and Tumor Biology Center, Karolinski Institute, Stockholm Sweden; ‡Howard Hughes Medical Institute and Division of Rheumatology, Department of Medicine, Washington University School of Medicine, 660 S Euclid, St Louis MO 63110 USA.

Natural killer (NK) cells vigorously proliferate during viral infections. During the course of murine cytomegalovirus (MCMV) infection, this response becomes dominated by the preferential proliferation of NK cells that express the activation receptor Ly49H. The factors driving such selective NK cell proliferation have not been characterized. Herein, we demonstrate that preferential NK cell proliferation is dependent on KARAP/DAP12 mediated signaling following the binding of Ly49H to its virally- encoded ligand, m157. Ly49H signaling through KARAP/DAP12 appears to directly augment NK cell sensitivity to low concentrations of pro-proliferative cytokines such as IL-15. The impact of Ly49H mediated signaling on NK cell proliferation is masked in the presence of high concentrations of pro-proliferative cytokines that non-selectively drive all NK cells to proliferate.





A065Evidence for human natural killer cell differentiation in secondary lymphoid tissue. Aharon G. Freud, Akihiko Yokohama, Brian Becknell, Hsiaoyin C. Mao, and Michael A. Caligiuri. The Ohio State University Comprehensive Cancer Center, 2001 Polaris Parkway, Columbus, OH USA.

The anatomical site of natural killer (NK) cell development has not been definitively established. Human lymph nodes (LN) are selectively and highly enriched for both mature CD56(bright) NK cells and their CD34(+) NK precursors, suggesting that human NK development may occur in secondary lymphoid tissue (SLT). A corollary to this hypothesis is that the necessary intermediate populations spanning the continuum of NK differentiation from precursor to progeny would similarly be enriched within SLT. In this study, we identified and functionally characterized four putative NK developmental intermediates based on the surface expression of CD34, c-kit, and CD94, each found within the parafollicular T cell rich regions of human SLT. CD34(+)c-kit(-) CD94(-) cells, termed stage 1 intermediates, did not differentiate into mature NK cells when incubated in IL-15 alone. However, in the presence of IL-15 plus IL-3, IL-7, FL and the MS-5 stromal cell line, stage 1 cells were capable of robust NK cell differentiation. In addition, stage 1 intermediates were also capable of T cell and dendritic cell (DC) differentiation under appropriate in vitro culture conditions. CD34(+)c-kit(+)CD94(-) stage 2 intermediates readily proliferated and differentiated into CD56(bright) NK cells in response to IL-15 alone and were also capable of DC differentiation. However, in contrast to stage 1 cells, stage 2 intermediates showed a marked reduction in potential for T cell differentiation. CD34(-)c-kit(+)CD94(-) stage 3 intermediates were completely devoid of both T and DC differentiation potential in vitro. These cells proliferated in response to IL-15 and all expressed the pan-NK receptor, NKR-P1A, suggesting that they are committed to the NK lineage. However, following short-term stimulation ex vivo, stage 3 cells were incapable of producing IFN- as measured by intracellular flow cytometry and ELISA. Further, stage 3 cells lacked intracellular perforin and displayed no perforin or Fas ligand-dependent natural cytotoxicity. In stark contrast, CD34(-)c-kit(dim/-)CD94(+) stage 4 intermediates produced IFN-, were uniformly CD56(bright)perforin(+), and killed in a perforin and Fas ligand-dependent fashion. When stage 3 and stage 4 cells were cultured in the presence of IL-15 and activated autologous SLT T cells, stage 3 cells gave rise to stage 4 cells whereas stage 4 cells retained the CD34(-)c-kit(dim/-)





CD94(+)CD56(bright) phenotype. Therefore, stage 3 cells are immature NK and stage 4 cells represent functionally mature and terminally differentiated CD56(bright) NK cells. In summary, we have developed a new model of human NK cell development consisting of four novel intermediate populations in SLT, providing direct evidence that SLT may be the site of human NK cell development in vivo.





A066Paradoxic inhibition of human natural interferon-producing cells by the activating receptor NKp44. Anja Fuchs, Marina Cella, Takayuki Kondo and Marco Colonna. Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110 USA.

Natural killer (NK) cell-mediated cytotoxicity is triggered by multiple activating receptors associated with the signaling adaptor protein DNAX activation protein 12/killer cell-activating receptor-associated protein (DAP12/KARAP). Here, we show that one of these receptors, NKp44, is present on a subset of natural interferon-producing cells (IPCs) in tonsils. NKp44 expression can also be induced on blood IPCs after in vitro culture with interleukin 3 (IL-3). Crosslinking of NKp44 does not trigger IPC-mediated cytotoxicity but, paradoxically, inhibits interferon (IFN-) production by IPCs in response to CpG oligonucleotides. We find that IPCs in tonsils are in close contact with CD8+ T cells and demonstrate that a subset of memory CD8+ T cells produces IL-3. Therefore, IL-3-mediated induction of NKp44 on IPCs may be an important component of the ongoing crosstalk between the innate and adaptive immune response that allows memory CD8+ T cells to control the IPC response to virus.





A067Ly49B is expressed on macrophage-like cells. Frances Gays, Jonathan G. Aust, Jane Falconer, *Delyth Reid, **Noriko Toyama-Sorimachi, and Colin G. Brooks. The Medical School, School of Biomedical Sciences, Newcastle, Tyne NE2444 UK., *The Edward Jenner Institute, Compton, U.K., and **Tokyo Medical and Dental University, Shinjuku-ku, Toyoma 1-31-1, Tokyo 162-8655 Japan

A series of monoclonal antibodies against Ly49B were raised by immunizing rats with cells transfected with Ly49B. These antibodies reacted with Ly49B but not with any of the other Ly49 molecules known to be expressed in C57 mice. Multicolour immunofluorescence studies revealed that about 1.5% of adult spleen cells expressed Ly49B, but none of the Ly49B-expressing cells were NK cells. Instead, Ly49B was found on three distinct subpopulations of CD11b+ spleen cells that expressed low, intermediate, and high levels of Gr1. In the bone-marrow up to 15% of CD45+ cells expressed Ly49B, and three distinct subpopulations of Ly49B+ cells could be defined by light scatter characteristics and expression of CD11b, Gr1, and other markers. Amongst most of these populations, Ly49B appeared to be expressed in a uniform non-stochastic manner. Co-staining of spleen and bone-marrow cells with appropriate mAbs revealed that Ly49B and Ly49Q were expressed on largely non-overlapping subpopulations of cells, most Ly49B+ cells being B220-/lo and most Ly49Q+ cells being B220hi. Cultured macrophages derived from bone marrow and spleen constitutively expressed little or no Ly49B but substantial levels were induced within 24hr by culture with LPS and IFN. Immunohistochemistry revealed that in spleen expression was predominantly in the red pulp on at least three morphologically distinct populations including PMN cells. A few positive cells were seen within the marginal zones, T and B cell areas. An analogous situation was seen in lymph nodes where expression was predominantly in the medullary (macrophage) regions. In thymus, Ly49B was expressed on scattered cells in the cortex with greater numbers in the medullary regions and corticomedullary junctions. Alveolar macrophages and Kupffer cells were moderately labelled, while intestinal lamina propria APCs were strongly positive. Epidermal Langerhans cells appeared negative for Ly49B, although some dermal APCs were positive. Brain parenchyma, kidney, skeletal and cardiac muscle were essentially negative although some brain choroid plexus macrophages were moderately labelled. In conjunction with previously published results for





Ly49Q, these findings demonstrate that the Ly49 molecules encoded by the genes at either end of the Ly49 cluster are expressed in a quite different manner to those encoded in the centre of the cluster and raise important questions concerning the function of Ly49 receptors on myeloid and dendritic cells.





A068Multiple cytokines regulate the NKC-encoded receptor repertoire of mature NK cells and T cells. Frances Gays, Kimberley Martin, Rupert Kenefeck, Jonathan G. Aust, and Colin G. Brooks. The Medical School, School of Biomedical Sciences, Newcastle, Tyne NE2444 UK

Mature NK cells comprise a highly diverse population of lymphocytes that express different permutations of receptors to facilitate recognition of diseased cells and perhaps pathogens themselves. Many of these receptors, such as those belonging to the NKRP1, NKG2, and Ly49 families are encoded in the NK gene complex (NKC). It is generally thought that these NKC-encoded receptors are acquired by a poorly understood stochastic mechanism that operates exclusively during NK cell development, and that following maturation the repertoire is fixed. However, we report here a series of observations that demonstrates that the mature NK cell repertoire in mice can in fact be radically remodelled by multiple cytokines. Thus, both IL2 and IL15 selectively induce the de novo expression of Ly49E on the majority of mature NK cells. By contrast, IL4 not only blocks this IL2-induced acquisition of Ly49E, but reduces the proportion of mature NK cells that express pre-existing Ly49 receptors and abrogates the expression of NKG2 receptors while leaving the expression of several NKRP1 receptors unaltered. IL21 also abrogates NKG2 expression on mature NK cells and selectively downregulates Ly49F. IL4 and IL21 additionally cause dramatic and selective alterations in the NKC-encoded receptor repertoire of IL2-activated T cells but these are quite different to the changes induced on NK cells. Collectively these findings reveal an unexpected aspect of NKC receptor expression that has important implications for our understanding of the function of these receptors and of the genetic mechanisms that control their expression.





A069Novel genes in the centromeric part of the human NK gene complex. Hormas Ghadially, Susanne Sattler, Irene Michl, Manuela Kropik, Frank Kalthoff and Erhard Hofer. Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria.

The centromeric part of the human natural killer (NK) gene complex spans a region of approximately 0.5 Mb on the short arm of chromosom 12. It contains the NKG2/CD94 NK receptor and the LOX/DECTIN myeloid receptor gene families. In addition to the known members of these two families several additional novel genes, some related and others unrelated to the lectin receptor families, became recently apparent in this genomic region. We have tested the expression of these genes in comparison to the other family members with an emphasis on dendritic cells and NK cells. The members of the myeloid receptor family, in particular the genes encoding LOX-1, DECTIN-1 and CLEC-1, were found to be highly expressed in immature dendritic cells and to be downregulated upon induction of maturation using a wide panel of different maturation stimuli. We found DECTIN-1 to be the most strongly downregulated of these molecules. Additionally, DECTIN-1 is alternatively spliced and we observed differential regulation of splicing during the maturation of DC dependent on the stimulus. For some members of this family it has been reported that they bind among others to surface structures of monocellular parasites providing some evidence that this whole subfamily of lectin-like receptors may be involved in pattern recognition, antigen uptake and activation of dendritic cells. On the other hand for some members preliminary evidence for binding to leukocyte surface structures has also been obtained suggesting additional functions.





A070

Contribution of NK Cells to Containment of SIV in Naïve and Vaccinated Macaques. Luis D. Giavedoni, M. Shannon Keckler, Laura M. Parodi, and Vida L. Hodara. Southwest National Primate Research Center, Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, TX 78227.

Live-attenuated vaccines are the most effective vaccines in the SIV/rhesus macaque animal model. Although unlikely to be used in humans, these vaccines offer the possibility of studying correlates of protection against infection with virulent virus. We studied the contribution of the innate and adaptive immune systems in protection against infection with pathogenic SIVmac251 in four rhesus macaques immunized with a live-attenuated virus and in four naïve animals. To reduce the confounding effect of repeated sedations of the animals while obtaining samples, we utilized a novel Tether system. This system permitted for repeated sampling of small blood volumes during the acute phase of infection in the absence of sedation or restraining. Blood NK cells, dendritic cells, T cells, B cells, monocytes, and SIV-specific tetramer-binding cells were studied by polychromatic flow cytometry (PFC). PFC was also used for analyzing variations in the level of memory (CD27, CD28, CD95), activation (CD69) and proliferation markers (Ki67), and also for activating NK receptors. CTLs were analyzed by IFN- ELISPOT, and antibodies by antigen-specific ELISA. A total of 22 cytokines and chemokines were detected simultaneously in plasma with the Luminex system. Vaccinated macaques were protected from infection with challenge virus, whereas all the naïve ones became infected and two of these animals progressed rapidly into simian AIDS. After challenge, immunized animals did not show significant changes in the levels of myeloid and plasmacytoid DC, CD4 T cells, CD8 T cells, B cells, proliferation markers, and cytokine/chemokines in plasma. The number of tetramer+ CD8 T cells and IFN--producing cells increased slightly but steadily after challenge, whereas no significant variation was observed for anti-SIV Gag and Env antibody titers. Naïve animals, on the other hand, had dramatic elevations in proliferating NK cells and less pronounced changes in CD8 T cells; the appearance of anti-SIV CTL and antibodies was delayed compared to immunized animals, reflecting the lack of immunological memory in these animals. As seen in other studies, rapid progressors did not mount a stable anti-SIV immune response. Animals that controlled the infection had elevated plasmatic concentration of MIP-1.





These studies indicate that cellular components of both the innate and adaptive immune systems of animals inoculated with a live-attenuated SIV vaccine respond to and control infection with virulent virus. However, there may be other mechanisms affording protection that remain to be identified.





A071Multiple cytokine-gene transduction of activated natural killer (A-NK) cells. Stephen R. Goding, Qin Yang, Patricia Rice, Lisa Bailey & Per H. Basse. University of Pittsburgh Cancer Institute, The Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh PA 15213, USA.

We have previously shown that IL-2 activated natural killer (A-NK) cells localize selectively to tumor sites upon adoptive transfer into tumor bearing animals. If supported by sufficient amounts of IL-2, the A-NK cells efficiently kill the tumor cells in the infiltrated tumors. Without exogenous IL-2 support, however, the NK cells die very rapidly in the host and have, as expected, no therapeutic effect. While we believe that adoptive A-NK cell therapy may be successfully used for treatment of patients with disseminated cancer, the high, often toxic amounts of exogenous IL-2 needed to support the transferred A-NK cells hinders translation of this therapeutic approach, in its current form, to the clinic. We therefore tested whether other cytokines, such as IL-12 and IL-18 could maintain viability and functionality of the A-NK cells. We found that neither cytokines could serve as survival factors for A-NK cells. However, if supported by IL-2 in combination with either IL-12 or IL-18, a 100-fold lower amount of IL-2 was needed to support the A-NK cells, presumably due to upregulation of CD25 on the A-NK cells, enabling them to express the high affinity IL-2 receptor. Indeed, A-NK cells cultured with both IL-2 and IL12 or IL-2 and IL18 were able to survive, localize to, and kill tumors when supported by greatly reduced amounts of IL-2. Although no obvious toxicity was observed, even the reduced IL-2 usage induced a substantial and potentially toxic proliferation of host lymphocytes in both lymphoid and non-lymphoid organs. To totally eliminate the need for exogenous IL-2, we adenovirally transduced A-NK cells with the gene for IL-2. To our disappointment, the IL-2 transduced A-NK cells survived almost as poorly without exogenous IL-2 as non-transduced A-NK cells when deprived of exogenous IL-2. However, when we co-transfected the A-NK cells with both the IL-2 and the IL-12 (or IL-18) genes, the survival and proliferation of the A-NK cells was greatly enhanced. A-NK cells transduced with all three cytokine genes grew even better than non-transduced A-NK cells maximally supported by exogenous IL-2 (6,000 IU/ml). We believe that these double and triple gene-transduced A-NK cells will localize to and kill tumors without the need for support by exogenous IL-2. Furthermore, since the A-NK cells apparently can be transduced with multiple cytokine-genes simultaneously, we predict that, in addition to enhancing the A-NK cells’ own





tumor killing activity, we can use cytokine-gene transduced A-NK cells to manipulate the cytokine environment of tumors to attract and activate endogenous immune cells such as NK cells, T lymphocytes and dendritic cells.





A072The CD85j/ Leukocyte Inhibitory Receptor-1 distinguishes between conformed and 2m-free HLA-G molecules. Tsufit Gonen-Gross, Hagit Achdout, Tal I. Arnon, Roi Gazit, Noam Stern, Václav Horejsí and Ofer Mandelboims

The Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel,and Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

For a proper development of the placenta, maternal natural killer (mNK) cells should not attack the fetal extravillous cytotrophoblast (EVT) cells. This inhibition of mNK cells is partially mediated via the nonclassical MHC class I molecule HLA-G. Recently, we demonstrated that HLA-G forms disulfide-linked high molecular complexes on the surface of transfected cells. Here, we demonstrate that HLA-G must associate with 2m for its interaction with CD85J/LIR-1. Although HLA-G free heavy chain (FHC) complexes are expressed on the surface, they are not recognized and possibly, interrupt with CD85J/LIR-1 and HLA-G interaction. The formation of these complexes on the cell surface might represent a novel mechanism developed specifically by the HLA-G protein aimed to control the efficiency of the CD85J/LIR-1-mediated inhibition. We also show that endogenous HLA-G complexes are expressed on the cell surface. These findings provide novel insights into the delicate interaction between EVT cells and NK cells in the decidua.





A073Enhanced immune responses and hematopoiesis in Ly49Q-deficient mice. Marie-Line Goulet1, Nassima Fodil2, Angela D. Troke1, Noriko Toyama-Sorimachi3, Etienne Rousselle1, Silvia M. Vidal2, and Andrew P. Makrigiannis1.1Laboratory of Molecular Immunology, Institut de Recherches Cliniques de Montréal, Montreal, 3775 University Street, Room 512 QC, H3A2B4 Canada2Department of Human Genetics, McGill University, Montréal, QC, Canada3Department of Gastroenterology, International Medical Center of Japan, Tokyo, 162-8655 Japan

Most Ly49 are expressed on NK, NKT, and T cells. However, Ly49Q, one of the framework Ly49, is expressed on plasmacytoid dendritic cells (pDC). Like NK cells, pDC are known to be potent anti-viral effectors during infections. To determine the role of Ly49Q in pDC development and function, the Ly49q coding region was disrupted with a neomycin cassette by homologous recombination in embryonic stem cells. Mice homozygous for the neomycin insertion (Ly49qneo/neo) are viable and display no outward physical abnormalities. Using flow cytometric analysis, Ly49Q was found not be expressed on pDC from various organs in Ly49qneo/neo mice. Also, the presence of the neomycin cassette did not affect the expression of the surrounding NKG2 and Ly49 genes. Analysis of hematopoietic lineages revealed that Gr-1+ and CD11b+ bone marrow cells, which have been shown to express low levels of Ly49Q in WT mice, are significantly increased in KO mice relative to WT. All other lineages, including pDC, were found to be present at normal levels. To determine, the effect of Ly49Q absence during immune responses, Ly49qneo/neo mice were challenged with MCMV. In various backgrounds, Ly49qneo/neo mice consistently displayed a modest, but significant, decrease in splenic viral titres compared to Ly49Q-sufficient mice. Furthermore, in vitro stimulation experiments with agonists of Toll-like receptors expressed by dendritic cells showed that cultured pDC from Ly49qneo/neo mice expressed significantly higher levels of MHC class II and the co-stimulatory CD86 antigens than WT-derived pDC. In contrast to normal mDC, pDC can migrate across the HEV present in lymphoid organs during infections. However, migration of pDC to spleen and lymph nodes in mice challenged with MCMV and P. acnes were found to be similar in both WT and knockout mice. Collectively, these data suggest that Ly49Q is necessary for normal bone marrow development and that, during immune responses, Ly49Q acts as a negative regulator of pDC activation, but not migration.





Supported by a New Investigator Award and grant from the CIHR (APM).





A074Regional Variations in NK Cell Receptor Expression in Ireland. Kieran Guinan 1 , Rodat Cunningham2, Derek Middleton2 and Clair Gardiner1. 1 School of Biochemistry and Immunology, Trinity College, Dublin 2, Republic of Ireland. 2 Tissue Typing Laboratory, Belfast City Hospital, Lisburn Road, BT9 7T5, Northern Ireland.

NK cells express a variety of receptors which have evolved in humans to identify and kill virally infected and tumor cells. There is a tremendous degree of diversity within the 16 KIR genes identified and inheritance patterns vary greatly between populations. Although Ireland is a small island with relatively little admixture, internal regional genetic variation exists e.g. with respect to Y-chromosome markers and HLA types. In this preliminary study, we investigated the potential for regional variation between Northern Ireland and Republic of Ireland populations (n=15 and 20 respectively) in terms of expression of NK cell receptors. Although differences were seen between individual receptor expression patterns, no significant differences were seen between the two populations in terms of KIR2DL1, KIR2DL2/3 or KIR3DL1 (DX9 and Z27) genotypes or phenotypes. In accordance with previous studies, the DX9 antibody gave rise to a variety of cell surface phenotypes, which correlated with the allele(s) involved. We can now report that the KIR3DL1*009 allele binds the DX9 antibody with a bright staining pattern. In addition, a binding phenotype described as “very dim” on staining with Z27 antibody supported claims that this antibody may bind KIR3DS1. Expression patterns of CD94, NKG2a and LILRB1(LIR1/ILT2) were similar between the two cohorts. We did find some potential differences in NKp30 expression between the two groups: while variation in the percentage of NK cells positive for NKp30 was large (range 2-80%), a mean of 28% of CD3-/56+ dim cells from Northern donors contrasted significantly with 40% from the Republic (p=0.0314). NKp44 was expressed at higher levels on CD3-/56+ bright (mean 12%) than CD3-/CD56+ dim cells (mean 2.5%). In both groups, variation between donors in the percentage of NKp46 positive cells was striking with values ranging from 18-98% on CD3-/56+ dim cells. A high proportion of CD3-/56+ bright cells were NKp46 positive (86-100%). While frequencies of genes and alleles are likely to differ between the two populations, the phenotypic manifestation of this will become even more apparent as donor numbers are increased and the contribution of allotype at a locus is considered.





A075Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus (HCMV)-infected fibroblasts. Mónica Gumá 1 , Matthias Budt2, Andrea Sáez1, Tamara Brckalo1, Hartmut Hengel3, Ana Angulo4, Miguel López-Botet1. 1Molecular Immunopathology Unit, Universitat Pompeu Fabra, Dr. Aiguader 80, 08003-Barcelona; 2Robert Koch-Institut, Division of Viral Infections, Berlin; 3Institute for Virology, Heinrich-Heine-University, Düsseldorf; 4Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona.

CD94/NKG2C+ NK cells are increased in individuals seropositive for human cytomegalovirus (HCMV+), suggesting that the viral infection may shape the NK cell receptor (NKR) repertoire. To address this question, we analysed the distribution of NK cell subsets in PBL co-cultured with HCMV-infected fibroblasts. A substantial increase of NK cells was detected by day 10-12 in samples from a group of HCMV+ donors, and CD94/NKG2C+ cells outnumbered the CD94/NKG2A+ subset. Fibroblast infection was required to induce the preferential expansion of CD94/NKG2C+ NK cells, that was comparable regardless of the origin of fibroblasts (allogeneic or autologous) and the virus strain. A CD94-specific mAb abrogated the effect, supporting an involvement of the KLR. Purified CD56+ populations stimulated with HCMV-infected cells did not proliferate, and the expansion of the CD94/NKG2C+ subset was only detected in the presence of exogenous IL-15. Experiments with HCMV deletion mutants indicated that the response of CD94/NKG2C+ NK cells was independent of the UL16, UL18 and UL40 HCMV genes, but was impaired when cells were infected with a mutant lacking the US2-11 region that preserved HLA class I expression. Taken together the data support that the interaction of CD94/NKG2C with HCMV-infected fibroblasts, concomitant to the inhibition of HLA class I expression, promotes a preferential outgrowth of CD94/NKG2C+ NK cells, enhancing their responsiveness to IL-15.





A076C-MYC induces KIR expression via a novel control region upstream of the conventional adult KIR promoter. Becky Haack, Valarie McCullar, Todd Lenvik, Michelle Pitt, Stephen Anderson*, Jeffrey S. Miller. University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455 and *SAIC-Frederick, Immunology Bldg, 560, Room 31-93, Frederick, MD 21702 USA.KIR determine whether NK cells will be alloreactive against targets. Although epigenetic control is thought to play a role in KIR expression, the mechanism of KIR repertoire formation is unknown. Recently, a probabilistic transcriptional mechanism has been shown to control the acquisition of mouse Ly49 receptors using a switch region upstream of each Ly49 promoter (S. Anderson). Based on this information, the DNA upstream of known KIR genes was scanned for similar motifs to test the hypothesis that similar control mechanisms are operant in human NK cells. A putative C-MYC/AML binding site was found approximately 1Kb upstream of the traditional KIR promoters. Initiation of transcription in this region was observed by RNAse protection, and spliced KIR mRNAs originating in the upstream region were cloned. To look at the effects of c-myc on KIR expression, we transduced NK92 cells using retroviral murine stem cell virus vectors containing either C-MYC/eGFP or eGFP alone. Cells expressing high levels of c-myc were 18% KIR positive as compared to 0.9% of control cells. To further look at the effect of c-myc on KIR from primary cells, we transduced CD34+ progenitors isolated from umbilical cord blood with C-MYC/eGFP and eGFP vectors. eGFP+ cells were plated on the murine fetal liver line AFT024 and cultured with IL-15, IL-7, IL-3, Flt3 ligand, and c-kit ligand, all known to induce NK cell differentiation. Although c-myc is an oncogene, it did not promote autonomous growth of NK cells in the absence of exogenous cytokines. Proliferation was significantly increased in single cell progenitors over-expressing c-myc with 739.3x103 NK cell progeny compared to 0.56x103 from each control cell (p<0.0001) after 21 days. At this early time point, 21.68 ± 3.25% of c-myc cells were KIR positive while 0.0% of control cells were KIR positive (p<0.0001) suggesting that c-myc targets KIR expression. Furthermore, bulk-transduced cells over expressing c-myc had 3.9 times the level of early non-coding transcripts originating from the upstream promoter as compared to eGFP cells. A direct effect of c-myc on the upstream promoter was supported by ChIP assays demonstrating increased binding to the c-myc/AML site. The presence of these upstream transcripts directly correlates with an increase of conventional KIR transcripts as shown by Q-RT-PCR and an increase of KIR expression as shown





by flow cytometry. These data show that c-myc may play an important role in the differentiation of NK cells as well as KIR acquisition. Experiments are currently in progress to look at the effects of the competing AML site on KIR expression and how non-coding RNA affects adult KIR expression. Understanding the mechanisms of KIR expression may allow novel strategies to manipulate NK cells for therapeutic benefit.





A077NK cells are differentially activated by TLR3 and TLR7/8 agonists. Orla M. Hart1, Veronica Athie-Morales2, Geraldine M. O’Connor1 and Clair M. Gardiner1. 1School of Biochemistry and Immunology, and 2Xoma Ireland Limited, School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.

Natural Killer (NK) cells express receptors that allow them to recognize pathogen and activate effector functions such as cytotoxicity and cytokine production. Among these receptors are the recently identified Toll-like receptors that recognize conserved pathogen structures and initiate innate immune responses. We demonstrate that human NK cells express TLR3, TLR7 and TLR8 at both mRNA and protein level, and that these receptors are functional. TLR3 is expressed at the cell surface where it functions as a receptor for poly (I:C) in a lysosomal independent manner. TLR7/8 signalling is sensitive to chloroquine inhibition indicating a requirement for lysosomal signalling as shown for other cell types. Comparison of the kinetics of signalling through TLR3 and TLR7/8 in NKL cells shows that although both lead to activation of the transcription factor NF-kappaB, TLR3 signalling results in the characteristic late phase NF-kappaB activation, while TLR7/8 signalling results in early phase NF-kappa B activation. Both R848, an agonist of human TLR7 and TLR8, and poly (I:C) activate NK cell cytotoxicity against Daudi target cells. However, IFN-gamma production, another important effector function of NK cells in response to infection, is differentially regulated by these TLR agonists. In contrast to poly (I:C), R848 stimulates significant IFN-gamma production by NK cells. This is accessory cell dependent and is inhibited by addition of a neutralising anti-IL-12 antibody. Moreover, stimulation of purified monocyte populations with R848 results in IL-12 production and reconstitution of purified NK cells with monocytes results in increased IFN-gamma production in response to R848. In addition, we demonstrate that while resting NK cells do not respond directly to R848, they can be primed to do so by prior exposure to either IL-2 or IFN-alpha. Therefore, although NK cells can be directly activated by TLRs, accessory cells make a major contribution to effector functions such as IFN-gamma production and cytotoxicity.





A078Using PCR-SSP technique for analyzing KIR mRNA expression profiles in NK cells. Ute Heider1, Anja Cremer1, Stefan Tomiuk2, Andreas Arendt1, Jürgen Schmitz1, Andreas Bosio2, Volker Huppert 1 , and Christian Biervert.1

1Miltenyi Biotec GmbH, Friedrich-Ebert Strasse 68, D-51429 Bergisch Gladbach, Germany; 2Memorec Biotec GmbH, Stoeckheimer Weg 1, 50829 Cologne, Germany.

Natural killer (NK) cells comprise approximately 10% of peripheral blood lymphocytes. They have the ability to kill tumor and virally infected cells without previous stimulation. The activity of NK cells is regulated by Killer Ig-like receptor (KIR) molecules through interaction with specific HLA class I molecules on target cells, predominantly HLA-C. NK cells are negatively regulated by inhibitory KIRs which recognize the self MHC class I molecules. The lack of self MHC-specific molecules because of down-regulation during virus infection results in target cell lysis by the NK cells. The same effects are observed when NK cells are faced with mismatched allogeneic targets (missing self hypothesis). Consistent with this hypothesis, a positive outcome of KIR/HLA disparity has been demonstrated in the setting of haploidentical stem cell transplantation (Ruggeri et al. 2002). Reduced rates of leukemia relapse are observed when the haploidentical graft possesses KIRs for which the recipient has no ligand. The KIR locus, containing a family of polymorphic and highly homologous genes, maps to human chromosome 19q13.4. There is remarkable diversity in the number and the type of KIR genes present on independent KIR haplotypes. In general, KIR haplotypes contain 7-12 genes plus 2 pseudogenes. Extra heterogeneity is provided at the expression level: different subsets of NK cells express different KIRs, even within one individual. Recently, it was shown that KIR genotyping alone does not seem to be sufficient for donor KIR assessment because of the lack of gene expression in approximately one-fourth of the individuals for one of the inhibitory KIRs that recognize the three major groups of MHC class I ligands (Leung et al. 2005). KIR phenotyping by flow cytometry using monoclonal antibodies is insufficient due to the lack of specific monoclonal antibodies. For trustworthy analysis, one has to combine KIR genotyping with mRNA expression profiling and flow cytometry.Here we use a new set of sequence specific primers (SSP) for PCR and RT-PCR. We show that these primers are well suited to be used both in KIR genotyping and mRNA expression profiling. The primers of each KIR gene (15





genes and two pseudogenes) cover all allelic variants annotated so far by the IPD KIR Sequence Data Base (status quo July 05), leading to a more representative result than with published primers. We discuss the importance of supplementation of KIR genotyping by mRNA expression profiling and flow cytometry.





A079IL-15 alters expression of the pro-apoptotic factor bid in primary human NK cells. Deborah L. Hodge, Matthew D. Buschman, William Bere, and Howard A. Young. LEI, CCR, NCI-Frederick, Frederick MD USA 21702

IL-15, a member of the four -helical bundle cytokine family, is a key regulator of NK cell development, function, and survival. The positive impact of IL-15 on cell survival is demonstrated in IL-15 transgenic mice as these mice, at any early age, exhibit expansion of NK and CD8+ T cell populations. A mechanism by which IL-15 enhances cell survival is through up-regulation of two anti-apoptotic factors, bcl-2 and bcl-xl. IL-15 mediated survival is not limited to control of anti-apoptotic proteins in that we have observed that IL-15 dramatically decreases the expression of a critical pro-apoptotic factor, bid. Downregulation of bid is not due to alterations in transcriptional or posttranscriptional activities as bid mRNA expression remains constant following IL-15 treatment of NK cells. Addition of the proteasome inhibitor lactocystin or an E1-ubiquitin ligase inhibitor to IL-15-treated NK cells reverses the IL-15-mediated decrease in bid accumulation. IL-15 activates multiple intracellular pathways in NK cells that include P38 MAPK, PI3K-AKT, and MEK-ERK signaling pathways. Our data demonstrate that IL-15-mediated degradation of bid is blocked by the P38 MAPK and PI3-K inhibitors, SB 203580 and LY 294002, respectively, but not by the MEK pathway inhibitor PD 980590. Collectively, these data suggest that IL-15 directs posttranslational control of bid through a mechanism involving P38 MAPK and PI3K-AKT activation of the ubiquitin-proteasome degradation pathway. The ability of IL-15 to control multiple pro- and anti-apoptotic pathways gives additional credence to IL-15 as a critical regulator of NK cell survival.





A080Multiple roles for CCR2 in mediating inflammatory cell trafficking to liver during MCMV infection. Kirsten L. Hokeness and Thais P. Salazar-Mather. Brown University, Box G, 8-6, Providence, RI, 02912 USA

Recruitment of immune effector cells to localized sites of viral infection during murine cytomegalovirus (MCMV) is dependent upon a highly coordinated network of cytokines and chemokines. Our studies have clearly demonstrated that initiation of inflammatory responses in liver is dependent upon rapid induction of interferon (IFN)-/ resulting in production of monocyte chemoattractant protein-1 (MCP-1) by resident macrophages. Interactions between MCP-1 and its sole receptor, inflammatory CC chemokine receptor (CCR2), are required for trafficking of macrophages to liver resulting in induction of macrophage inflammatory protein- (MIP-). These events promote natural killer (NK) cell inflammation and local delivery of IFN- required for antiviral defense. Innate protection of MCMV has been extensively studied whereas little is known about the initiation of adaptive immune responses. Of particular importance is the generation of CD8+ T cell responses. Kinetics of CD4+ and CD8+ T cell accumulation in liver during MCMV infection was determined. Percentages of CD4+ T cells remain similar in uninfected and infected animals, whereas CD8+ T cell accumulation increases steadily from uninfected mice to peak levels at day 7 post infection. As activated Th1 cells can express CCR2, the effects of CCR2 and MCP-1 on T cell accumulation were evaluated in liver during late MCMV infection. Despite the coordinated effort of MCP-1 and CCR2 in initiation of innate responses, here we describe differential roles for CCR2 and MCP-1 on the recruitment of CD8+, but not CD4+ T cells, to liver at both day 5 and day 7 post infection. Concurrent with these findings, MCP-1 production in liver peaks approximately 38 hours post infection, after which protein levels decline rapidly and are below levels of detection during times of peak T cell accumulation. Trafficking studies as well as CCR2 mRNA expression demonstrate that this defect observed in CCR2- animals can be directly attributed to CCR2 functions on the CD8+ T cell. In addition, lack of CCR2 results in defective Th1 responses marked by decreases in IFN-. Together these data demonstrate that CCR2 but not MCP-1 is required for establishing and maintaining appropriate adaptive immune responses following MCMV infection in liver.





A081Lack of Killer Immunoglobulin-like Receptor (KIR) Ligand More Predictive of Protection from Relapse than KIR Ligand Incompatibility in Recipients of Unrelated Hematopoietic Cell Transplantation (HCT) for Hematologic Malignancies. Katharine C. Hsu, Mari Malkki, Ted Gooley, Bo Dupont, Effie Petersdorf on behalf of the International Histocompatibility Working Group HCT-KIR Component. Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, NY, NY, 10021 USA and Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98912 USA.

The relationship between donor inhibitory KIR and recipient HLA has been proposed as the basis for KIR-driven alloreactivity by donor natural killer (NK) cells, leading to higher overall survival (OS) and lower relapse in HLA-mismatched transplants for hematologic malignancy. The “KIR ligand incompatibility model” predicts NK alloreactivity when donors with HLA genotypes comprising class I ligands for inhibitory KIR are paired with recipients lacking the same class I ligands. Population frequencies for inhibitory KIR2DL2/3,-2DL1, and -3DL1 demonstrate that nearly all individuals have a complete complement of inhibitory KIR, in contrast to population frequencies of the corresponding HLA class I ligands (HLA-C1, -C2, or -Bw4), whose frequencies deviate greatly from 100%. This leads to the frequent situation of HLA-C or –Bw6 homozygosity and lack in the patient of at least one KIR ligand for donor inhibitory KIR (“missing KIR ligand”). To clarify their relevance to transplant outcome, we examined both the KIR ligand incompatibility and missing KIR ligand models using data provided from the International Histocompatibility Working Group. In this study, we examined the HLA genotypes of 1765 unrelated HLA-matched and -mismatched donor-recipient transplant pairs, segregating pairs based on KIR ligand incompatibility or based on lack of KIR ligand as predicted by HLA-C or –B epitope homozygosity. All patients received myeloablative conditioning followed by infusion of a T-replete allograft as treatment for AML, CML, or ALL. AML patients lacking KIR ligand demonstrated a significantly lower relapse rate (p=0.001). To stratify for ethnic disparities in KIR-HLA combination frequencies, analysis was then restricted to the non-JMDP group (n=1204), which demonstrated that in HLA-mismatched transplants (n=623), patients with “missing KIR ligand” had significantly less relapse (p=0.004). Comparison of the “missing KIR ligand” and “KIR ligand incompatibility” algorithms was performed for patients mismatched at HLA-B and/or HLA-C. There was no beneficial effect with KIR ligand incompatibility, but there was significantly less AML relapse with missing KIR ligand (p=0.008). Analysis of non-JMDP pairs and restriction to HLA-C mismatched pairs revealed consistent results. We were not able to demonstrate any beneficial effect of KIR ligand incompatibility on OS or relapse in this analysis. In contrast,





this analysis supports lack of KIR ligand as predicted by HLA-B or –C epitope homozygosity in the recipient as a significant contributing factor to transplant outcome.





A082Anti-tumor antibody-H60 fusion creates a novel class of antibody molecule that harnesses the NKG2D receptor dependent cell cytolytic activity. Zhilan Hu, Gerald Nakamura, Jiabing Ding, Henry Chiu, Richard Erickson, Asja Praetor, Austin Gurney, Henry Lowman and Sherman Fong. Genentech, Inc, 1 DNA way, South San Francisco, CA 94080. USA.

The engagement of the NKG2D receptor on natural killer cells (NK) and on T cells by NKG2D binding ligands on tumor cells lead to tumor target cell destruction. To design an antibody that can directly activate NK cell-mediated killing via NKG2D, we fused murine ligand H60 or human ligand MicB, to the C-terminus of anti HER2 (4D5), a murine IgG2a antibody specific for the cellular proto-oncogene p185HER2/neu. In order to knockout CD16 mediated cytotoxicity of NK cells, we also mutated the Fc piece of anti HER2-MicB antibody. This Fc mutated anti HER2*-MicB fusion selectively engages mouse NKG2D receptor and induces activation and the lysis equivalent to that exhibited by CD16. The non Fc-mutated anti HER2-MicB fusion could augment the killing of BT474 cell by murine NK cells through engaging both CD16 and NKG2D.





A083Involvement of NK1.1 antigen in antibody formation triggered by GlcNAc-coated dendrimer. Katarina Hulikova, Miloslav Pospisil, Jan Svoboda, Marketa Kuldova, Luca Vannucci and Anna Fiserova. Institute of Microbiology, Laboratory of Natural cell Immunity, Academy of Sciences of the Czech Republic, Videnska 1083, Prague, Czech Republic.

Activated NKT and NK cells, beside other functions, were reported to regulate antibody formation either via direct contact with B lymphocytes or cytokine secretion. NK and NKT cells dispose with wide repertoire of carbohydrate-binding, activating as well as inhibitory cell surface receptors belonging to C-type lectin superfamily (NKR-P1, NKG2D, Ly-49). We investigated whether N-acetyl-ß-D-glucosamine-coated polyamidoamine dendrimer (GlcNAc8) which exerted high affinity to the recombinant NKR-P1A molecule, modulate immunoglobulin (Ig) secretion. In previous studies we have demonstrated that GlcNAc8 exhibited therapeutic efficacy in experimental tumor models, reducing cancer growth and prolonging survival time of animals. For this purpose, either sheep red blood cells (SRBC) or keyhole limpet hemocyanin (KLH) were employed as antigens in the presence or absence of GlcNAc8, administrated to healthy and B16F10 melanoma-bearing mice. The immune response to SRBC was assessed by plaque-forming cell assay and serum KLH-specific antibody production by ELISA. To reveal the mechanism by which GlcNAc8 treatment influences antibody formation and to identify the involved receptor(s), we measured Ig levels after depleting CD49b+, NK1.1+ or NKG2D+ cell subpopulations. The phenotype of cells in the peripheral blood, lymphoid organs and tumor microenvironment was analyzed by flow cytometry, focusing on NK, NKT cells and B lymphocytes. The number of SRBC-specific IgM and IgG producing plasma cells in the spleen was significantly increased after repeated GlcNAc8 injections in healthy, but not in tumor-bearing mice. The same tendency in Ig levels was observed after application of KLH as the antigen. In further experiments with KLH-challenged melanoma-bearing mice, we detected an increase of B16F10-specific serum IgM levels after GlcNAc8 administration. FACS analysis demonstrated NKT (CD49b+CD8+) and B220+CD49b+ cell increment, raise of CD86 expression on B lymphocytes as well as CD5 activation marker on T, NK and NKT cells in the spleen. Depletion of NK1.1 positive cells almost completely blocked in vitro Ig secretion after simultaneous antigen and GlcNAc8 stimulation, whereas depletion of NKG2D and CD49b positive cells caused only partial reduction in comparison with undepleted controls.





We can conclude that GlcNAc8 activates regulatory NKT and NK cells through NK1.1 receptor, consequently triggering B lymphocyte differentiation into antigen-presenting cells and plasma cells producing antibodies, including specific anti-tumor IgM.This work was supported by the grants: IAA 500200509 Czech Academy of Sciences, GA CR 310/03/H147 and Institutional Research Concept AV0Z50200510





A084The regulation of NK cell homeostasis and function by CD45. Nicholas Huntington, Yuekang Xu, David Tarlinton and Stephen Nutt. The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.

CD45 is expressed on all hematopoietic cells, including NK cells, and functions by dephosphorylating the inhibitory C-terminal tyrosine of Src-family kinases, favoring full kinase activity. We have recently reported that CD45 is pivotal in eliciting a precise subset of NK cell responses. CD45-deficient mice, whilst displaying normal cytotoxic function, have a selective signalling block following stimulation of NK receptors, such as Ly49D, that associate with immunoreceptor tyrosine activation motifs (ITAMs). This defect results in the failure to fully activate Syk and severely impairs cytokine and chemokine production (1). Surprisingly, CD45-/- mice also have 10-fold more peripheral NK cells suggesting that signalling through a receptor regulated by CD45 is important for NK cell homeostasis. While early development in the bone marrow appeared normal, analysis of cell proliferation in mixed bone marrow chimeric mice revealed an intrinsic increase in cell turnover rate of splenic CD45-deficient NK cells. Flow-cytometric analysis of CD45-/- NK cells showed relatively normal marker expression with the notable exception of the dramatic up-regulation of killer-cell-lectin-like receptor G1 (KLRG1). KLRG1 is thought to be an inhibitory receptor that is expressed on a proportion of naive NK cells and is induced by viral infection. Analysis of C57BL/6 and CD45-/- NK cells showed that KLRG1 was absent from bone marrow progenitors and was expressed specifically on a subset of mature MacIhigh NK cells in the spleen. BrdU labelling revealed that the majority of the proliferating cells were KLRG1-. Culture of splenic NK cells in vitro with IL-15 showed a preferential proliferation, and cytokine production, by KLRG1- cells, whereas the transfer of KLRG1-, but not KLRG1+, NK cells into alymphoid hosts allowed homeostatic expansion and the up-regulation of KLRG1

after 2 weeks. Interestingly, analysis of the NK cell compartment in multiple tissues revealed that KLRG1+ NK cells were preferentially recruited to the blood and lung, while KLRG1- NK cells predominated in the lymph node and peritoneal cavity. These data suggest that KLRG1 expression defines a distinct stage in late differentiation with decreased proliferative potential that is negatively regulated by CD45 to maintain NK cell homeostasis.





1. Huntington ND, Xu Y, Nutt SL, Tarlinton DM (2005). J Exp Med. 201:1421-1433





A085Identification of a ligand for killer cell lectin-like receptor G1 (KLRG1). Masayuki Ito, Takuma Maruyama, Naotoshi Saito, Kazuo Yamamoto and Naoki Matsumoto. Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Frontre-Scien. Bioscience Bldg, Suite 602, 5-1-5 Kashimanoha, Kashiwa 277-8562 Japan

KLRG1 is originally identified in rat as the mast cell function–associated antigen (MAFA), of which ligation by a specific antibody inhibits a secretion of pro-inflammatory mediators by rat mucosal mast cells. In human and mouse, KLRG1 is expressed on subsets of NK and T cells. Infection of mice with murine CMV increases frequency of KLRG1+ populations of NK cells. KLRG1 engagement by an anti-KLRG1 antibody inhibits NK cell effecter functions. To date, physiological ligand(s) and functions of KLRG1 are unknown. Here, we report the identification of a ligand for mouse KLRG1. To explore the ligand(s) for KLRG1, we generated fluorescently labeled soluble KLRG1 tetramers. The human and mouse KLRG1 tetramers bound several human and mouse cell lines, respectively. These observations were confirmed using human or mouse chimeric KLRG1 reporter systems. To identify the KLRG1-ligand(s), we constructed a cDNA library from a cell line expressing a putative KLRG1-ligand(s) and cloned a cDNA encoding KLRG1-L by expression cloning using the KLRG1 tetramer as a probe. Mouse KLRG1-L-transduced BW5147 cells bound the mouse KLRG1 tetramer and also stimulated the mouse KLRG1 reporter cells. These results indicate that KLRG1-L is a ligand for mouse KLRG1.

To investigate the functional consequence of the ligation of KLRG1 by KLRG1-L, we retrovirally transduced a mouse NK cell line that does not express KLRG1, with KLRG1 and examined its capacity to kill the KLRG1-L-transduced BW5147 targets. Expression of KLRG1 reduced the ability of the NK cells to kill the KLRG1-L-transduced targets. The reduction was canceled by the addition of an anti-KLRG1 antibody, indicating that ligation of KLRG1 on NK cells by KLRG1-L on the targets inhibits NK cell cytotoxicity to the targets. Our studies indicate that KLRG1 binds KLRG1-L and that KLRG1 ligation with

KLRG1-L inhibits NK cell cytotoxicity. As KLRG1-L has totally different structure from MHC class I, KLRG1 should contribute to NK cell target recognition independent of MHC class I.





A086Production of chemokines and proinflammatory cytokines by bronchial epithelial cells in response to infection with Francisella tularensis. Thomas R. Jerrells, Debbie Vidlak, and Melanie R. Norton. University of Nebraska Medical Center, 986495 Nebraska Medical Center, 986495 Nebraska Medical Center, Omaha NE 68198-6495 USA.

Francisella tularensis is a small, facultative intracellular, gram-negative coccobacillus that is the etiologic agent of tularemia. The most severe infection with F. tularensis is a pneumonitis characterized by a robust inflammation in the lungs. We hypothesized that the initial response to a pneumonic infection is mediated, at least in part, by chemokines and cytokines produced by the epithelial cells in the lungs. To test this hypothesis, a cell culture model of pulmonary epithelial cell infection by F. tularensis was developed that used Beas2B bronchial epithelial cell line and A549 alveolar epithelial cell line. Cytokine production was assessed by using a specific ELISA (OptEIA, BD Biosciences) or a flow cytometric assay (CBA proinflammatory and cytokine kits, BD Biosciences). F. tularensis replicated in both cell lines, as well as in HeLa cells. Although the A549 cells supported bacterial replication, there was no increased production of any cytokines or chemokines that were evaluated. In fact, this cell line constitutively produced high levels of cytokines. Beas2B cells responded to infection by producing tumor necrosis factor (TNF)-, interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1), but with different kinetics. Proinflammatory cytokines (TNF and IL-6) peaked 4 h after infection and declined to background concentrations from 72 to 96 h of infection. Chemokine (IL-8 and MCP-1) concentrations peaked at 24 and 48 h of infection and were demonstrable at 96 h. Mediator production by Beas2B cells did not require infection of the cell because heat-killed bacteria stimulated production of the same mediators as stimulated by the live organisms, albeit at lower levels. Pulmonary infection of mice with F. tularensis resulted in production of similar cytokines and chemokines in bronchial alveolar lavage fluid, which was associated with infiltration of granulocytes and mononuclear cells into the lungs. Data obtained to date support the suggestion that interaction of pulmonary epithelial cells and F. tularensis results in proinflammatory innate host responses. Although F. tularensis contains lipopolysaccharide (LPS), study results obtained by others show that LPS from this organism or the intact organism does not induce cytokine production by mechanisms similar to those involved in cytokine





production by LPS from enteric gram-negative bacteria, leading to the suggestion that bacterial components other than LPS induce a robust cytokine production in the lungs of infected individuals. In summary, our study results support the suggestion that pulmonary epithelial cells are important contributors to inflammation of the lung associated with pneumonic tularemia. Further in vivo studies are needed to provide proof of principle for this hypothesis.





A087NK cell education and tolerance in mice with no, single or multiple MHC class I molecules. Sofia Johansson, Maria Johansson, Eleftheria Rosmaraki, Gustaf Vahlne, Mali Salmon-Divon, Francois Lemmonier, Klas Kärre, Ramit Mehr and Petter Höglund. Strategic Research Center for studies of Integrative Recognition in the Immune System (IRIS),Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, Nobels Väg 16, Stockholm, Sweden.

The ability of murine NK cells to reject cells lacking expression of specific MHC class I alleles results from an in vivo education process that depend on interactions between Ly49 receptors on NK cells with self MHC class I on other cells. In this project, NK cell tolerance is investigated at three levels of resolution. First, the molecular control of NK cell tolerance is studied by comparing RNA and proteins from MHC class I-deficient and sufficient NK cells using genomic and proteomic techniques. Initial results indicate several differences, the analysis of which will hopefully reveal molecules controlling NK cell tolerance. Secondly, the impact of individual MHC class I alleles on NK cell education and tolerance is studied in mice expressing single MHC class I alleles or combinations of two or more alleles. Transplantation experiments paralleled by analyses of activating and inhibitory receptors in these mice, suggest that the “educating impact” of an individual MHC class I molecule is determined by the number of NK cells (or NK cell receptors) it interacts with as well as by the strength of each MHC class I/Ly49 receptor interaction. Thirdly, mathematical and computational analyses are used to simulate NK cell repertoire development, fitting the results from simulations to data on Ly49 receptor expression in single MHC class I mice. These data supports the two-step selection model over the sequential model. Altogether, our multidisciplinary approach to NK cell tolerance suggests that NK cell education is strongly determined by individual Ly49/MHC class I interactions in a process containing qualitative as well as quantitative events.





A088Structure/Function Studies of Human NKG2x CTLD NK Receptors. Brett Kaiser*, Juan Carlos Pizarro*, Daniel E. Geraghty†, Thomas Spies† & Roland K. Strong*Divisions of Basic Science* and Clinical Research†, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Seattle, WA 98109, USA

The NKG2x–CD94 family of C-type lectin-like immunoreceptors (x = A, B, C, E & H) mediates surveillance of MHC class Ia cell-surface expression, often dysregulated during infection or tumorigenesis, by recognizing the MHC class Ib protein HLA-E. HLA-E specifically presents peptides derived from class Ia leader sequences; thus, its normal cell-surface expression corroborates normal MHC class Ia expression. The affinities and interaction thermodynamics between three heterodimeric NKG2x–CD94 receptors (NKG2A, NKG2C & NKG2E) and complexes of HLA-E with four representative peptides show that inhibitory NKG2A–CD94 and activating NKG2E–CD94 receptors bind HLA-E with indistinguishable affinities, but with significantly higher affinities than the activating NKG2C–CD94 receptor. Despite minor sequence differences, the peptide presented by HLA-E significantly influenced the affinities; HLA-E allelic differences had no effect. These results, along with comparable studies of NKG2D–ligand interactions, reveal important constraints on the integration of opposing activating and inhibitory signals driving NK cell effector functions and draws distinctions between NK and T cells. The more distantly-related, homodimeric NKG2D receptor, unlike the NKG2x-CD94 receptors, uses unusual mechanisms of recognition degeneracy to bind structurally-distinct families of inducible MHC class I-like proteins: MICA/B and ULBPs. These interactions mediate important anti-viral and -tumor activities of NK cells. NKG2D is also found on subsets of T cells. Studies of the interactions between MIC-responsive clones of TCRs and MICA/B, and the ternary interactions between NKG2D–MIC– TCR, show that V1 TCRs can directly bind to MICA/B and reveal the interplay between receptors in putative ternary complexes. MICA also displays homophilic binding, potentially affecting signaling to cells bearing MIC-specific receptors.





A089NKG2D gene silencing by lentivirus-mediated delivery of siRNA results in abrogation of NK cell effector functions. Christian P. Kalberer, Vanessa Baeriswyl and Aleksandra Wodnar-Filipowicz. Experimental Hematology, Department of Research, University Hospital Basel, Hebelstrasse 20, Basel, Switzerland.Activating NK cell receptor NKG2D is constitutively expressed by human NK cells. Ligand binding to NKG2D leads to cell proliferation, cytokine production and cytotoxicity. NKG2D expression is regulated by the cytokine environment and has direct consequences for NK cell activity. To study the function of NKG2D in NK cell cytotoxicity and cytokine production independent of cytokine treatment, we used the lentiviral gene transfer system for stable silencing of NKG2D by small interfering (si) RNA. Towards this goal we constructed a lentiviral vector containing GFP and a NKG2D-specific siRNA expression cassette under the control of the H1 promoter. The cloning strategy was published previously by our laboratory (Schomber et al., Blood, 2004; 103:4511-4513). Human peripheral blood (PB) NK cells were transduced at MOI=30, GFP+ cells were purified by FACS cell sorting and expanded in vitro. The knock-down efficiency of NKG2D was 70-85% at the protein level, as assessed by FACS analysis, and 80-95% at the mRNA level, as assessed by quantitative RT-PCR, compared to untransduced NK cells. These NKG2D levels were comparable to those in freshly isolated resting PB NK cells. The silencing of NKG2D expression was specific since the expression of other NK cell receptors such as the natural cytotoxicity receptors was not affected. We next examined whether silencing of NKG2D had an effect on NK cell function. The cytotoxicity of GFP+ NK cells against the human Daudi cell line was consistently reduced by 15-20% over a wide range of effector to target ratios (20:1 to 0.6:1) compared to control NK cells. This result was confirmed in re-directed killing assays. While cytolysis of P815 cells by control NK cells was strongly increased when target cells were labeled with anti-NKG2D antibodies (29% vs. 57%), the cytotoxicity of genetically modified NK cells was equally low against labeled and unlabeled P815 cells (30% vs. 31%). The potential to produce IFN- was measured by stimulating NK cells with ULBP1-Fc fusion protein. The NKG2D-specific response was virtually abrogated in GFP+ NK cells while the IL-12/IL-18 dependent response was not affected (<0.2 vs. 30 ng/ml IFN-. Control NK cells produced 5.5 and 32 ng/ml IFN-, respectively. Altogether, these results demontrate that, despite residual cell surface expression of NKG2D, genetically modified NK cells are insensitive to





ULBP-Fc or antibody triggering indicating that a minimal expression level is required for NKG2D-mediated stimulation. Our results may explain why freshly isolated NK cells with low NKG2D levels are refractory to stimulation. This model of siRNA-mediated stable down-modulation of NKG2D expression provides strong arguments for the importance of NKG2D cell surface levels in eliciting NK cell responses.





A090Natural killer (NK) cells in C57BL/6 (BL/6) mice infected with Herpes simplex virus type 1 (HSV 1). Lorne F. Kastrukoff and Allen S. Lau. Department of Medicine, University of British Columbia, Dept of Medicine; Koerner Pavilion, KRC Hospital, 2211 Weisbrook Rail, Vancouver, BC V6T L23 Canada.

Rationale: NK cells are important in the immune response to murine cytomegalovirus (MCMV) but their role in HSV 1 infection remains controversial. Previously, we reported an interaction between NK cells and cytotoxic T-lymphocytes (CTL) in BL/6 mice following mucosal (lip) inoculation with HSV 1 and suggested a role for NK cells in the induction of HSV gB498-505 specific CTL (Kastrukoff et al. 2004). In this study, we examine the effect of NK cell depletion on HSV 1 infection in BL/6 mice. Methods: BL/6 ♀ mice, 8-10 weeks of age, were infected with HSV 1 strain 2 via a mucosal route. Mice were pretreated with either -asialoGM1 (-ASGM1) or -PK136 (-NK1.1) IP. Mice were sacrificed on day 1, 3, 6, 9, 12, 15 PI and the lip along with peripheral and central nervous system tissue removed. Tissue was homogenized and plaque assayed for infectious virus. Results: In mice receiving 100 g of -NK1.1 on day –1, 0, and 7 PI, viral titers increase in the lip and the duration of active infection is prolonged in the trigeminal ganglia (TG) compared to controls. When 200 g of -NK1.1 is given on the same days, not only are viral titers increased in the lip and the duration of active infection in the TG prolonged, but also viral titers are increased, to a limited extent, in the TG and brainstem. The administration of 200 g of -NK1.1 on day –4, –1, 0, and 7 results in an increase in viral titers in the TG and brainstem by 10 fold. Furthermore, viral titers are increased in the TG by 1000 fold when 200 g of -NK1.1 is given on day –4, and 300 g on day –1 and 0. Pretreatment of mice with 200 g of -ASGM1 on day –1, 0, and 7 PI, increases viral titers in the lip and prolongs the duration of active infection at this site. In addition, virus is isolated from the cerebellum. Conclusions: The results indicate that NK cells limit viral replication in the lip and TG following mucosal infection with HSV 1. As doses of -NK1.1 previously reported to affect CTL, results in increased viral titers in the TG and brainstem, NK cells may also contribute to limiting viral replication at these sites through the induction of HSV gB498-505

specific CTL. Studies with -ASGM1 raise the possibility that additional cell populations expressing this marker contribute to the control of HSV 1 during later stages of infection in the lip and central nervous system.









A091NK cells efficiently prevent engraftment of donor stem cells after tolerigenic bone marrow transplantation. Leslie Kean, Kelly Hamby, Thomas Pearson, Christian Larsen, Departments of Pediatrics and Surgery, Emory University, Atlanta GA 30322Introduction: Immunologic tolerance remains an elusive goal of transplantation. In mice, mixed-chimerism and donor-specific tolerance can be induced by blocking the CD28/CD40L T-cell costimulatory pathways after bone marrow transplant (BMT). However, large doses of marrow (~1x109 cells/kg) are required, and these regimens have not yet been successfully translated to clinical practice. There is a growing body of evidence that NK cells may play a central role in the failure of low doses of donor bone marrow to engraft, but the mechanisms underlying NK alloreactivity remain to be determined. Methods: (1) BMT in the presence of CD28/CD40L T cell costimulation blockade was performed using C57BL/6 (B6) recipients and Balb/C donor bone marrow. The role of host-anti-donor NK alloreactivity in preventing engraftment was determined by specifically depleting B6 NK cells. The contribution of the NK cell-surface receptor, LFA1 to NK alloreactivity was determined with the anti-LFA1 blocking antibody M17/5.2. (2) An in vivo NK alloreactivity assay was developed that should allow the investigation of the mechanism of NK alloreactivity and the molecular mediators of this process. In this assay, CFSE-labeled B6 splenocytes were adoptively transferred into B6xBalbC F1 progeny. As such, alloreactivity was specifically mediated by NK cells. NK alloreactivity was measured flow-cytometrically by the disappearance of the CFSE-labeled B6 population.Results: Transient depletion of recipient NK cells resulted in increased donor stem cell survival and the induction of stable mixed-chimerism and tolerance despite BMT with low doses (<2x106 cells) of donor bone marrow. This effect was specific to allogeneic donor cells: depletion of NK cells did not increase engraftment of syngeneic bone marrow. Blocking the adhesion molecule, LFA-1 recapitulated the effects of whole-scale NK depletion. Newly emergent NK cells exhibited significantly lower expression of the donor-specific activating receptor, Ly49D, and these NK cells did not exhibit in vivo alloreactivity. These results suggest that the NK repertoire in the mixed-chimeric setting exhibited donor-specific tolerance.

Using the in vivo hybrid resistance NK alloreactivity assay, we measured 80% NK-specific target killing 8 days after adoptive transfer. Significantly less killing occurred at 2, 4, and 6 days. Pre-sensitizing the recipient for 4 days increased the efficiency of killing—from 50% to 80%, suggesting a potent activation phenomenon required for efficient NK allorecognition and/or cytotoxicity.





Implications: These results reveal the importance of NK alloreactivity in the acquisition of mixed-chimerism after BMT, and suggest that clinical approaches to tolerance-induction transplantation may require mechanisms to control NK alloreactivity.





A092Ly49P recognition of MCMV infected cell in the presence of H2k haplotype points to a novel mechanism of NK cell mediated innate resistance to cytomegalovirus infection. Agnieszka Kielczewska 1 ,2, Marie-Pierre Desrosiers1,2, J-C Loredo-Osti1,2, Sonia Girard Adam1,2, Melissa B. Lodoen3, Kenneth Morgan1,2,4, Lewis L. Lanier3 and Silvia M. Vidal1,2,5

1Department of Human Genetics, McGill University, Montreal, Quebec, H3A 1B1, Canada;2McGill Centre for the Study of Host Resistance, McGill University, 1650 Cedar Avenue, Montreal, Quebec, H3G 1A4, Canada;3Department of Microbiology and Immunology, the Biomedical Sciences Graduate Program, and the Cancer Research Institute, University of California San Francisco, 513 Parnassus Avenue, Box 0414, San Francisco, CA 94143-0414, USA4Department of Medicine, McGill University, Montreal, Quebec5Department of Microbiology and Immunology, McGill University, Montreal, Quebec, H3A 2B4, Canada

We have utilized the model of infection with mouse cytomegalovirus (MCMV) in order to characterize host/pathogen mechanisms determining innate resistance. Linkage analyses in F2 progeny from MCMV-resistant MA/My (H2k) and MCMV-susceptible BALB/c (H2d) mouse strains indicated that only the combination of alleles encoded by a gene in the Ly49 cluster on chromosome 6, and one in the major histocompatibility complex (H2) on chromosome 17, is associated with virus resistance. Moreover, we showed that this resistance mechanism was dependent on recognition of MCMV infected cells of H2k background by an activating Natural Killer (NK) receptor Ly49P MA/My. Furthermore, this activation was blocked using anti-H2-Dk antibodies, but not by anti-H2-Kk, indicating that the H2-Dk molecule plays a role in MCMV-dependent Ly49P activation. In addition, the genetic interaction was further confirmed in a cross between susceptible mouse strains FVB/N (H2q, Ly49p present) and BALB.K (H2k, Ly49p absent), where progeny with a similar Ly49p/H2k combination were resistant. Finally, in an in vitro assay, cells of H2d background when transfected with H2-Dk cDNA and infected with MCMV were also able to stimulate the Ly49P receptor. Above results support the existence of a novel NK cell mechanism implicated in MCMV-resistance, which depends on the functional interaction of the Ly49P receptor and the MHC class I molecule, H2-Dk, on MCMV-infected cells.









A093

Licensing of natural killer cells by the interaction of host MHC class I molecules with inhibitory receptors. Sungjin Kim 1 , Jennifer Poursine-Laurent1, Steven M. Truscott2, Lonnie Lybarger2†, Yun-Jeong Song1, Liping Yang1, Anthony R. French1,3, John B. Sunwoo1,4, Suzanne Lemieux5, Ted H. Hansen2 & Wayne M. Yokoyama1,2. 1Howard Hughes Medical Institute, Departments of Medicine, 2Pathology and Immunology, 3Pediatrics and 4Otolaryngology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110 USA. 5Institut national de la recherche scientifique, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.

Natural killer (NK) cells have potent capacity to kill cellular targets and produce cytokines, providing innate defense against tumours and pathogens. These potentially self-destructive functions can be kept in check by inhibitory receptors that recognize target cell major histocompatibility complex (MHC) class I molecules and block activation. However, other mechanisms must control NK cell tolerance for self as illustrated by the absence of NK cell auto-reactivity in MHC class I-deficient mice and humans. Here we show that the MHC class I-specific inhibitory receptors play a surprisingly positive role in NK cell maturation. Using multiple approaches including a single chain MHC trimer transgenic mouse and retroviral transduced receptor expression, we demonstrate that only NK cells expressing receptors for self-MHC acquire functional competence. This “licensing” process requires signaling through the ITIM in these receptors. Thus, licensing results in two types of self-tolerant NK cells; licensed NK cells bearing inhibitory receptors for self, and unlicensed NK cells that are not functionally competent, illustrating an NK cell tolerance mechanism.





A094In vitro modulation of NK cell activity and expression of activating and inhibitory receptors with interferon-alpha, retinoic acid and IL-2 in metastatic melanoma patients. Gordana M. Konjević1,2, Katarina M. Mirjacic, Ana R. Radovanovic, Viktor S. Jovic, Nada M. Babovic, Ivan V. Spucic. 1School of Medicine, Belgrade, 2Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia and Montenegro. As NK cells are the major effector subpopulation of the innate antitumor immune response, in this study, we evaluated the cytotoxic activity of this subpopulation and the expression of NK cell activating (CD161, NKG2D) and inhibitory (CD158a, CD158b) receptors prior and following predictive in vitro treatments. The study was performed on freshly isolated and 18 h in vitro treated peripheral blood lymphocytes (PBL, 2.5x106/ml) with interferon- (IFN, 250U/ml), 13-cis retinoic acid (RA, 10-6M), their combination and rh IL-2 (200U/ml), obtained from 40 metastatic MM patients, prior to therapy and 20 healthy controls. Analysis of NK cell cytotoxic activity and the expression of CD161 and NKG2D activating receptors of freshly isolated PBL of MM patients showed significant decrease compared to healthy controls. IFN, IFN and RA and IL-2, unlike RA alone, gave a significant increase in NK cell activity of MM patients. Contrary to the lack of any significant effect in healthy controls, in MM patients, IFN, alone, also induced a significant increase in CD161 activating receptor expression. The performed treatments gave no change in the expression of CD158b, while RA, alone, induced significant decrease in the expression of the inhibitory CD158a antigen. Further investigation showed that IRF-1, as a transcription factor, is involved in the obtained effect of IFN and IL-2. As NK cell activity is regulated by the balance of activating and inhibitory signals, in this study it is shown that IFN-induced increase in NK cell cytotoxic activity of MM patients is the consequence of the up-regulation of the activating signals, mediated through increased expression of CD161, rather than of the decrease in inhibitory receptors. This predictive in vitro investigation gives an insight into some aspects in the mechanism involved in the effect of applied immunomodulating agents and suggests useful novel immunological parameters for monitoring during immunotherapy in MM patients.





A095Targeting IL-2 To The Endoplasmic Reticulum Confines Autocrine Growth Stimulation To NK-92 Cells. Kyriakos V Konstantinidis1, 2, Evren Alici1, 2, Alar Aints1, Birger Christensson3, Hans- Gustaf Ljunggren2 and Sirac Dilber1

1Division of Hematology, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden

2Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden 3Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden

Anti-tumor effects mediated by adoptively transferred natural killer (NK) cells are dependent on the presence of interleukin-2 (IL-2). IL-2 is considered to be a survival factor for NK cells and an enhancer of their cytotoxic potential. However, systemic administration of IL-2 is frequently impeded by undesirable side effects, such as high toxicity and non-localized administration. Genetic modification of NK cells expressing IL-2 in a localized and controlled manner could be a powerful tool for overcoming these obstacles. Consequently, we have cloned the IL-2 gene using PCR and designed constructs that target IL-2 to specific subcellular compartments. The IL-2-dependent NK-92 cell line was used to verify the functionality of the subcellularly targeted IL-2 constructs. IL-2 targeted specifically to the ER was sufficient to support growth of NK-92 cells. In such cell lines, IL-2 was verified to be localized to the endoplasmic reticulum. IL-2 was not detected in the supernatant and growth of non-IL-2-modified NK-92 cells was not supported during co-culturing experiments. IL-2-transduced NK-92 cell lines showed comparable functional activity and cytotoxicity to parental NK-92 cells.We demonstrate the ability of ER-retained IL-2 to provide autocrine growth stimulation to NK-92 cells, without secretion of the cytokine to the extracellular compartment. Therapy with IL-2 gene-modified autoactivating NK cells may avoid side effects imposed by exogenously administered IL-2.





B001HETEROGENEOUS HUMAN NATURAL KILLER CELL RESPONSES TO PLASMODIUM FALCIPARUM-INFECTED ERYTHROCYTESDaniel S. Korbel*, Kirsty C. Newman*, Katerina Artavanis-Tsakonas*, Karina L. McQueen†, Paul J. Norman†, Catarina R. Almeida‡, Daniel M. Davis‡, Peter Parham† and Eleanor M. Riley*, §

*Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK; † Departments of Structural Biology, and Microbiology and Immunology, Stanford University Medical School, Stanford, CA 94305-5126, USA, ‡Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK

Human natural killer cells can respond rapidly to Plasmodium falciparum-infected red blood cells (iRBC) to produce IFN-. Here we have examined the heterogeneity of this response amongst malaria-naive blood donors. Cells from all donors become partially activated (upregulating CD69, perforin and granzyme) upon exposure to iRBC but cells from only a subset of donors become fully activated (additionally upregulating CD25, IFN- and surface expression of LAMP-1). Whilst both CD56dim and CD56bright NK cell populations can express IFN- in response to iRBC, CD25 and LAMP-1 are upregulated only by CD56dim NK cells and CD69 is upregulated to a greater extent in this subset; by contrast, perforin and granzyme A are preferentially upregulated by CD56bright NK cells. NK cells expressing IFN- in response to iRBC always co-express CD69 and CD25 but rarely LAMP-1, suggesting that individual NK cells respond to iRBC either by IFN- production or cytotoxicity. Furthermore, physical contact with iRBC can, in a proportion of donors, lead to NK cell cytoskeletal reorganisation, suggestive of functional interactions between the cells. When taken together with data indicative of associations between NK responses to iRBC and KIR genotype, these observations imply that individuals may vary in their ability to mount an innate immune response to malaria infection with obvious implications for disease resistance or susceptibility.





B002A large multiprotein complex of ~1.2 mDa with WIP at its core, formed during NK cell activation is altered by inhibitory signaling. Konrad Krzewski1, Xi Chen1, Jordan S. Orange2, Jack L. Strominger1

1 Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave, Cambridge MA, 021382 University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Division of Immunology 3615 Civic Center Blvd, ARC-1216F Philadelphia, PA 19104

The activity of Natural Killer (NK) cells, a subset of lymphocytes involved in protection against microbial pathogens and tumors, is mediated through interaction of NK cell surface receptors with their ligands, expressed on the surface of target cells. One group of such receptors, known as killer cell immunoglobulin-like receptors (KIR), bearing immune tyrosine-based inhibitory motifs (ITIM) that bind the tyrosine phosphatases, negatively regulates NK cell activity. Although ligands for inhibitory KIR are well studied, little is known about downstream signals leading to inhibition of NK cell cytotoxicity. The actin cytoskeleton could be a plausible target of KIR inhibitory receptors, as its rearrangements are absolutely necessary for formation of the activation synapse and NK cell cytotoxicity. A key regulatory protein involved in actin polymerization is the Wiskott-Aldrich Syndrome protein (WASp). WASp activity is modulated by variety of adaptor and regulatory proteins, including the WASp interacting protein (WIP). WIP has been suggested to take part in actin polymerization through regulation of WASp activity. Here, we show that WIP integrates actin remodeling signals by forming a novel multiprotein complex of ~1.2 mDa consisting of WIP, WASp, actin and myosin IIA. The complex formed during NK cell activation is altered by KIR2DL1 inhibitory signaling. Recruitment of actin and myosin IIA to the WIP-WASp complex observed during interaction of NK cell line with a susceptible target cell line is greatly decreased after induction of an inhibitory signal. The formation of the complex is independent of WASp, as both actin and myosin IIA were recruited to WIP in the absence of WASp. The recruitment of actin and myosin IIA correlates with increased WIP phosphorylation, mediated by PKC. Furthermore, introduction of WIP RNAi to the YTS NK cell line results in knock-down of WIP expression and leads to almost complete inhibition of the NK cell cytotoxic activity. These data suggest that KIR inhibitory signaling directly or





indirectly affects proteins involved in regulation of cytoskeleton rearrangements and that WIP plays an essential role in regulation of NK cell activity.





B003The role of A2A adenosine receptor in NK cell-mediated cytotoxicity. Marketa Kuldova 1 , Jan Svoboda1, Hana Kovaru2, Frantisek Kovaru3, Igor Splichal1, and Anna Fiserova1. 1Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, Prague, Czech Republic, 21st Faculty of Medicine, Charles University, Katerinska 32, Prague, Czech Republic, 3University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, Brno, Czech Republic.

The function of NK cells predetermines their broad array of molecular structures, where the substantial role plays adenosine receptors (ARs). This is especially exerted when system undergoes maturation, infection, stress, or tumor growth. High amount of extracellular adenosine present in solid tumors or inflammatory sites serves as negative feedback regulation that may contribute to tumor survival or initiation of antigen-specific immune responses.

The aim of our study to assess the participation of adenosine A2A receptor in NK cell effector function under suppressed immune response in the presence or absence of antigenic stimulation was compared with healthy controls. To follow the effect of A2A agonist on imunocompromised animals, the restrain stress model in mice and immature immune system in newborn piglets were chosen. For combined immunosuppresion with antigenic stimulus, Salmonela typhimurium loaded piglets, and tumor-bearing hosts (lung carcinoma patients, C6 glioma or colorectal carcinoma in rats and B16F10 melanoma in mice) were employed. NK cell and specific CTL-mediated cytotoxicity was measured by standard 51Cr-release assay after in vitro addition of A2AR agonist (CPCA).

In concordance with literature data, the suppressive effect of A2AR agonist was determined in all tested healthy subjects. This inhibitory effect on NK cell-mediated cytotoxicity was amplified in tumor-bearing hosts, whereas CTLs function was not influenced. CPCA stimulates NK cell activity in stressed animals. The same tendency was found in newborn piglets with naive immune system. On the other hand, the inflammatory response of immature immunity was counteracted by CPCA.

These results account for the important role of A2A receptors in NK cells effector function. Since the decreased NK cell activity in the absence of antigenic stimuli was enhanced by A2AR agonist, the antigen loading reverse this effect. Correspondingly, when both immunosuppressive and antigenic agents are presented, CPCA evoked a more pronounced inhibition of cytotoxicity. Thus the





action of A2AR agonist seems to be dependent on the initial state of immune response as well as on antigenic challenge.

This work was supported by the grants 524/05/0267, 524/04/0102, IAA5020403 and Institutional Research Concept AV0Z50200510





B004Extensive polymorphisms of LILRB1 (ILT2, LIR1) and their association with HLA-DRB1 shared epitope negative rheumatoid arthritis. Kimiko Kuroki1,2, Naoyuki Tsuchiya, Mitsunori Shiroishi1, Linda Rasubala1, Yumi Yamashita3, Kunio Matsuta4, Toru Fukazawa5, Makio Kusaoi5, Yoshinori Murakami6, Masafumi Takiguchi7, Takeo Juji8, Hiroshi Hashimoto5, Daisuke Kohda1, Katsumi Maenaka1, Katsushi Tokunaga2

1Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, 2Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, 3Department of Medicine II, Hokkaido University, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan, 4Matsuta Clinic, 3-28-16-1901 Tanito, Nishitokyo, Tokyo 155-0032, Japan, 5Department of Rheumatology and Internal Medicine, Juntendo University, 3-1-3 Hongo, Bunkyo-ku, Tokyo 113-8421,Japan, 6Tumor Suppression and Functional Genomics Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan, 7Division of Viral Immunology, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan and 8Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, 1-1-3 Shibadaimon, Minato-ku, Tokyo 105-8521, JapanLeukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1/LIR1/ILT2), is an inhibitory receptor broadly expressed on leukocytes, and recognizes HLA-class I and human cytomegalovirus UL18. LILRB1 is encoded within the leukocyte receptor complex on 19q13.4, previously implicated in systemic lupus erythematosus (SLE). In the present study, we performed a polymorphism screening and association analysis with SLE and rheumatoid arthritis (RA). In the 5' portion of LILRB1, three haplotypes containing four nonsynonymous substitutions within the ligand binding domains and two single nucleotide polymorphisms within the promoter region were identified and designated as PE01-03. In the 3' portion, two haplotypes (CY01, 02) containing a nonsynonymous substitution of the cytoplasmic region were identified. Significant association with susceptibility to SLE or RA was not observed; however, among the subjects not carrying RA-associated HLA-DRB1 shared epitope (SE), LILRB1.PE01/01 was significantly increased in RA (odds ratio 2.05, P = 0.019, Pc = 0.038). Gross difference was not observed in the crystal structures, thermostabilities and binding affinities to HLA-class I ligands among LILRB1.PE01-03 haplotype products; however, surface expression of LILRB1





was significantly decreased in lymphocytes and monocytes from the carriers of PE01 haplotype. These findings demonstrated that LILRB1 is highly polymorphic, and is associated with susceptibility to RA in HLA-DRB1 SE negative subjects, possibly by insufficient inhibitory signaling in leukocytes. In addition, these observations suggested that the polymorphisms of LILR family members may be substantially involved in the diversity of human immune responses.





B005The novel inhibitory NKR-P1C receptor and Ly49s3 identify two complementary, functionally distinct NK cell subsets in rats. 1Lise Kveberg, 1Camilla J. Bäck, 2Ke-Zheng Dai, 1Marit Inngjerdingen, 1Bent Rolstad, 3James C. Ryan, 2John T. Vaage, and 2Christian Naper. 1Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway; 2Institute of Immunology (IMMI), Rikshospitalet University Hospital, Oslo, 0027 Norway; and the 3Veterans Affairs Medical Center, the Northern California Institute for Research and Education and the University of California, San Francisco, CA USA.

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors (KLRs) which in mice bind members of the genetically linked C-type lectin related (Clr) family, while the distal region encodes Ly49 receptors for polymorphic MHC class I-like molecules. While certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C+ NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a non-polymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C+ NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3+ subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different KLR repertoires.





B006Ly-49 receptor recognition of the rat classical class I molecule, RT1-A1c, is determined by B-pocket conformation. Kerry J. Lavender and Kevin P. Kane. University of Alberta, 6-60 Heritage Medical Research Centre, Edmonton, AB Canada.

Mouse and rat Ly-49 receptors recognize one or more specific allele products of class I MHC (CI). However, motifs on CI molecules that determine recognition by one Ly-49 receptor but not another remain unidentified. The original crystal structure of Ly-49A and H-2Dd depicted two interaction sites: site 1, a highly polymorphic region at the end of the peptide binding groove, initially favoured as conferring allelic specificity, and site 2, a highly conserved region including residues under the peptide binding groove, the alpha 3 domain and the beta 2 microglobulin. Site 2 is now the accepted interaction site between Ly-49 receptors and CI ligands but being so highly conserved, it has been difficult to determine how Ly-49 receptors discriminate between different CI allele products at this site. We previously demonstrated that the mouse inhibitory Ly-49GBALB/c and activating Ly-49W receptors recognize the xenogeneic rat CI molecule, RT1-A1c, in an allele specific manner. We found xenogeneic recognition, like previously demonstrated syngeneic recognition, occurs at site 2. These site 2 residues lie below the polymorphic B-pocket, which is responsible for binding the P2 anchor residue of the bound peptide. This led us to consider that the polymorphic structure of the B-pocket may determine the conformation of conserved site 2 residues lying beneath the peptide binding groove, thereby increasing or decreasing their availability for interaction with different Ly-49 receptors. Single mutants of the RT1-A1c B-pocket, altering either charge or shape resulted in loss of xenogeneic mouse recognition and syngeneic recognition by the PVG rat Ly-49i2 receptor. An additional, conservative mutation in the floor of the B-pocket also disrupted recognition of RT1-A1c by the mouse and rat receptors. In contrast, non-conservative mutagenesis of non-anchor binding pockets of the CI molecule, both on the sides and floor of the peptide binding groove, resulted in no loss of recognition. Loss of recognition was not due to lowered expression of mutants or inability to load peptide as is demonstrated through surface staining and peptide elutions from B-pocket mutants, respectively. In summary, mutagenesis of polymorphic residues in the anchor binding B-pocket results in loss of recognition of RT1-A1c, possibly through alterations in the side chain conformation of solvent exposed residues at





site 2, while mutagenesis of other non-anchor binding pockets within the peptide binding groove had no effect on Ly-49 receptor recognition. These findings give insight into the fundamental nature of Ly-49 discrimination of CI ligands, indicating a possible reliance on conformational features dictated by the polymorphic peptide anchor binding pockets of the CI molecule.





B007TGF-1 down-regulates NKG2D expression via endocytic and degradative pathways.June-Chul Lee1 and Dae Seog Heo1, 2

1Cancer Research Institute and 2Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul, 110-744, Korea.

NKG2D triggers cytotoxicity upon binding to MHC class I-related NKG2D ligands (NKG2DLs). However, despite expression of NKG2DLs on malignant tumors, NK cells in cancer patients are impaired. We have shown previously that TGF-1 produced in advanced cancer patients was primarily responsible for down-modulation of NKG2D and poor lytic activity of NK cells. However, the mechanism underlying this impairment is not clearly understood. In this study, we investigated the molecular basis for NKG2D down-regulation by TGF-1. In IL-2-activated human NK cells treated with TGF-1, total content of NKG2D decreased in parallel with surface NKG2D in a dose and time-dependent manner. No significant difference in mRNA level was observed between normal and TGF-1-treated cells, indicating that TGF-1 did not affect transcriptional process. In addition, TGF-1 did not affect normal protein turnover process or stability of NKG2D. Rather, TGF-1 appeared to facilitate endocytosis and degradation of NKG2D, since inhibition of lysosomal function by bafilomycin A1 or folimycin resulted in marked recovery of total NKG2D expression. Interestingly, this impaired NKG2D down-modulation by TGF-1 was not associated with activation of MAPK signaling pathway, as blocking Erk, JNK, or p38 MAPK with their specific inhibitors did not prevent NKG2D down-modulation by TGF-1. Thus, other signaling pathways, not involving MAPK, underlie NKG2D down-modulation by TGF-1. Taken together, our data provide the first piece of evidence that TGF-1 controls normal trafficking of NKG2D by facilitating endocytosis and lysosomal degradation.





B008Regulation of TNF- production during murine cytomegalovirus infection by NK cell effector functions. Seung-Hwan Lee, Marc Dalod, Jennifer Louten, Kwang-Sin Kim, and Christine A. Biron. Molecular Microbiology and Immunology, Brown University, 171 Meeting Street, Providence, Rhode Island, 02912, USA.

Tumor necrosis factor- (TNF-) is a major mediator of inflammation and immunity. Even though TNF- is found in serum during murine cytomegalovirus (MCMV) infection, the cellular sources and mechanisms of regulation for its production have not been characterized. The studies reported here demonstrated that plasmacytoid dendritic cells, characterized as CD11bdull, CD11c+, B220+, Ly6C+ and pDCA-1+, were the major producers of TNF- during early MCMV infection in immunocompetent mice. Because of their important immunomodulatory effects, the role of NK cells in controlling TNF- production was investigated. At day 5 of MCMV infection, NK cell-depleted and E26 (deficient in NK and T cells) mice had high TNF- production whereas only residual TNF production was observed in control mice. The NK cell-deficient conditions were accompanied by dramatic increases in disease severity when the mice were challenged with high MCMV doses. To dissect the contribution of particular NK cell functions, TNF- production was analyzed in mice mutated in either the interferon receptor (IFN-R) or the perforin gene. High production of TNF- was only observed in the absence of perforin function, suggesting that NK cytotoxicity was critical for the regulation of TNF- production. Flow analyses identified mature macrophages, i.e. F4/80+ and CD11b+ cells, as major TNF- producers. The numbers of F4/80+CD11b+ macrophages were also dramatically increased in perforin-deficient as compared to immunocompetent mice, and many activation markers, including CD40, CD80, CD86, as well as MHC class I and II, were highly expressed on these cells. Purified F4/80+ macrophages were shown to be responsible for most of the TNF- produced by isolated populations after overnight culture, and had activated morphology on cytospin preparations. Thus, macrophages were highly activated and produced massive TNF- in the absence of NK cytotoxic activity. These results indicate that the presence of NK cells regulates the number and activation status of macrophages during MCMV infection as well as their TNF- production. This regulation is dependent on cytotoxic function, and appears to be important in protecting against overwhelming TNF- production as well as cytokine-associated disease. The understanding of the immunomodulatory consequences of NK cell functions provides new insights into the different mechanisms of pathogenesis and approaches for therapeutic intervention to improve health during viral infections. Supported by NIH Grant RO1 CA41268, and Post-Doctoral Fellowships from the Canadian Institute for Health





Research and the Cancer Research Foundation. MD's current address is Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS-Univ, Mediterrancee, F-13288 Marseille Cedex 09, France.





B009Regulation of the Expression of NKG2D Ligands MULT-1 and H60 by MCMV. Tihana Lenac1, Astrid Krmpotic1, Milena Hasan1, Ivan Bubic1, Anne Halenius2, Zsolt Ruzsics3, Jurica Arapovic1, Hartmut Hengel2, Martin Messerle4, Ulrich H. Koszinowski U.H.3, Stipan Jonjic1

1Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, 2Institute for Virology, Heinrich-Heine-University Dusseldorf, Dusseldorf, Germany, 3Max von Pettenkofer Institute, LMU, Munich, Germany, 4Medical Faculty, Martin Luther University of Halle-Wittenberg, Halle, Germany,

NK cells are of crucial importance in the early control of murine cytomegalovirus (MCMV) infection. One of the most potent activating NK cell receptor is NKG2D that recognizes different MHC class I-like molecules. Up to now, three NKG2D ligands have been identified in mice: RAE-1, H60 and MULT-1. We have previously shown that MCMV down-regulates NKG2D ligands from the surface of infected cells and identified MCMV m152/gp40 as a regulator of NKG2D ligands (Krmpotic A. et al, Nat Immunol, 3:529, 2002). Subsequent studies identified RAE-1 as the target for m152/gp40-mediated down-regulation (Lodoen M. et al, J Exp Med, 197: 1245, 2003). Recently, we identified two additional proteins encoded by viral m145 (Krmpotic A. et al, J Exp Med, 201: 211, 2005) and m155 (Hasan M. et al, J Virol, 79: 2920, 2005) genes, which are responsible for the down-regulation of NKG2D ligands MULT-1 and H60, respectively. Our results indicated the involvement of an additional, so far unknown, MCMV gene in the regulation of H60. The importance of m145 and m155 genes in the evasion of NK cells was confirmed also in vivo, underlying the significance of the escape of NKG2D signaling for viral survival and maintenance. Acquisition of EndoH resistance and preserved half-life of H60 and MULT-1 in MCMV infected cells indicate that viral inhibitors affect expression of these proteins only after their exit from the ERGIC/cis-Golgi compartment. In an attempt to identify potential mechanisms by which m145 and m155 interfere with their cellular targets, the co-localization of H60 and MULT-1 proteins with different cellular compartments have been investigated. MULT-1 and H60 co-localize with TGN in uninfected and infected cells confirming that in infected cells both proteins can reach this compartment. The co-localization of MULT-1 and H60 with endo-lysosomal compartment is increased in infected cells. Furthermore different inhibitors of





endocytosis and protein degradation were used. We showed that the downmodulation of surface MULT-1 in MCMV infected cells is mediated via clathrin dependent endocytosis and that both ligands, H60 and MULT-1, are subject of protein degradation in lysosomes.





B010Interleukin-15 Prevents Concanavalin A-induced Liver Injury by Down-regulating IL-4 and IL-5 Production of hepatic NKT cells. Bofeng Li, Rui Sun, Haiming Wei, Zhigang Tian. Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 443 Huangshan Road, Hefei 230027, China.

The hepatic natural killer T (NKT) cells play essential roles in Con A-induced liver injury via Fas/Fas-L pathway as well as releasing a variety of pro-inflammatory cytokines including IL-4, IL-5, IFN-, TNF-. IL-15 is a key cytokine in the proliferation, survival and homeostasis of NKT cells, however, the role of IL-15 in NKT cell-mediated liver injury remains unclear. In this study, we found that pretreatment with IL-15 dose-dependently prevented Con A-induced liver injury. Significantly lower serum ALT levels were detected in mice treated with IL-15, particularly at 12 h and 24 h after Con A injection. Surprisingly, 80% of mice pretreated with IL-15 at 6 h prior to Con A injection survived, while 100% of mice injected a lethal dose of Con A without IL-15 pretreatment died within 12 h. Liver histological studies revealed massive necrosis in the liver of mice 24 h after treatment with Con A alone, whereas mice pre-treated with IL-15 showed minor or slight damage. TUNEL assay also showed significant hepatocyte apoptosis in the liver of mice treated with Con A alone, which was markedly alleviated by IL-15 pretreatment. Various antibodies, including anti-mouse-CD3, -CD4, -CD8, -ASGM1 antibodies, were used to eliminate the corresponding murine lymphocyte subpopulation, indicating that the protective effect of IL-15 on Con A-induced hepatitis is not mediated by NK- or CD8+ T cell-dependent mechanisms. Accordingly, adoptive transfer of hepatic NKT cells isolated from PBS treated mice restored Con A-induced hepatitis, while adoptive transfer of hepatic NKT cells pretreated with IL-15 either in vivo or in vitro failed to restore Con A-induced liver injury. These results indicated that IL-15 protected against Con A-induced hepatitis via NKT cells-dependent mechanism.

NKT cells can secrete a large amount of IL-4 and IL-5 which have been shown to play essential roles in Con A-induced liver injury. IL-15 pretreatment reduced serum IL-4 level by 30% and 50% at 2 h and 3 h and also serum IL-5 level at 3, 6, and 12 h after Con A injection. IL-15 pretreatment completely suppressed intracellular contents of IL-5 and IL-4 of NKT cells by flow cytometry. It has been reported that depletion of eosinophils completely prevented Con A-induced liver damage and IL-4 and IL-5 played an essential role in recruiting eosinophils into liver. In our study, ALT levels and infiltration of eosinophils into the liver were greatly decreased by anti-IL-4 or anti-IL-5 Ab. NKT cells could not express IL-5 mRNA and the expression of





eotaxin-1 (CCL11) and eotaxin-2 (CCL24) were reduced with IL-15 pretreatment in liver by RT-PCR analysis. We also found that EPO activity (eosinophils infiltration) in the liver was much lower in IL-15 pretreatment group.[his work was supported by Natural Science Foundation of China (#30125038, #30230340, #30328022) and 973 project (#2001CB510009; #2003CB515501)]





B011Changes in NK cell phenotype and function in HIV-1 infected patients treated with antiretroviral therapy with or without IL-2. Brian R. Long1, Christopher P. Loo1, Jakob Michaelsson1, Gerald Spotts1, Frederick M. Hecht1 and Douglas F. Nixon1 1Gladstone Institute of Virology and Immunology, University of California San Francisco, San Francisco, CA 94158, USANatural Killer (NK) cells play an important role in the human innate immune response by identifying and attacking virally infected or malignantly transformed cells. NK cells are granular lymphocytes that release cytokines and chemokines, often in response to reduced levels of MHC class I expression and/or detection of cellular stress-induced proteins. Subpopulations of NK cells are defined by the expression of CD56 and CD16 on the cell surface. CD56bright NK cells secrete large amounts of cytokines, particularly IL-2 and IFN, and these cytokines play an integral part in communication between the innate and adaptive immune system. CD56dim, CD16+ NK cells exhibit increased cytotoxicity and effector function and are the subset largely responsible for lysis of target cells. Recently, a population of CD56-, CD16+ NK cells has been described that is increased in HIV-1 infected individuals and displays an anergic phenotype. NK cells express a heterogeneous mix of both activating and inhibitory receptors that serve to further define their function. These receptors include the killer cell immunoglobulin-like receptors (KIR), C-type lectins that heterodimerize with CD94 (NKG2A, NKG2C and NKG2D), and the natural cytotoxicity receptors Nkp30 and Nkp46. The integration of signals from this assortment of receptors determines whether or not these cells become activated. Antiretroviral therapy (ART) has been used successfully to suppress the levels of HIV-1 in infected patients. IL-2 administration has been shown to substantially increase the population of CD4+ T cells. However, little is currently known regarding the effect of either or both of these treatments on the function of NK cells. We utilized multiparametric flow cytometric analysis to evaluate peripheral blood NK cells in a cohort of 60 HIV-1 infected patients that were either untreated (n=20), treated with ART (n=20), or treated with ART + IL-2 (n=20). NK cells from these patients were analyzed for expression of 14 separate markers expressed on the surface of NK cells, as well as for their ability to produce perforin, IL-2 and IFN. The detection of surface CD107a, an indicator of degranulation, was used to assess cytotoxic effector function. Our results demonstrate phenotypic and functional alterations in the NK cell populations amongst the three groups of patients. Most





notably, NK cells from patients receiving ART + IL-2 displayed less cytotoxicity in response to HIV peptides (p<0.05), and produced decreased amounts of IL-2 relative to both untreated and ART only treated patients (p<0.01). Our results indicate that treatment with IL-2 may inhibit cytokine secretion and function of NK cells in HIV-1 infected individuals.





B012Expansion of NK cells with restricted receptor expression and aberrant lineage marker expression in a healthy elderly subject. Charles T. Lutz, Mikel B. Moore, and Zoya B. Kurago. University of Kentucky and University of Iowa, 800 Rose Street, Lexington, KY 40536-0298, USA

Subject 15 was an 82 year old female recruited into a cohort of healthy elderly subjects. . Except for allergic rhinitis and hypothyroidism, she had not been hospitalized or had any serious illness in the previous 45 years. Eighteen months after initial study, Subject 15 had no sign of lymphadenopathy, splenomegaly, or hepatomegaly and no evidence of lymphoproliferative disease on the peripheral blood smear. We identified an unusual cell population that did not express four major mononuclear lineage markers (CD3, CD14, CD19 and CD56). These “quadruple negative” cells (QN) bound mAb GL183, but not HP-3E4 or other available anti-KIR mAb, consistent with restricted KIR2DS2 expression. The QN cells uniformly expressed CD94 without NKG2A, although

NKG2A was co-expressed on 23% of the CD94+ NK cells. These observations

provide evidence that the GL183+CD94+NKG2A- QN cells are clonally related. Although we did not detect CD56 expression with two mAb, the QN cells resembled NK cells by expression of perforin, CD2, CD7, CD8, CD16, CD57, CD94, CD122, and KIR. Upon stimulation the QN cells produced IFN-. The QN cells did not express CD3, CD4, CD8, TCR, or TCR, ruling out T lineage cells. However, the QN aberrantly expressed CD5, a marker usually associated with T cells and some B cells. The QN cells killed K562 target cells about 3-fold

less actively than CD56+ NK cells. The QN cells had ADCC activity that was 10-fold more active than NK cells from the same subject. During in vitro culture, highly purified QN cells retained restricted KIR expression, natural cytotoxicity, and ADCC, but gradually lost CD5 expression and gained heterogeneous CD56 expression, providing additional evidence of NK lineage. A second blood donation after an interval of 18 months showed the QN population to be stable in Subject 15. We conclude that NK cells with restricted KIR and CD94/NKG2 expression and aberrant lineage marker expression expanded in a healthy elderly research subject in the absence of lymphoproliferative disease.





B013Production of soluble activating Ly-49 receptors as tools for studying receptor-ligand interactions. Brian J. Ma and Kevin P. Kane. Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.

Ly-49 receptors play an important role in the regulation of murine NK cell function. NK cells kill cells that do not express self MHC class I molecules. This “missing self” mechanism works through inhibitory Ly-49 receptors that recognize self MHC class I resulting in inhibition of activation. In addition to the inhibitory Ly-49 receptors, there are activating Ly-49 receptors. Although the normal physiological functions of these activating receptors are not fully elucidated, they can recognize class I MHC molecules and may also play a role in the recognition of viral ligands. There are a number of activating Ly-49 receptors, such as Ly-49P and W, that are known to recognize the same ligands as inhibitory Ly-49 receptors. The existence of inhibitory- activating receptor pairs is not unique to the Ly-49 receptor family as they also exist in other NK receptor families. The functional relationship between these highly similar Ly-49 receptor pairs is currently unknown. We previously cloned the activating Ly-49W receptor which recognizes H-2Dd and H-2Dk and is highly similar in the lectin-like domain to inhibitory Ly-49G. We also cloned the activating Ly-49P receptor which recognizes H-2Dd and is highly similar to the inhibitory Ly-49A receptor. Through a series of refolding screens we have found conditions to refold conformationally correct extracellular domains of recombinant Ly-49W and Ly-49P. These tools will be useful in studying the interaction of these activating receptors with their respective ligands and may provide a tool to screen for previously uncharacterized ligands.

This work was funded by: Alberta Heritage Foundation for Medical Research and the Canadian Institutes of Health Research.





B014Impacts of the signaling adaptor protein, BCAP, on murine NK cell function. Alexander W. MacFarlane IV1, Tetsuo Yamazaki2, Tomohiro Kurosaki3, and Kerry S. Campbell1, 1Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA, 2Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575 Miyagi, Japan, and 3RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehirocho, Tsurumi-ku,, Yokohama, Kanagawa, 230-0045, Japan.

B cell adaptor for phosphatidylinositol 3-kinase (BCAP) was previously identified as a signaling adaptor protein that can recruit phosphatidylinositol 3-kinase (PI3K) after B cell antigen receptor stimulation. BCAP has been shown to influence maturation, proliferation, apoptosis and translational activities in B cells. Previous reports demonstrated that BCAP is not expressed in T lymphocytes. We have discovered, however, that BCAP is strongly expressed in murine and human NK cells and can also recruit PI3K upon tyrosine phosphorylation in NK cells. We have further identified several impacts on the phenotype and function of natural killer (NK) cells that develop in BCAP-deficient mice when compared to normal C57Bl/6 mice. One prominent finding is a doubling of the percentage of NK cells in the spleens of BCAP-deficient mice. Freshly isolated or IL-2-stimulated NK cells from BCAP-deficient mice also exhibit enhanced cytotoxicity toward several target cell lines. While proliferative responses of the NK cells were unchanged, we observed increased resistance to apoptosis of BCAP-deficient NK cells upon withdrawal of IL-2 from cultures. These results suggest that the increased number of NK cells in these mice is due to enhanced survival. In stark contrast, BCAP-deficient B cells were previously found to be more sensitive to apoptosis and exhibited impaired proliferative responses. In summary, our results suggest that BCAP may function as a negative regulator of PI3K in NK cells to influence cytotoxicity and apoptosis.Supported by NIH grants R01-CA083859, R01-CA100226, and T32-IA07492.





B015CD83+CCR7+ NK Helper Cells: IL-18-induced Helper Pathway of NK Cell DifferentiationRobbie B. Mailliard,1 Sean M. Alber2, Hongmei Shen3,4, Simon C. Watkins2,4 , John M. Kirkwood5, Ronald B. Herberman,6 and Pawel Kalinski1,4,6. Departments of 1Surgery, 2Cell Biology and Physiology, 3Radiation Oncology, 4Immunology, 5Medicine, University of Pittsburgh; and the 6University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.

In addition to their cytotoxic activities, Natural Killer (NK) cells can also play immunoregulatory functions. Here, we describe a distinct “helper” pathway of NK cell differentiation in which human CD56+CD3- NK cells acquire a unique CD56+/CD83+/CCR7+/CD25+ phenotype and display high migratory responsiveness to the lymph node-associated chemokine CCL21, a strongly enhanced ability to produce IFN upon exposure to Dendritic Cell (DC)- or T Helper (TH) cell-related signals, and pronounced abilities to promote IL-12p70 production in DC and the development of TH1 responses of CD4+ TH cells. This helper pathway of NK cell differentiation, is not associated with any enhancement of cytolytic activity, and is uniquely induced by IL-18, but not other NK cell-activating factors. Also, prostaglandin (PG)E2, a factor which induces a similar CD83+/CCR7+/CD25+ lymph-node-homing phenotype in maturing DC, inhibits the development of this NK cell type. The current data demonstrate independent regulation of the “helper” versus “effector” pathways of NK cell differentiation and novel mechanisms of immunoregulation by IL-18 and PGE2.





B016MCMV encoded MHC-I like proteins that do not require 2m or peptide for cell surface expression. Janet Mans*+, Kannan Natarajan+, Caroline T. Tiemessen* and David H. Margulies+

+Molecular Biology Section, Laboratory of Immunology, NIAID, NIH, 10 Center Drive, Bethesda, MD USA; * Department of Virology, Witwatersrand University, 7 York Road, Parktown 2193 and AIDS Unit, NICD, Private Bag X4, 2131 Sandringham, Johannesburg, South Africa

As part of a strategy to evade host immunity and establish a latent infection, the genome of mouse cytomegalovirus (MCMV) encodes several proteins that exhibit an MHC-I like structure. Viral MHC-I like molecules have been shown to inhibit natural killer cells, either directly by binding to an inhibitory receptor e.g., the interaction of MCMV m157 with Ly49I, or indirectly by downregulating the expression of ligands for the activating receptor NKG2D e.g., the intracellular interaction of MCMV proteins m145, m152 and m155 with Mult-1, Rae 1 and H-60, respectively. To focus on those MCMV MHC-I like molecules that are likely to be expressed on the surface of virally infected cells and engage host receptors, we have used a FLAG-tag approach to examine surface expression in transfected mammalian cells. Additionally, to aid in the structural characterization of viral MHC-I like molecules, we have also investigated the requirements for peptide and/or 2-microglobulin (2m) association of members of the m145 family of glycoproteins. To analyze the requirement of the viral proteins for 2m, we transfected the FLAG-tagged constructs in 2m positive (R1.1) and negative (R1E) cells and compared the levels of surface expression by anti-FLAG staining and FACS analysis. The absence of 2m did not influence the level of M37, m144, m151 or m153 surface expression on R1E cells. In a similar approach, we investigated the requirement for peptide by comparing the level of surface expression of the viral proteins on TAP deficient RMA-S cells to that on TAP sufficient RMA cells. Equivalent surface expression was observed for M37, m151 and m153 in the both cell types, indicating that peptide is not essential for surface expression. Thus the surface expression of these viral molecules is independent of both 2m and peptide. The role of M37, m151 and m153 in NK and T cell receptor immunoevasion is being investigated.





B017NK cells regulate anticryptococcal activity via Interleukin 10 in an autocrine fashion. Kaleb J. Marr 1 , Jeremy C. Wiseman1, and Christopher H. Mody1, 2. Departments of 1Medical Science and 2Microbiology and Infectious Diseases, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada.

NK cells have important roles in the innate response against tumors cells and viruses. Recently, it has been determined that NK cells also possess direct antimicrobial activitiy. In particular, NK cells have the capacity to exert their microbiocidal effect against the opportunistic pathogen Cryptococcus neoformans. However, the mechanism of NK cells anticryptococcal activity has not been elucidated. It is hypothesized that NK cells are able to recycle from one target to another. In this study, the ability of NK cells to recycle from one fungal target to another has been demonstrated using an NK-like human cell line (YT). Not only can NK cells inhibit the growth of subsequent yeast after initial contact, they seem to inhibit growth better. It was then hypothesized that NK cell can regulate their anticryptococcal activity in an autocrine fashion. This was confirmed by inhibiting de novo protein secretion with brefeldin A. It was found that new protein secretion is important for NK cell anticryptococcal activity. A microarray was performed and of the hundreds of genes up regulated; interleukin (IL) 10 was a potential candidate for regulating NK cell anticryptococcal activity. IL-10 is typically thought of as an immunosuppressive cytokine; however, it has been shown to possess the ability to activate NK cells. The transcriptional up regulation of IL-10 in NK cells after fungal contact was confirmed by real time PCR. It was subsequently demonstrated that exogenous IL-10 has the ability to enhance the anticryptococcal effects of NK cells. Also, inhibition of IL-10 via an anti-IL-10 neutralizing antibody reduced the NK cell cytotoxicity against C. neoformans. This demonstrates that NK cells can recycle from one fungal target to another and that this ability may be regulated by IL-10 in an autocrine fashion.





B018Localization of a binding site for the killer cell receptor KLRG1 to the N-terminal two domains of its ligand. Takuma Maruyama1, Masayuki Ito1, Seiko Nakamura2, Kimiko Kuroki2, Katsumi Maenaka2, Kazuo Yamamoto1 and Naoki Matsumoto1. 1Dept. Integrated Bioscience, Grad. Sch. Frontier Sci., University of Tokyo, Chiba, Japan. 2Div. Struct. Biol., Med. Inst. Bioreg., Kyushu University, Fukuoka, Japan.

The killer cell lectin-like receptor G1 (KLRG1) is an inhibitory receptor expressed on natural killer (NK) cells and activated CD8+ T cells. KLRG1 is a type II transmembrane protein that forms a disulfide-linked homodimer and contains a C-type lectin-like domain in the extracellular region and an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic domain. Recently, we have identified KLRG1-L as a ligand of KLRG1.

Majority of killer cell lectin-like receptors (KLRs) with known ligands bind MHC class I or MHC class I-related molecules with exception of NKR-P1s, which bind the other lectin-like molecules Clrs. As KLRG1-L has totally different structure from MHC class I or C-type lectins, mode of ligand recognition by KLRG1 should be dissimilar to those by the other KLRs. To understand the molecular interaction between KLRG1 and KLRG1-L, we sought to identify a KLRG1-binding site on KLRG1L.

To examine KLRG1-L region that is involved in KLRG1-binding, we prepared a series of domain deletion mutants of KLRG1-L and examined their interaction with KLRG1. As the extracellular region of KLRG1-L is composed of five domains (1 - 5), the KLRG1-L domains 1, 2, 3, 4 and 5 were independently deleted to construct the 1 - 5 mutants. Wild type KLRG1-L and the domain deletion mutants were then expressed on BW5147 cells, which naturally don’t express KLRG1-L, and were tested for binding of KLRG1-tetramers. Wild type KLRG1-L and the mutants 3, and 5 bound KLRG1-tetramers to a similar extent. The 1 mutant showed no detectable binding of KLRG1-tetramers, while the 2 showed minimum binding, even though 1 and 2 were expressed on the cell surface at comparable level to that of wild type.Our results indicate that KLRG1 recognizes the domains 1 and 2 of KLRG1-L and also suggest that the domain 1 mainly contributes to the binding, while the domain 2 has a supplementary role. The current studies provide the basis to further investigate the molecular interaction between KLRG1 and KLRG1-L.





B019CD94/NKG2A inhibits natural killer cell activation by depolymerizing actin at the immunological synapse. Madhan Masilamani, Connie Nguyen, Francisco Borrego and John E. Coligan. Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852.

CD94/NKG2A is an inhibitory receptor expressed by human natural killer (NK) cells and a subset of T cells that recognizes human leukocyte antigen E (HLA-E) on potential target cells. Upon ligand binding, CD94/NKG2A is phosphorylated leading to the binding and activation of tyrosine phosphatase SHP1, which initiates signals required to suppress NK cell activation. Using RBL-2H3 cells transfected with CD94/NKG2A-EGFP, we have previously shown that the exclusion of lipid rafts at the inhibitory NK cell immunological synapse (iNKIS) suppresses the formation of raft dependent activation synapse (aNKIS). In this study we show that the enrichment of CD94/NKG2A at the iNKIS is not dependent on the integrity of the actin cytoskeleton, and in fact actin is excluded from the site of contact of CD94/NKG2A with HLA-E coated beads (iNKIS). In contrast, FcRI, an activation receptor, induced actin patch formation. While cholesterol depletion had no effect on CD94/NKG2A accumulation or actin exclusion at the iNKIS, latrunculin-A treatment abolished lipid raft exclusion at the CD94/NKG2A iNKIS. These data indicate that the lipid raft exclusion at the iNKIS is an active process and an intact cytoskeleton is required to maintain lipid rafts outside the inhibitory synapse. Crosslinking CD94/NKG2A induced Vav1 dephosphorylation, most likely by the SHP1 recruited to the ligated CD94/NKG2A. Consequently the signal transduction events leading to the phosphorylation of ezrin-radixin-moesin proteins are interrupted. This results in the inhibition of actin polymerization. Minimally, this prevents the actin dependent recruitment of additional activation receptor complexes to the target cell contact site, thereby maintaining the signaling balance toward inhibition.





B020Functional Redundancy between the transcription factors T-bet and Eomes: Implications for the development of NK and NKT cells. Jennifer L. Matsuda*, Qianjun Zhang*, Amy R. Howell†, Laurent Gapin*

*Integrated Department of Immunology, National Jewish Medical and Research Center, University of Colorado Health Science Center, 1400 Jackson Street, Denver, CO, 80206. †Department of Chemistry, Unit 3060, University of Connecticut, 55 North Eagleville Road, Storrs, CT 06269-3060.

CD1d-restricted V14i NKT cells are innate like lymphocytes that play a critical early role in regulating various disease conditions. V14i NKT precursor cells develop in the thymus, but their maturation process and late fate specification to the NKT cell lineage remains poorly defined. Mice with a targeted deletion of T-bet, a T-box transcription factor critical for Th1 cell differentiation, have a profound, stem cell intrinsic defect in their ability to generate mature V14i NKT and NK cells. We analyzed the molecular profiling of V14i NKT cells in the course of their development and showed that the T-box transcription factor T-bet is sufficient to promote V14i NKT cell maturation. In addition, Eomes, a paralogue of T-bet expressed in NK and CD8 memory T cells, can also invoke V14i NKT cell maturation. By regulating the expression of Th1-associated cytokines, chemokines, chemokine receptors and molecules involved in cytolysis, T-bet defines the unique lineage attributes of mature V14i NKT cells.

Altogether, these results extend the functional redundancy between T-bet and Eomes and implicate a fundamental role for these transcription factors in innate or effector lymphocytes such as NKT cells, NK cells and CD8 memory T cells.





B021HIV-1 Infection and Innate Immunity: Disrupted Cross-Talk Between NK Cells and DCs in Viremic Individuals and the Role Of the CD56neg/CD16pos NK Cell Subset. D. Mavilio1-2, G. Lombardo1, D. Kim1, S.Ortolano1, M. Daucher1, A.M. O’Shea1, C. Kovacs3, D.Follman1, E. Marcenaro2, A. Moretta2 and A.S. Fauci1.NIAID, NIH, Bethesda, MD, USA, 208921

DiMeS, University of Genova, Italy,161322

Department of Medicine, University of Toronto, Ontario, Canada, M5S1A14

Background: Reciprocal interactions of Natural Killer (NK) cells and Dendritic Cells (DCs) require both cell to cell contact through different NK cell receptors and the secretion of several cytokines in order to select/promote optimal DCs that will induce the priming of the adaptive immune response. The effects of HIV viremia on NK cell phenotype and function lead to an impaired ability to kill HIV-1 autologous infected cells, as well as tumor target cells. Moreover, HIV viremia is associated with the appearance of an unusual CD56neg/CD16pos NK cell subset that is iNKRspos/NKG2Adim-neg/NCRsdim-neg and is also highly defective in cytolytic activity and cytokine secretion (IFN-, TNF- and GM-CSF).

Methods: NK cells and DCs were isolated from PBMCs of 15 healthy HIV-1 uninfected subjects, 15 viremic and 15 aviremic HIV-infected subjects receiving antiretroviral therapy. Immature (i)DCs were generated from monocytes stimulated with rIL-4 and GM-CSF for 6 days; mature (m)DCs were generated by overnight stimulation with LPS and IFN-. Autologous NK cells were obtained by negative selection of PBMCs, enriched for CD56pos/CD16pos and CD56neg/CD16pos NK cell

subsets, respectively, and activated with rIL-2. We tested mDC cytokine secretion by ELISA; activation of NK cells driven by mDCs in a Mixed Lymphocyte Reaction; and the ability of NK cells and NK cell subsets to kill iDCs by a Cr51 release assay and fluorescence microscopy.

Results: NK cells isolated from HIV+ viremic, but not HIV+ aviremic, individuals were dramatically impaired in their ability to kill autologous iDCs in vitro, as compared to those of uninfected donors. This defect was associated with significantly reduced NK cell expression of NKp30 and decreased NK expression and secretion of TNF-related apoptosis-inducing ligand (TRAIL) and s-TRAIL, respectively. In addition, the CD56neg/CD16pos NK cell subset predominantly accounted for the lack of iDC killing, as the CD56pos/CD16pos NK cells appeared to be relatively intact in their cytolytic activity. Moreover, mDCs from viremic HIV-1 infected subjects produced lower levels of IL-12, and this likely led to a lack of activation/proliferation/IFN- secretion of NK cells, which, in turn, failed to kill iDCs.





Also, the very low level of secretion of IL-10 by mDCs could potentially enhance HIV-1 replication. Conclusion: The disrupted NK-DC interactions occurring in HIV-1 infected viremic individuals, driven predominantly by the CD56neg/CD16pos NK cell subset, may lead to a defective clearance of HIV-infected cells and likely influence the type and strength of the adaptive immune response to HIV.





B022Identification of a LAT Independent Signaling pathway for Ly49D/DAP12 that requires the expression of Syk. Daniel W. McVicar, Gillian Whittaker, Laura Quigley, David Reynolds, and Deborah Burshtyn. Lab of Experimental Immunology, NCI-Frederick, MD 21702 and Dept. of Medical Microbiology and Immunology, University of Alberta, Edmonton Alberta, Canada. The Linker of T cell Activation (LAT) has been characterized as an integral protein in TcR-mediated activation. A substrate of Zap-70, LAT phosphorylation leads to downstream events such as Ca2+ mobilization and Ras activation through its association with PLC1, Vav, Slp-76, Grb2 and the 85 kDa subunit of PI3K in T cells. LAT is also present in NK cells, becomes tyrosine phosphorylated during natural killing and may mediate PLC1 activation during FcR-mediated NK cell killing. LAT -/- mice, however, exhibit normal killing and ADCC leaving the role of LAT in NK cells poorly defined. Here we demonstrate that LAT is downstream of the DAP12 pathway in both mouse and human NK cells. Moreover, we utilize a variant of RNKD that has a severe reduction in Ly49D-mediated calcium mobilization, RNKDLS, to demonstrate the existence of a LAT-independent pathway to calcium mobilization that requires expression of Syk. We show that RNKDLS cells express significantly lower levels of both LAT and Syk than parental RNKD cells. Reconstituting RNKDLS with LAT via vaccinia virus infection restores calcium mobilization. Similarly, RNKDLS cells reconstituted with Syk also mobilize calcium efficiently even with their low levels of LAT. Consistent with this data, Ly49D-mediated calcium mobilization is largely intact in NK cells of LAT-/- mice. Taken together our data suggest that, DAP12 signals efficiently in the absence of Syk only when LAT is present. We therefore, suggest that DAP12 preferentially utilizes a Syk-based signaling system that can couple through multiple adaptors. In Syk-/- NK cells Zap70 compensates, but the pathway now is wholly dependent on LAT. The flexible use of multiple signaling pathways employing various adaptors may allow individual NK cell activation receptors to send biochemically and functionally unique signals via differential use of the various cascades.





B023Human Nk Cell IFN- Production Is Regulated By Endogenous TGF-Sarah K. Meadows, Mikael Eriksson, Amorette Barber, and Charles L. SentmanDepartment of Microbiology & Immunology, Dartmouth Medical School, Lebanon, NH 03756 U.S.A.

NK cells are an important component of innate immunity, and they have the ability to produce large quantities of IFN- and promote CTL and Th1 cell development, antigen presentation, and macrophage activation. TGF- is believed to be an important immunoregulatory molecule that limits or prevents immune cell activation. In this study we examined the ability of endogenous TGF- to alter human NK cell responses to monokines and TLR agonists. We have demonstrated that blocking the action of endogenous TGF- led to an increase in the percentage of human NK cells that produced IFN- and an increased production of IFN- per cell in response to stimulation by IL-12, IL-15, and IL-18. Blocking TGF- alone did not induce IFN- production by NK cells, but it did result in a signifcant increase in NK cell IFN- production under suboptimal stimulation conditions. TLR agonists, polyI:C and zymosan, stimulated IFN- production by human NK cells within a mixed cell population when TGF- was inhibited. Our findings also suggest that TGF- associated with other cells limited NK cell activation. These data have implications for vaccine and therapeutic strategies where innate IFN- production is desirable and for long-term therapy with TGF- inhibitors in man.





B024Murine Embryonic Liver Differentiates Human Stem Cells into a Spectrum of NK Precursors and Polyclonal KIR+ NK Cells. Jeffrey S. Miller, Karen Brungard, Valarie McCullar. University of Minnesota Cancer Center. 420 Delaware Str. SE, Minneapolis, MN 55455 USAWe have shown that a murine fetal liver cell line (AFT024) and human cytokines (IL-15, IL-7, IL-3, Flt3-L and c-kit ligand) are needed to induce NK cell differentiation and KIR acquisition. To understand the level of maturation where these factors orchestrate NK cell development, a switch culture was designed to separate early and late events. Cord blood CD34+/Lin-/CD38- stem cells were cultured on AFT024 for 28 days. Use of IL-3 or Flt3-L alone resulted in minimal growth. In contrast, we show that NK cell differentiation can occur, albeit at low frequency, with a combination of IL-3 and Flt3-L, in the absence of IL-15. These early NK cells were negative for both CD94 and KIR. These conditions also allowed accumulation of CD56- NK cell precursors. CD34+CD7-, CD34+CD7+ and CD34-CD7+ cells were detected in cultures lacking IL-15. Each precursor was tested in secondary cultures containing AFT024 with IL-15 alone, +IL-3, or +IL-3 and Flt3L. After an additional 2-4 weeks, NK cells differentiated from each distinct cell population. Few NK cells resulted from IL-15 alone, these were predominantly KIR negative. Addition of IL-3 or IL-3+Flt3L significantly increased the absolute number of NK cells and the acquisition of CD94 heterodimers and KIR. We next explored other stromal cell lines in attempt to identify novel factors important in early NK cell maturation. A novel cell line derived from murine embryonic liver (EL08-1D2), identified for its ability to support expansion of mouse stem cells, was compared to AFT024. To test the differential capacity of these microenvironments, single cord blood stem cells were plated on the two feeders supplemented with all cytokines. After 4 weeks, EL08-1D2 induced 125,852±1400 NK cells from a single stem cell versus AFT024 where significantly less resulted (23,143±8117). KIR+ NK cells were also significantly greater with EL08-1D2 (3689±801 vs. 799±491), always in a polyclonal pattern. NK cell development and KIR acquisition were dependent on direct contact with EL08-1D2. Increased development could be from greater differentiation, proliferation or both. Cord blood stem cells were cultured in direct contact with EL08-1D2 under primary culture conditions with IL-3 and Flt3-L but in the absence of IL-15. All CD56- NK cell precursors developed with greater frequency on EL08-1D2 than AFT024. In conclusion, EL08-1D2, derived from a primitive microenvironment during mouse ontogeny,





efficiently recapitulates NK cell development by inducing NK cell differentiation and proliferation. IL-3 and Flt3-L, and not IL-15, allows the isolation and study of distinct NK cell precursors. Direct contact with EL08-1D2 induces KIR acquisition suggesting that unique environmental factors conserved between mouse and man contribute to the extrinsic signals which lead to KIR acquisition.





B025

Negative Impact of KIR-Ligand Mismatch on Transplant-Related Mortality (TRM) in Umbilical Cord Blood Transplant (UCBT) Recipients. Jeffrey S. Miller, John E. Wagner, Daniel J. Weisdorf, Juliet N. Barker, Harriet Noreen, Ye Tan, Martin Maiers, Michael R. Verneris, Bruce R. Blazar, Claudio G. Brunstein, from the Blood and Marrow Transplant Program, University of Minnesota, 420 Delaware Str SE, MMC 806, Minneapolis, MN 55455 USA.

Umbilical cord blood (UCB) is frequently considered as a suitable alternate source of hematopoietic stem cells (HSC) for both pediatric and adult patients who require HSC transplant for treatment of high-risk or relapsed hematologic malignancy. As mismatched killer Ig-like receptor ligands (KIR-L MM) has been associated with anti-host alloreactive natural killer (NK) cell activity and reduced risk of acute myeloid leukemia relapse in recipients of T-cell depleted haploidentical HSC , we hypothesized that GVL after UCBT may also be mediated by NK alloreactivity. We therefore assessed the effect of KIR-L MM in 243 recipients of UCB transplanted at the University of Minnesota between 1998 and 2004 for whom HLA-A, -B, -C and DRB1 typing was available for both patient and UCB unit(s). Median age, weight, and follow-up were 27 yrs (range, 0.2-69), 64.6 kg (range, 3.8-120.2), and 1.1 yr (range, 0.5-6.6), respectively. For recipients of double UCBT (n =106), we analyzed the KIR-L assignment of the engrafting unit only. KIR-L MM in the GVH direction was established using the algorithm of Ruggeri and Velardi and was found in 70 (29%) donor-recipient pairs. Probability of 2-year survival was 54% (95%CI:42-66%) vs. 47% (95%CI:38-56%) (p=0.88) in KIR-L MM vs. KIR-L matched pairs, respectively. Incidence of relapse at 1-yr was 17% (95%CI:8-26%) vs. 31% (95%CI:24-38%)(p=0.12). Incidence of graft failure and grade II-IV acute GVHD was 11% (95%CI:3-18%) vs. 10% (95%CI:6-15%) (p= 0.97) and 50% (95%CI:37-63%) vs. 50% (95%CI:42-58%) (p=0.67), respectively. Notably, the incidence of transplant-related mortality (TRM) was higher among recipients of KIR-L MM grafts [26% (95%CI:15-26%) vs. 13% (95%CI:8-18%), p=0.01]. Outcomes were not affected by the intensity of the preparative regimen, type of UCB graft (single or double), or by diagnosis (AML, CML or MDS). In summary, in the setting of UCBT, KIR-L MM is associated with increased TRM with no obvious beneficial effect on engraftment, relapse risk or survival as previously demonstrated in recipients of TCD haploidentical HSC. Differences in NK cells reconstitution, presence of T-cells and/or use of immunosuppression may interfere with any





potential effect alloreactive NK cells in the setting of UCBT. While additional studies are still needed, results to date fail to support a specific search for UCB units with a KIR-L MM.





B026Retrovirus-mediated expression of the IL2-receptor beta chain (CD122) in hematopoietic stem cells facilitates the in vitro expansion and differentiation of natural killer cells. Anjali Mishra, Karl Welte, Christoph Klein

Murine NK cells are notoriously difficult to expand in vitro, thus limiting the possibility to assess NK cell based therapies in murine cancer models. To circumvent this hurdle, we hypothesized that genetically modified hematopoietic stem cells might be used as a source for amplified NK cell cultures. We focussed on the IL2-receptor beta chain CD122, an early marker expressed on committed NK progenitor cells. We generated the retroviral vector CMMP-CD122 and characterized ectopic CD122 expression in BAF/3 cells. CMMP-CD122-IRES-GFP, but not the control vector CMMP-GFP, conferred IL2-responsiveness in BAF/3 cells. Next, we transduced purified Sca1+ckit+lin- hematopoietic stem cells with pseudotyped retroviral vectors. Gene transfer efficiency was 24% (CMMP-CD122-IRES-GFP) and 21% (CMMP-GFP), respectively. After an initial culture period of 4 days in a cytokine cocktail consisting of SCF, Flt3L, and IL2, CD122 cells expanded significantly more (10 fold) compared to GFP-transduced control cells (3 fold). CD122-transduced cells, but not control-transduced cells, continued to proliferate in the presence of IL2 as semiadherent, hypergranular cells for at least 6 months (NKL cells). FACS analysis revealed the following immunophenotype: NK1.1+, CD3-, CD4-, CD8-, CD19-, CD25+, CD45+, CD49b+, CD94+, NKG2D+, Mac-1low B220-, c-kit+, perforin I+, granzyme B+. Furthermore, expression of perforin I+, granzyme B+ and FasL was documented by RT-PCR. NKL cells also produced Interferon-gamma and TNF-alpha, as assessed by ELISA. Functionally, NKL cells displayed strong cytotoxic activity against YAC cells and various murine tumor cell lines. In summary, we here provide evidence that large numbers of NK-like cells can be differentiated from genetically engineered murine HSC. NKL cells could be a useful tool to dissect molecular mechanisms of NK cell differentiation and to study the effects of adoptive NK cell transfer in murine disease models.





B027STAT regulation and function in the NK cell IFN- response to acute viral infections. Takuya Miyagi, M. Pilar Gil, and Christine A. Biron. Department of Microbiology and Immunology, Brown University, 69 Brown Street, Providence, RI 02912, USA.Innate cytokines regulate NK cell responses to infections. Type 1 IFNs, i.e. IFN-, activate cytotoxicity. Under immunocompetent conditions of viral infections, however, they do not induce NK cell IFN-. The signal transducers and activators of transcription (STATs) control a wide range of biological responses to cytokines with IFN- signals primarily induced through activation of STAT1/STAT2 heterodimers or STAT1/STAT1 homodimers. Other STAT molecules can be activated, however, and another cytokine, IFN-, primarily uses STAT1/STAT1 homodimers to signal. Type 1 IFNs and IFN- both induce STAT1 expression. Our laboratory has reported that an IFN--mediated inhibition of NK cell IFN- production is STAT1 dependent. The studies reported here were undertaken to extend characterization of the regulation of NK cell IFN- responses as well overall STAT1 expression to involvement of STAT2. The experiments were carried-out examining responses to lymphocytic choriomeningitis virus (LCMV) infections in immunocompetent, STAT1-deficient and STAT2-deficient mice. Immunocompetent mice produce high type 1 IFN but low spontaneous NK cell IFN- levels during LCMV infections. ELISAs of serum samples demonstrated that the absence of either STAT2 or STAT1 resulted in increased IFN- production at the earliest times after infection, i.e. up to day 2. However, a sustained and extended production in STAT1-deficient was not observed in STAT2-deficient mice. Western blot analyses of total STAT1 protein expression demonstrated that although leukocyte populations from STAT2-deficient mice had delayed induction of STAT1 protein, the molecule was induced to near-normal levels by day 2.5 after infection. Flow cytometric analyses detecting total STAT1 and IFN-expression within individual NK cells isolated at different times after infection showed that the ability to express IFN- was associated with low STAT1 IFN- expression decreased in STAT2-deficient cells as the STAT1 levels increased. ELISA analysis of IFN- in the media cultured with natural IFN- demonstrated that the type 1 IFN could induce IFN- production in activated populations having low but not high STAT1 levels. Thus, induction of STAT1 is key in blocking a pathway for type 1 IFN induction of IFN-, and STAT2 is critical for this induction in response to IFN-. However, the ability of IFN- to induce a STAT2-independent pathway promoting





STAT1 expression appears to act to promote a tight regulation of type 1 IFN induction of IFN- when the cytokine is present. Taken together, the observations significantly extend the understanding of how regulation of STAT expression acts to shape immune responses in vivo. The authors thank Kathryn Doiron for technical assistance. Supported by NIH RO1 Grants CA41268 and AI55677.





B028CD100 on NK cells enhance the killing of target cells expressing CD72 by increasing the adhesion to the target cells. Sa'ar Mizrahi and Ofer Mandelboim. The Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel

NK cells are able to kill tumor and virus-infected cells without the need of prior antigen stimulation. The killing of these target cells is regulated by inhibitory, lysis and co-stimulatory receptors that are expressed on the surface of NK cells, many of them are still unknown. Here, we show that the CD100 (Semaphorin 4D), a 150kD transmembrane protein expressed on the surface of activated NK cells mediate the Killing of target cells by binding to its ligand CD72. We show that CD100 is not involved directly in the killing process but rather increasing adhesion between NK cells and their target cells which lead to a more efficient killing of the target cells. These observations demonstrated for the first time a direct role for new family of proteins- the semaphorins, in mediating killing of target cells by NK cells. We demonstrate a functional role for CD100 in NK cell killing and suggest a mechanism by which increased killing of tumor cells expressing CD72 such as B cell lymphomas is expected.





B029The AP-1 subunit JunB determines NK cell-mediated target cell killing by regulation of the NKG2D-ligand RAE-1 Norman Nausch*, Lore Florin†, Bettina Hartenstein†, Peter Angel†, Marina Schorpp-Kistner†, Adelheid Cerwenka* Division of Innate Immunity, † Division of Signal Transduction and Growth Control, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, email: [email protected]

The activating receptor NKG2D and its ligands RAE-1 play an important role in the NK, + and CD8+ T cell-mediated immune response to tumors. Expression levels of RAE-1 on target cells have to be tightly controlled to allow immune cell activation against tumors but to avoid destruction of healthy tissues.Here, we report that cell surface expression of RAE-1 is greatly enhanced on cells lacking JunB, a subunit of the transcription complex AP-1. Furthermore, tissue-specific junB knock-out mice respond to TPA, a potent AP-1 activator, with markedly increased and sustained epidermal RAE-1 expression. Accordingly, junB-deficient cells are efficiently killed by NK cells and induce IFN- production.Our data indicate that the transcription factor AP-1, which is involved in tumorigenesis and cellular stress responses, regulates RAE-1. Thus, up-regulated RAE-1 expression due to low levels of JunB could alert immune cells to tumors and stressed cells.





B030

Increase in natural killer cells in tocotrienol supplemented nude mice. Kalanithi Nesaretnam and Kanga Rani Selvaduray.Malaysian Palm Oil Board, P.O.Box 10620, Kuala Lumpur 50720, Malaysia

Vitamin E supplementation has been shown to contribute in immunoregulation, antibody production and resistance to implanted tumours. The aim of this study was to evaluate the effect of tocotrienols, a vitamin E isomer on the cytotoxicity of natural killer (NK) cells. NK cells are important effectors in immunity to tumours, intracellular bacteria, parasites and viruses. Four week old athymic nude mice were injected with 1x106 MDA-MB-231 human breast cancer cells into the right mammary fat pad. The tumour size and incidence was measured weekly. The animals were divided into two groups. One group was orally administered tocotrienol-rich fraction (TRF) at a dose of 1mg/day. The other group was the control. Blood lymphocytes from control and TRF supplemented nude mice were prepared and NK cells measured using a flow cytometer. At necropsy, 12 weeks after the tumour cell injections, there was a significant decrease in tumour size and incidence in the TRF treated nude mice compared to the control mice at all time points. The time of appearance of tumour in the TRF was also delayed. Metastasis to the lungs in the TRF supplemented group was lower than in the control group. Single colour flow cytometry analysis using various leukocyte marker antibodies demonstrated that NK cells were significantly increased in the TRF treated group (88.1± 5.1) compared to the control (44.4± 4.3). We did not observe any significant changes in the number of B-lymphocytes or -TCR T-lymphocytes. These demonstrations suggest the involvement of tocotrienols and NK cells in mediating immunity in cancer.





B031Function of NKG2D in NK cell-mediated rejection of mouse bone marrow grafts. Kouetsu Ogasawara 1 ,2, Jonathan Benjamin1, Rayna Takaki1, Joseph H. Phillips3, and Lewis L. Lanier1

1Department of Microbiology & Immunology and the Cancer Research Institute, University of California, San Francisco, 513 Parnassus Ave. HSE 1001, Box 0414, San Francisco, California 94143-0414, USA2Department of Intractable diseases, Division of Clinical Immunilogy, The Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjyuku-ku, Tokyo, 162-8655 Japan3 Schering Plough BioPharma, 901 California Avenue, Palo Alto, California 94304, USA

Irradiation-resistant NK cells in a F1 recipient can reject parental bone marrow (BM), and host NK cells can also prevent engraftment of allogeneic BM. We show that repopulating BM cells in certain mouse strains express the RAE-1 proteins, which are ligands for the activating NKG2D NK cell receptor. Treatment with a neutralizing NKG2D antibody prevented rejection of parental BALB/c BM in (C57BL/6 x BALB/c) F1 recipients, and allowed engraftment of allogeneic BALB.B BM in C57BL/6 recipients. Additionally, BM from RAE-1 transgenic C57BL/6 mice was rejected by syngeneic animals, but accepted with anti-NKG2D treatment. If other stem cells or tissues up-regulate expression of NKG2D ligands after transplantation, NKG2D may contribute to graft rejection in immunocompetent hosts.





B032An inhibitory receptor, Ly49Q, is a useful marker for defining development of murine plasmacytoid dendritic cells. Omatsu, Y.1), Iyoda, T.1), Kimura, Y.1), Ishimori, M.1), Toyama-Sorimachi, N.2), and Inaba, K.1) 1) Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. 2) Department of Gastroenterology, Research Institute, International Medical Center of Japan, Toyama, 1-21-1, Shinjuku-ku, Tokyo 162-8655, Japan

Inhibitory receptors play critical roles for fine tuning of immune responses. A number of inhibitory receptors have been identified on various types of cells. Ly49Q is an ITIM-bearing inhibitory receptor belonging to Ly49 family that is known as one of subfamilies of NK receptors. Ly49Q has several unique features different from other known NK receptors. Ly49Q expresses on professional antigen presenting cells including macrophages and dendritic cells but not on NK and NKT cells. Expression of Ly49Q on antigen presenting cells is enhanced by type I and type II IFNs, suggesting a particular role of this molecule in regulation of functions of antigen presenting cells in viral infection and/or inflammation.

We found that different expression levels of Ly49Q define sequential developmental stages of plasmacytoid dendritic cells (PDCs), which are known as the cells producing very high levels of type I IFN. PDCs lacking Ly49Q are immature in terms of lower production of cytokines, including IFN-, IL-6 and IL-12p70, upon TLR-dependent stimulation compared with Ly49Q+ PDCs, but Ly49Q- PDCs can produce TNF-. Our results revealed that an early developmental stage of PDCs, with a distinct cytokine profile, can be identified by the expression of Ly49Q.





B033Presence of “super-self” or “missing self” – or both? Reevaluation of four early experiments on NK recognition from 1976-1989. Anders Örn, MTC, Karolinska Institutet, Box 280, SE-171 77, Stockholm, Sweden

An analysis will be presented based on the results from four sets of experiments, (2 published and 2 non-published) on NK target cell recognition. These experiments were performed in Uppsala, Pasadena and Stockholm during the first 15 NK years and include experiments on:

1. Target-binding in the mouse - the lacking negative control for thymocytes (non-published data, 1976-77)

2. Natural killer cells mediate lysis of embryonal carcinoma cells lacking MHC. Stern et al. Nature. 1980 May 29;285(5763):341-2.

3. Transfection of H-2 genes (Class 1 MHC genes and pseudogenes + Qa) into Hepa-cells and L-cells. (non-published data, 1983-86)

4. PL-C treatment of Molt-4 and U-937 cells makes these cells temporary resistant to NK killing. Uggla et al. Scand J Immunol. 1989 Jan;29(1):83-9.

The presented data have implications for our understanding on the nature of NK target structures and on the possible presence of NK cells in thymus. Finally a wild speculation on NK cell function will be presented and discussed.





B034Activating Ly-49 receptors regulate LFA-1 mediated adhesion by NK cells. Mohammed S. Osman, Deborah N. Burshtyn and Kevin P. Kane. 659 Heritage Medical Research Center, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB Canada.

The ligands of activating Ly-49 receptors are class I MHC proteins and viral orthologs of MHC proteins. Activating Ly-49 receptors associate with the ITAM-containing signaling adapter protein DAP-12, and trigger NK cell lysis of target cells. To study the mechanism of Ly-49 mediated target lysis, we stably expressed mouse Ly-49D or Ly-49P activating receptors on the heterologous rat RNK-16 cell line. Both Ly-49D and Ly-49P confer specific lysis of H-2Dd expressing cell lines. This Ly-49 mediated lysis was LFA-1 dependent. In addition, we found that Ly-49P transfectants, but not parental RNK-16 cells, formed tight conjugates with target cells expressing the Ly-49P ligand H-2Dd in an LFA-1 dependent manner. RNK-16 cells expressing an Ly-49P mutant (R57L) unable to associate with DAP-12 did not form conjugates with H-2Dd expressing target cells, nor lyse these cells. Thus, DAP-12 dependent signaling is required upon Ly-49P engagement to facilitate effector:target cell conjugate formation. Furthermore, crosslinking of wildtype Ly-49P or Ly-49D on RNK-16 transfectants with soluble antibodies to these receptors triggered rapid adhesion of the RNK-16 transfectants to purified and immobilized mouse ICAM-1. No binding to ICAM-1 was observed with RNK-16 control cells or with cells transfected with the R57L mutant under similar conditions of antibody treatment. These results demonstrate for the first time that activating Ly-49 receptors can control the adhesive properties of a co-expressed receptor, LFA-1. Furthermore, our results indicate that this regulation is mediated through DAP-12 in a mechanism that may be analogous to TcR-mediated inside-out signaling. Thus, an important component of activating Ly-49 function may be the rapid activation of LFA-1 adhesion to enhance conjugate formation with target cells and enable additional engagement of Ly-49 and other receptors, thereby determining the fate of target cells in the encounter.





B035SHIP promotes repertoire diversity required for normal NK function. Kim H. T. Paraiso,1 Joseph Wahle,1 Caroline Desponts,1 Wayne Yokoyama,2 Masaru Taniguchi,3 Charles Sentman,4 Laurent Brossay5 and William G. Kerr.1 1H. Lee Moffitt Comprehensive Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Ave., Tampa, FL USA; 2Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO, 63110 USA; 3RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan; 4Dartmouth Medical School, Lebanon, NH, USA; 5Brown University, Providence, RI, USAAs part of the innate immune system, natural killer (NK) cells play a critical role in the acute rejection of allogeneic bone marrow (BM) grafts. Previously, we found that that the NK repertoire of SHIP-/- mice on a mixed 129Sv/C57BL6 background was highly disrupted with a single NK subset comprising 70-80% of the peripheral NK compartment. As a consequence of this repertoire disruption, SHIP-/- hosts were shown to engraft fully mismatched allogeneic BM transplants from Balb/c (H-2d) and A/SW/Sn (H-2s) mice. Here we extend our findings to show that H-2b SHIP-/- hosts also permit engraftment of BM from H-2f, H-2k, H-2p, H-2r, and H-2u donors. We recently completed backcrossing the SHIP mutation to the F10 generation on the C57BL6 background and thus we reexamined the NK repertoire in this defined genetic background. On this background we find that the NK repertoire is severely disrupted such that most inhibitory and activating receptors are under-represented in the peripheral NK compartment. The receptors that we find to be significantly under-represented include: CD94, Ly49A, Ly49C, Ly49D, Ly49I, Ly49G2, Ly49H, Ly49I, KLRG1, KLRE-1 and NKG2D. Interestingly, peripheral SHIP-/- NK cells on the B10.D2 (H-2d) background express Ly49A at higher levels than WT, while the remaining receptors are under-represented. Ly49F is unchanged by the SHIP mutation while the surface density of 2B4 is increased approximately two-fold. This under-representation of activating and inhibitory receptors leads to a peripheral NK compartment that has reduced number of receptors per NK cell or reduced combinatorial diversity. Intriguingly, the repertoire of immature NK cells in the BM does not show the same trend. Here we show that this reduced combinatorial diversity in the NK compartment has different consequences for different functions. First, we show that the NK compartment is mature in SHIP-/- mice in that they retain their ability to acutely reject missing self BM grafts. However, SHIP-/- mice on a BL6 background are still unable to mediate acute rejection of





allogeneic BM grafts from BALB/C mice. In addition, preliminary NK killing assays show that IL2-activated NK cells from BL6 SHIP-/- LAK cells are altered in their ability to kill a syngeneic RMA cell line that expresses the NKG2D tumor receptor target, Rae-1. These results demonstrate that SHIP’s role in promoting combinatorial diversity of the NK repertoire is critical for certain NK functions, but not all.





B036NF-B/p65 affects NK cell development by regulating the activation of Ly49 genes. Véronique Pascal,1 Mehrnoosh Abshari,2 and Stephen K. Anderson2 .1Laboratory of Experimental Immunology, NCI-CCR, and 2Basic Research Program, SAIC-Frederick, Frederick, MD 21702-1201, USA. Developmental stages of murine NK cells have been characterized in mice based on the acquisition of receptors for the MHC class I molecules such as CD94 and the members of the Ly49 family. Each NK cell selectively expresses a subset of the complete Ly49 repertoire. We have recently described a novel Ly49 promoter, Pro1, involved in the induction of Ly49 expression in immature NK cells. Biochemical studies demonstrated a drastic decrease of Pro1 transcriptional activity in an immature NK cell line (LNK) after mutation of the NF-B binding sites of Ly49g Pro1. Co-transfection of p65 with Ly49g Pro1 in LNK cells decreases the transcriptional activity of the core promoter. Moreover, incubation of LNK cells with the proteosome inhibitor MG132 leads to increased p65 protein levels and decreased Pro1 transcriptional activity. Therefore, expression of p65 seems to inhibit Pro1 transcription due to the formation of a stable p50/p65 NF-B complex. In addition, analysis of the Ly49 repertoire in NF-B/p50 KO mice reveals a decrease in the proportion of NK cells expressing a given Ly49 molecule, and an increase in the size of the immature CD94+ Ly49- NK cell compartment. This maturational block is observed in the bone marrow, spleen and liver with a similar altered pattern of developmental stages, contrasting with the phenotype found in control mice. Preliminary results also suggest an immaturity of NK cell functions such as a decrease of their killing activity. All together, these results support the hypothesis that NF-B/p65 expression represents a key step in the maturation process of NK cells by playing a major role in the regulation of the Ly49 repertoire on NK cells.





B037Analysis of the receptor-ligand interactions occurring between NK and freshly derived leukemic cells. Daniela Pende 1 , Grazia M. Spaggiari2,3, Stefania Marcenaro2, Stefania Martini1, Maria C. Mingari1,4, Alessandro Moretta3,5 and Lorenzo Moretta2,3,5. 1Istituto Nazionale per la Ricerca sul Cancro, L.go R. Benzi 10, 16132 Genova, Italy; 2Istituto Giannina Gaslini, Genova; 3Centro di Eccellenza per la Ricerca Biomedica, 4DOBIG and 5DIMES, University of Genova, Italy. By exploiting our current knowledge on inhibitory or triggering NK receptors and their ligands on target cells, we investigated the interactions between NK and myeloid (AML) or lymphoblastic (ALL) leukemic cells. The NK-mediated killing of leukemic cells by allogeneic NK cells is predictable either when leukemic cells display a substantial downregulation of HLA-class I molecules or in the presence of a “mismatch” between KIR expressed by donor’s NK cells and HLA-class I antigens expressed by leukemic cells. We observed a sharp downregulation of HLA-class I molecules in various leukemias and it was more frequent in AML than in ALL. We could identify, at the population level, the NK cell subset expressing KIR not engaged by the HLA-class I alleles of the patient (referred as alloreactive NK cells) in hypothetical NK donors, either siblings with a haploidentical HLA haplotype or unrelated donors. We showed that the size of alloreactive cell subset paralleled the degree of NK cytotoxicity against leukemic cells.Thanks to the availability of a complete set of mAb directed to the all known ligands for triggering NK receptors, we could analyze the leukemias for their surface expression. These included PVR (CD155) and Nectin-2 (CD112), which have been recently identified by our group as the ligands of the activating receptor DNAM-1 (CD226). We consistently found expression of PVR and Nectin-2 in AML, while these molecules were less frequent in ALL. Regarding the NKG2D ligands, MICA/B and ULBPs were either absent or weakly expressed. In most leukemias we found that both CD48 (i.e. the 2B4 ligand) and NTBA, which is characterized by homophilic interaction, were downregulated as compared with the normal peripheral blood mononuclear cells. We performed cytolytic assays to assess the involvement of each triggering receptor in the NK-mediated lysis of a given leukemia. We clearly showed that the major role was exerted by NKp46 and NKp30, confirming previous studies. Importantly, we could correlate the surface expression of PVR and/or Nectin-2 on leukemias with a role of DNAM-1 in inducing their NK-mediated lysis. Therefore these molecules represent





important functional markers. Consistent with the scanty detection of NKG2D ligands on leukemias, this receptor was very rarely involved in their lysis. The correlation found between marker expression and susceptibility to lysis may reveal useful for NK-based immunotherapy.





B038Dendritic Cell Recruitment and Activation of NK Cells in the Peritoneal Cavity. Catrine Persson1, James Di Santo2, Hans-Gustaf Ljunggren1 and Benedict Chambers1

1Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. 2 Department of Immunology, Inserm U 668,Unité des Cytokines et Dévelopement Lymphoide, Institut Pasteur, Paris France.

Recent in vivo studies have examined the role of dendritic cells (DC) in the activation of NK cells for tumor rejection and in the recruitment of NK cells to lymph nodes. In the present study, we have examined the recruitment and activation of NK cells following intraperitoneal injection of DC. Injection of untreated DC led to slight increase in the number of NK cells in the peritoneum when compared to mice injected with PBS. However, a two-threefold increase in the number of NK cells was observed in those mice receiving LPS-activated DC. To address whether NK cells were recruited or proliferated in the peritoneum, CFSE labeled-NK cells were adoptively transferred into NK cell deficient common chainxRAG2-/- mice. These experiments demonstrated that i.v. injected NK cells were recruited to the peritoneum while no enhanced proliferation of i.p. injected NK cells was observed. When NK cell cytotoxicity was compared between mice receiving untreated and LPS-treated DC, NK cells purified from mice receiving LPS-treated DC exhibited a two-threefold increase in cytotoxicity against YAC-1 tumor cells. This indicated that LPS-treated DC not only induced migration to the peritoneum but also activated the NK cells to kill. Interestingly, LPS-treated DC from mice lacking both CD80xCD86-/- exhibited reduced recruitment of NK to the peritoneum and cytotoxicity. A role for TNF in the NK cell recruitment was observed as mice lacking the p55 subunit of the TNF receptor also failed to recruit NK cells to the peritoneum. However, the recruitment of NK cells did not appear to be dependent on cytokines such as IL-2, IL-15 or IFN. Resident peritoneal T cells are not required in the recruitment of the NK cells to the peritoneum, as LPS-treated DC injected could also recruit and activate NK cells in RAG1-/- mice. This study then demonstrated that NK cells are recruited and activated in the peritoneum by DC. NK cell recruitment to the peritoneum has been associated with pathological condition such as endometriosis, therefore,





this model can be a useful tool to study the induction of NK cells in such diseases.





B039Flow cytometric detection of IL-2 and IL-12 induced IFN production in NK cells from chronic myeloid leukemia patients. Line Petersen 1 , Jesper Stentoft2, Peter Hokland2 and Marianne E. Hokland1. 1 Dept. of Medical Microbiology and Immunology, Aarhus University, The Bartholin Building, 8000 Aarhus C, Denmark. 2 Dept. of Hematology, Aarhus University Hospital, Tage-Hansens Gade, 8000 Aarhus C, Denmark.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a balanced translocation in a haematopoietic stem cell. This rearrangement forms the fusion gene BCR-ABL, which encodes a constitutively active tyrosine kinase that causes the leukemogenic phenotype. Imatinib (Gleevec), which selectively inhibits this oncogenic BCR-ABL fusion protein, is today the treatment of choice for CML. As part of a general immunosupression in CML patients, NK cell activity has been shown to be reduced. As the NK cell is recognized as an important component in the immune response against malignancies we wished to establish a method being able to evaluate the efficiency of NK cell subsets in CML patients receiving imatinib. Peripheral blood mononuclear cells from patients and normal controls were stimulated by employing IL-2 and IL-12. Using three-color flow cytometry, the production of IFN in the CD56bright NK cell subset was then measured. We demonstrated that this regulatory NK cell subset in the CML patients had a significantly lower IFN production than the normal controls. Interestingly, this result is in contrast to earlier publications, which show a normalization of IFN production in CML patients receiving IFN or hydroxyurea treatment. Current studies are being performed to determine whether or not imatinib treatment influences the NK cells.





B040HLA-G1s downregulates perforin mediated decidual NK cell killing activity via Stat3 suppression. Tobias G. Poehlmann, Andreas Schaumann, P. Le Bouteiller, Udo R. Markert

Objectives: HLA-G1s ist produced by fetal trophoblasts in the placenta. Natural killer (NK) cells express HLA-G receptors, but the ways of function are widely unclear. Aim of this investigation was to analyze the capacities of HLA-G1s to inhibit interleukin-2 (IL-2) induced killing activity in decidual NK cells. Methods: NK cells were isolated from the decidual layer of term placentae, cultivated for 24 h, stimulated or not with IL-2 and supplemented with various concentrations of HLA-G1s. For analysis of NK cell cytotoxicity K562 were used as targets and killing rate was measured by using flow cytometry. The expression of perforin as the major killing effector molecule was analyzed by Western Blots as well as Stat3 (signal transducer and activator of transcription 3), which is involved in the intracellular signalling pathway for perforin transcription. HLA-G1s induced NK cell apoptosis was determined by analysis of PARP cleavage and suppression of NK cell proliferation by analysis of transferrin receptor expression, both by using flow cytometry. RNAinterference was used to knock down Stat3 production in NK cells to confirm the role of Stat3 in perforin synthesis. Results: HLA-G1s reduces the cytotoxicity, perforin and Stat3 expression of stimulated and non-stimulated NK cells dose-dependently (1.6µg/ml – 0.00016µg/ml). At the same time it decreases NK cell proliferation, but only at the highest applied concentration apoptosis is increased. Conclusion: HLA-G1s is involved in protection of fetal trophoblast cells from maternal NK-cells.





B041HLA-G1 protects jeg-3 choriocarcinoma cells against nk cells.Tobias G. Poehlmann, Tobias Wengenmayer, Susann Busch and Udo R. Markert.Placenta-Lab, Department of Obstetrics, Friedrich-Schiller-Universität Jena, Bachstr. 18, 07743 Jena, Germany

Problem: Trophoblast cells and choriocarcinoma cells escape from NK cell killing by interaction of HLA-G with killing inhibitory recepeptors. The role of HLA-G isoforms is not yet exactly revealed.Methods of Study: RNAinterference (RNAi) was applied to Jeg-3 cells to knock down total HLA-G and HLA-G1 and soluble HLA-G1 (HLA-G5). Small interfering RNA (siRNA) and scrambled oligonucleotide controls were self-designed. Due to homologies in the HLA-G gene structure, siRNA to knock down further single isoforms seem to be not realizable. HLA-G knock down was analyzed by Western blot and flow cytometry. NK cells were isolated from peripheral blood of healthy donors. For cytotoxicity assays, Jeg-3 cells were stained with CFSE and coincubated with NK cells. Death rate was measured by flow cytometry.Results: HLA-G1 knock down was successfully performed. Increase of cytotoxicity after HLA-G1 knock down was similar to that after total HLA-G knock down..Conclusion: Among the different HLA-G isoforms, HLA-G1 plays a key role for protection of trophoblastic cells from NK cell cytotoxicity.





B042In celiac disease (auto)antibodies against tissue transglutaminase bind toll Like Receptor 4 (TLR4) and induce activation of monocytes. A. Puccetti*, G. Zanoni^, R. Navone°, R. Beri°, C. Bason°, G. Tridente^, R. Corrocher° and C. Lunardi°* Institute G.Gaslini, Genova and University of Genova, Via Marsano,10. 16132 Genova, Italy; ^ Department of Pathology, University of Verona, Piazzale Scuro, 37134 Verona, Italy; ° Department of Clinical and Experimental Medicine, University of Verona, Piazzale Scuro, 37134 Verona, Italy

Toll-like receptors (TLRs) are a family of pattern recognition receptors that evolved to detect microbial infection. These receptors recognize conserved molecular products derived from different classes of microorganisms, including Gram-positive and -negative bacteria, fungi, protozoa and viruses. Following recognition of ligands TLRs initiate signaling events that result in acute innate responses. Indeed recent evidence has highlighted the role of TLRs as key recognition structures of the innate immune system. The activation of TLRs initiates the production of inflammatory cytokines, chemokines, tissue destructive enzymes, and type I interferons. In addition, TLR signalling plays an important role in the activation and direction of the adaptive immune system by the upregulation of costimulatory molecules on antigen presenting cells. Considering the important role of TLR signalling as a critical link between innate and adaptive immunity it has been proposed that a dysregulation in TLR signalling might be associated with autoimmunity. TLR4 is an essential receptor for LPS recognition. Stimulation of macrophages with LPS results in the production of various cytokines such as TNF-a, IL-1, IL-6, IL-12, MIP-1a/b, and inflammatory effector substances.It has been shown that activation of APCs via innate immune receptors such as TLR4, can break self tolerance and trigger the development of autoimmunity even in genetically resistant strains of mice, suggesting that the development of autoimmune diseases is determined at least in part by the endogenous activation state of APCs. Celiac disease (CD) is a permanent gluten intolerance characterized by a vast array of clinical symptoms. Here we show that in CD autoantibodies directed against the prototypic autoantigen target of the disease, tissue transglutaminase, crossreact with TLR4 and induce monocyte activation upon engagement of this surface receptor. We have identified a subset of anti-tranglutaminase antibodies directed against a 12 aa epitope of the enzyme which are able to bind TLR4 expressed on the cell surface. Upon binding this receptor the antibodies induce activation of monocytes as confirmed by upregulation of CD83 and CD40 and of increased





secretion of IL6 anf IL12 in the cell supernatants. To our knowledge this is the first report of cell activation induced by an antibody via TLR.Our results indicate a connection between innate and acquired immunity in the pathogenesis of CD.





B043Natural Killer Cells Ameliorate Liver Fibrosis through Killing Activated Stellate Cells in RAE1/NKG2D- and TRAIL-dependent Manners. Svetlana Radaeva, Rui Sun, Khang-Van Nguyen, Zhigang Tian, and Bin GaoSection on Liver Biology, Laboratory of Physiologic Studies, National Institute onAlcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, R-25-12, Bethesda, MD 20892 USA

Natural killer (NK) cells kill foreign cells, transformed cells, and microbially infected autologous cells. In this investigation, we provide new evidence that NK cells are also able to kill autologous activated hepatic stellate cells (HSCs), which are central mediators in the development of liver fibrosis, via RAE1/NKG2D- and TRAIL-dependent manners. In a mouse model of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver fibrosis, NK cell activation by poly I:C, a TLR3 ligand, induced cell death to activated HSCs and attenuated the severity of liver fibrosis. The observed protective effect of poly I:C on liver fibrosis was diminished through either depletion of NK cells or by disruption of the IFN-g gene. Expression of RAE1, the NKG2D ligand, was undetectable on quiescent HSCs, whereas high levels were found on activated HSCs, which correlated with the resistance and susceptibility of quiescent HSCs and activated HSCs to NK cell lysis, respectively. Moreover, treatment with poly I:C enhanced the expression levels of NKG2D, TRAIL, and perforin on liver NK cells. Blocking NKG2D or TRAIL with neutralizing antibodies diminished markedly the cytotoxicity of poly I:C-activated NK cells against activated HSCs, while disruption of the perforin gene only weakly attenuated such cytotoxicity. These findings suggest that NK cells kill activated HSCs via RAE1/NKG2D- and TRAIL-dependent mechanisms, thereby ameliorating liver fibrosis.





B044Activation of NK cells by an endocytosed receptor for soluble HLA-G. Sumati Rajagopalan 1 , Yenan T. Bryceson1, S. Priya Kuppusamy1, Daniel E. Geraghty2, Arnold van der Meer3, Irma Joosten3, Eric O. Long1

1 Laboratory of Immunogenetics, National Institues of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 207, Rockville, Maryland, 20852 USA, 2 Fred Hutchinson Cancer Research Center, Seattle, Washington, USA, 3 Department of Blood Transfusion and Transplantation Immunology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.

Uterine tissue in early human pregnancy is characterized by extensive vascular remodeling, invasion of fetal trophoblast cells, and by an abundance of maternal natural killer (NK) cells. Trophoblast cells express membrane-bound and soluble isoforms of the non-classical major histocompatibility class I molecule HLA-G. How NK-trophoblast cell interactions influence vascular remodeling is unknown. Here we show that interferon-secretion by resting NK cells is induced by soluble, but not solid-phase antibodies to the killer cell immunoglobulin-like receptor (KIR) 2DL4, a receptor for HLA-G. KIR2DL4 is constitutively internalized into Rab5-positive compartments by a dynamin-dependent process. Soluble HLA-G was endocytosed into KIR2DL4-containing compartments in NK cells and in 293T cells transfected with KIR2DL4, as shown by confocal microscopy. The profile of genes upregulated by KIR2DL4 engagement on resting NK cells suggests a role for vascularization. IL-8 secretion was induced by transfection of KIR2DL4 into 293T cells, and occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. We propose that soluble HLA-G produced by fetal trophoblast cells activates maternal NK cells through endocytosis of KIR2DL4, thereby promoting vascularization during implantation.





B045Genetic mapping and analysis of a Cmv2.X quantitative trait locus. Marisela, Rodriguez, Pearl Sabastian and Michael G. Brown. Division of Rheumatology and Immunology, Departments of Internal Medicine and Microbiology, University of Virginia, Charlottesville, VA 22908

Innate MCMV immunity in NZW requires Ly49H-independent NK cell control. Classical genetics studies using NZW x NZB hybrid offspring demonstrated multiples genes contribute NK cell-mediated MCMV control. To identify principal New Zealand genes associated with host resistance or susceptibility, MCMV control traits determined for individual intercross offspring using quantitative real-time PCR to measure spleen MCMV levels were compared with their genome-wide genotypes using marker regression analysis. A significant (LOD = 3.3) quantitative trait locus (QTL) is linked with DXMit216 on chromosome X that controls ~10% of the genetic variance associated with MCMV control in this genetic system. Because much of chromosome X is highly conserved among humans and mice, we have sought to identify Cmv2.X through use of additional quantitative genetics strategies. Novel simple sequence length polymorphisms (SSLP) markers were designed that distinguish NZW and NZB chromosome X alleles. Of these, DXUva01 is tightly linked with DXMit216 and has been useful in establishing a Cmv2.X minimal genetic interval. Interestingly, DXMit216nzw F2 females displayed MCMV resistance more than 3 times more frequently than DXMit216nzw/nzb females. Curiously however, DXMit216nzb was associated with innate MCMV resistance in males more than 3 times more frequently than in DXMit216nzw males. Thus, Cmv2.X may exert its effect in a gender specific manner. Additionally, it is possible that more than one chromosome X gene contributes quantitative effects in MCMV control. Thus, it will be important to determine the role of Cmv2.X in NK cells during acute MCMV infection.





B046A role for epigenetics in mouse NKG2A expressionSally Rogers 1 , Arefeh Rouhi1,2, Fumio Takei1,3 and Dixie L. Mager1,2

1The Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, and the Departments of 2Medical Genetics and 3Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.

NKG2A is a C-type lectin-like inhibitory receptor detected predominantly on the surface of Natural Killer (NK) cells. It is expressed as a heterodimer with CD94 in both humans and mice, and binds non-classical MHC molecules to broadly monitor MHC class I expression. NKG2A is the major inhibitory receptor expressed by neonatal NK cells in mice, with acquisition occurring after CD94 but prior to NKG2E during fetal development. In contrast, very few neonatal NK cells express Ly49 receptors. The percentage of NK cells expressing NKG2A drops to around half in adult mice, concomitant with increasing expression of Ly49 receptors. A role for GATA-3 has been shown in regulating expression of human NKG2A, but the mechanisms determining expression of mouse NKG2A remain unknown, in part due to the fact that the transcriptional start sites appear to be different in the two species. However, there is an emerging role for epigenetics in regulating expression of NK receptors. In particular, it has been shown that DNA methylation status maintains the allele-specific expression of human Killer Immunoglobulin Receptors (KIR), and recent work in our laboratory implicates DNA methylation and histone modifications in regulation of Ly49 transcription.

Here we report the identification of a cluster of CpG motifs around the transcriptional start site of mouse NKG2A, and show that lack of methylation of these sites correlates with gene expression in ex vivo NK cells, cell lines and embryonic stem cell-derived NK cells. Furthermore, we show that acetylated histones H3 and H4 are associated with the CpG-rich region in NKG2A+ cell lines, but not in NKG2A- cell lines, providing evidence that histone modifications correlate with unmethylated DNA to produce an open chromatin structure. Treatment of an NKG2A negative cell line with the DNA methylase inhibitor 5-aza-2'-deoxycytidine in combination with the histone deacetylase inhibitor trichostatin A induced NKG2A transcription. Finally, we show that demethylation of the CpG rich region correlates with NKG2A expression during development of fetal NK cells from embryonic stem cells. This region is methylated in the undifferentiated stage (NKG2A-), CD34+ hematopoietic progenitor stage





(NKG2A-), and NKG2A- ES-derived NK cells, but is unmethylated in NKG2A+ ES derived NK cells.

This is the first report implicating epigenetics in regulating transcription of NKG2A, a receptor expressed by both human and murine NK cells. Moreover, our results suggest that epigenetics has a role in determining the NK receptor repertoire during development.





B047CD56brightCD16-KIR- NK cells of peripheral blood and lymph nodes display longer telomeres than circulating CD56dimCD16+KIR+/- cytotoxic NK cells and acquire cytolytic functions and KIRs upon activation. Chiara Romagnani, Antonella D’Agostino, Roberta Costa, , Lorenzo Moretta, Guido Ferlazzo.Clinical Immunology, German Rheumatism Research Centre, DRFZ, Schumannstrasse 21/22, 10117 Berlin, GermanyIstituto Giannina Gaslini, Largo Gaslini, 5, 16100, Genoa, Italy University of Genoa, Vial Benedetto XV, 16132, Genoa, Italy University of Messina, Via Consolare Valeria, 98100, Messina, Italy.

Human NK cells may be divided into a CD56dimCD16+KIR+/- subset and a CD56brightCD16-KIR- subset. In peripheral blood, CD56dim NK cells dominate, whereas CD56bright NK cells are greatly enriched in secondary lymphoid organs and, in a steady state, do not express KIRs, CD16 and perforin.The developmental relationship between these two human NK cells subsets

remains controversial. We have analyzed telomere length of NK cells isolated from peripheral blood and autologous lymph nodes and found that circulating NK cells have shorter telomeres than CD56brightCD16-KIR- counterpart of both blood and lymph nodes. Also, as already shown for lymph node NK cells, CD56brightCD16-KIR- can express KIRs and CD16 upon activation by a variety of stimuli, including IL-2, IL-15, IL7, IL12, IP10 and MIP1a. Under the same culture conditions they acquire cytotoxic properties comparable to peripheral blood CD56dimCD16+KIR+/- NK cells. Finally, we isolated NK cells from both human bone marrow and thoracic duct lymph and found that while the former were homogeneously CD56brightCD16-KIR-, the latter express CD16, KIRs and cytoplasmic cytotoxic granules similarly to the autologous peripheral blood NK cells. The same molecules were not expressed in NK cells harbored in autologous lymph nodes collected at the same time.Altogether, our results indicate that CD56brightCD16-KIR- and CD56dimCD16+KIR+/- NK cells may represent sequential steps of natural killer cell differentiation. These data also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.





B048DNA methylation pattern of Ly49a correlates with mono-allelic gene expression. Arefeh Rouhi*,†, Liane Gagnier*, Fumio Takei*,‡ and Dixie L. Mager*,†

*The Terry Fox laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada and the †Department of Medical Genetics and ‡Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.

Although structurally unrelated, the human killer cell immunoglobulin-like (KIR) genes and the rodent lectin-like Ly49 genes serve similar functional roles in natural killer (NK) cells. Moreover, both gene families display variegated, mono-allelic expression patterns established at the transcriptional level. DNA methylation has been shown to play an important role in maintenance of expression patterns of KIR genes, which have CpG island promoters. The potential role of DNA methylation in expression of Ly49 genes, which have CpG-poor promoters, is unknown. Here we show that hypomethylation of the region encompassing the Pro-2 promoter of Ly49a and Ly49c in primary C57BL/6 NK cells correlates with expression of the gene. Using C57BL/6 x BALB/c F1 hybrid mice, we demonstrate that the expressed allele of Ly49a is hypomethylated while the non-expressed allele is heavily methylated, indicating a role for epigenetics in maintaining mono-allelic Ly49 gene expression. Furthermore, the Ly49a Pro-2 region is heavily methylated in fetal NK cells but variably methylated in non-lymphoid tissues. Finally, in apparent contrast to the KIR genes, we show that DNA methylation and histone acetylation state of the Pro-2 region strictly correlate with Ly49a expression status. Our results support a role for epigenetic mechanisms in the maintenance of mono-allelic Ly49 expression. Moreover, these epigenetic mechanisms appear to differ from those governing KIR gene expression.





B049Modeling natural killer cell development and repertoire formation. Mali Salmon-Divon1, Sofia Johansson2, Maria Johansson2, Marjet Elemans1,2, Klas Kärre2, Petter Hoglund2, Ramit Mehr1,2. 1Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. 2Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm, Sweden.

NK cells are able to recognize and reject cells lacking expression of self MHC class I molecules. Inhibition of lysis is mediated by inhibitory receptors expressed by NK cells, such as the murine Ly49 receptors, which bind to MHC class I molecules. Most NK cells adapt to the self-MHC environment in a process ensuring that each NK cell will express at least one self-specific inhibitory receptor but not too many. Two models have been proposed to account for the development of a useful Ly49 repertoire. The two-step selection model proposes a random initial receptor expression combined with selection of cells expressing appropriate receptor compositions. The sequential model proposes that each NK cell sequentially expresses individual Ly49 receptors and continues to do so until an interaction of sufficient magnitude between a Ly49 receptor and self-class I MHC occurs. These two models predict different repertoire compositions under various conditions. To dissect the complexity of NK cell repertoire development, we have conducted mathematical modeling and computer simulation studies of each NK cell education model, fitting them to published (Salmon-Divon et al, Bull. Math. Biol. 65:199-218, 2003; Clin. Dev. Immunol. 10:183-192, 2003; Mol. Immunol. 42:397-403, 2004) and newly generated experimental data from mice expressing single MHC class I molecules. Our results favor the two-step selection model over the sequential model. Additionally, we have observed that murine NK cells downregulate the expression of the inhibitory Ly49 receptors when developing in the presence of MHC class I ligands. Using mathematical models, we have found that Ly49 downregulation in mice expressing several MHC class I proteins is dominated by the one binding most strongly to the MHC class I. This suggests that MHC class I saturates the binding ability of Ly49 receptors. A prediction from such a model would be that NK cells from a strong selecting background will be less capable of sensing the downregulation of more weakly binding MHC class I proteins. In addition, a model of saturating MHC class I also suggests a distinct threshold below which self MHC class must be downregulated before the cell is sensed as “missing self”. We are currently testing the validity of these and other predictions.









B050Regulation of 2B4 (CD244)-mediated NK cell activation by ligand-induced receptor modulationMina Sandusky, Birgitta Messmer and Carsten WatzlInstitute for Immunology, University Heidelberg, INF305, 69120 Heidelberg, Germany

Natural Killer (NK) cell activity can be stimulated by a variety of different surface receptors. 2B4 (CD244) is a member of the SLAM-related receptor (SRR) family and is important for stimulating human NK cell cytotoxicity and cytokine production. Here we show that stimulation of human NK cells by antibody-mediated 2B4 cross-linking results in a strong down-modulation of 2B4 surface expression. Incubating NK cells with target cells expressing the 2B4 ligand CD48 showed a similar effect. This down-modulation of 2B4 was CD48 dependent, as no modulation of 2B4 was observed when we used sensitive target cells that lacked CD48 expression. The down-modulation of 2B4 upon contact with CD48-positive target cells was observed in NK cell lines, purified human NK cells and NK cell clones and is accompanied by an internalization of 2B4. The modulation of 2B4 is dependent on activity of Src-family kinases, but cannot be blocked by inhibitors of PI3K or actin polymerization. This suggests that specific signals are necessary for the ligand-induced modulation of 2B4.

Inhibitory receptors can interfere with 2B4-mediated signals and NK cell activation. We therefore tested if inhibitory receptors can block the ligand-induced down-modulation of 2B4. Interestingly, co-engagement of KIR2DL1 by antibodies or HLA-Cw4-expressing target cells had no influence on the down-modulation of 2B4. This suggests that the signals necessary for the modulation of 2B4 expression are independent from inhibitory receptors. The lower surface expression of 2B4 after ligand-induced down-modulation resulted in reduced 2B4-mediated NK cell activation and cytotoxicity. The modulation of activating surface receptors may therefore be another mechanism for the fine-tuning of NK cell activity and may lead to the adaptation of NK cell cytotoxicity in tissues with high ligand expression.





B051Differentiation of hematopoietic stem cells to NK cells is accompanied by general chromatin decondensation of the KIR locus and sequential acquisition of KIR receptors Simeon Santourlidis, Julia Christ, Martina Sribar, Michael Punzel, Peter Wernet, and Markus Uhrberg. Institute for Transplantation Diagnostics and Cell Therapeutics, University Clinic of Düsseldorf, Moorenstr. 5, Düsseldorf, Germany.

Most members of the human KIR gene family are expressed in a clonally-distributed fashion, leading to repertoires of structurally and functionally diverse NK cells. In mature NK cells, KIR expression is epigenetically controlled through DNA methylation of CpG islands, which are hypomethylated in expressed and hypermethylated in silenced KIR genes. This study was set up to show, how KIR patterns are initially established in the course of NK cell differentiation. KIR promoters were found to be densely methylated in highly enriched hematopoietic stem cells (HSC) from cord blood, suggesting that those KIR genes that are induced in the course of NK cell differentiation are subject to demethylation. Consistent with the hypermethylated and thus inactive state of KIR genes, chromatin immunoprecipitation (ChIP) analyses in HSC revealed a histone signature, which is indicative of a repressive chromatin structure throughout the KIR locus. Moreover, MeCP2, a major methyl-CpG binding protein that is known to recruit histone deacetylases, was found by ChIP analysis to bind to KIR promoters in HSC, which might provide a mechanistic link between DNA methylation and chromatin condensation in HSC. Unexpectedly, in NK cells all KIR genes consistently exhibited active histone signatures irrespective of their expression status. Thus, DNA methylation appears to be the only detectable structural difference between expressed and silent KIR genes in NK cells. To follow the induction of KIR expression during NK cell development more closely, single HSC were differentiated into NK cells in vitro, employing a stroma cell-based culture system. RT-PCR analysis of NK cell progenitor clones, taken at different time points following induction of differentiation, revealed a sequential acquisition of receptors, consistently starting with KIR2DL4, followed by non-random expression of specific other KIR genes. The current results are supportive of an epigenetically driven process of sequential KIR expression, which has its starting point at the KIR2DL4 gene.





B052Killer Immunoglobulin like Receptor (KIR) genotype as an additional selection criteria in Matched Unrelated Donor selection for Haematopoietic Stem Cell Transplantation (HSCT) patients. Jennifer Schellekens*, Leo Verdonck‡ , Rowena C.A. Melchers*, Petra van der Weide*, Eefke Petersen‡, Erik H. Rozemuller*, Marcel G.J. Tilanus*.*Department of Pathology, ‡Department of Haematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands

The Killer Immunoglobulin like Receptors (KIR) are membrane bound receptors expressed on Natural Killer (NK) cells. The fourteen different KIRs identified thus far are variably present in the genotype of different individuals. The ligand for most of these receptors is HLA class I. Binding of the receptor to its ligand can, based on the receptor, yield an inhibitory or activating signal to target cell lysis. To evoke a cytolytic response the NK cells should express the right combination of KIRs on their membrane. Moreover, the presence or absence of certain ligands for these KIRs is crucial for this alloreactive response. The NK cells are ideal immune cells in the transplantation setting since they only induce a Graft versus Leukemia (GVL) effect without inducing a Graft versus Host Disease (GVHD). However, the current criteria for selecting an unrelated donor do not account for this potential NK alloresponse. In this study 23 HSCT patients and all their potential donors (N=70) are typed for the complete set of KIR genes by the Sequence Specific Typing (SSP) procedure. KIR genotypes are correlated with the HLA data and clinical data available for all these patients and donors. In this retrospective study the combined data indicate that in those five transplanted patients who died from a relapse, the selected donor contained less activating KIRs in its genotype than in those donors of patients surviving after transplantation. Since other donors with more activating KIRs were available for these deceased patients, an other donor would have been selected if KIR repertoire was included in the selection procedure. Also when the patients KIR repertoire was included in the donor KIR repertoire the chance of developing a Graft versus Host Disease (GVHD) was larger. This also indicates the importance of accounting for all the relevant data including the KIR repertoire in the donor selection.





B053KIR2DL4 gene polymorphism identified by sequencing based typing (KIR-SBT) and the construction of its allele database for direct sequence analysis. Jennifer Schellekens, Jessica Wijbenga, Marvin M. van Luijn, Erik H. Rozemuller, Marcel G.J. Tilanus. Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands

The killer cell immunoglobulin like receptors (KIR) on NK cells recognize defined groups of HLA class I alleles. By this mechanism the NK cells fulfil a significant role in the first line of defence against for example infectious agents and cancer. This allorecognition is important in the treatment of leukemia. For an appropriate effect against the leukemic cells, the expression level of both the KIR as the HLA molecule is of enormous impact. It is known that polymorphisms in the KIR gene may cause an altered expression of this KIR on the membrane. We studied the KIR2DL4 gene polymorphism by a newly developed direct sequencing (SBT) protocol. The absence of a descent database for KIR sequence analysis is a distinct problem. To facilitate routine KIR high resolution typing, and to gain insight in all polymorphic positions in the KIR2DL4 gene, we created a database that combined the exon ánd intron information with the current knowledge of the polymorphic positions. New polymorphisms, insertion and deletions have been characterized in 47 individuals. The routine SBT protocol is currently extended to other KIR genes and generation of the appropriate databases allows routine KIR SBT typing.





B054TRAIL mediates cytolysis of cultured dendritic cells in IL-2 propagated NK-/T-cell lines from NK deficient individuals suffering from hemophagocytic lymphohistiocytosis (HLH). E. Marion Schneider and Ying WangSektion Experimentelle Anaesthesiologie, University Clinic, Ulm; Germany; Department of Immunology, Fudan University, Shanghai, China

Introduction: NK deficiency is a hallmark in threatening HLH. In this disease presenting early in childhood, cells of the monocytic (Mo) and dendritic cell (DC) lineage massively phagocytose hematopoetic cells but spare lymphocytes. Mutations in granule exocytosis molecules such as Perforin, Munc13-4, Rab27a and Syntaxin 11 have been demonstrated to lead to familial HLH. In addition to fulminant generalized inflammation, plasma levels of the soluble alpha chain of the high affinity IL-2 receptor, sCD25 constitute the best disease marker for HLH. We propose, that high sCD25 interferes with the IL-2 dependent activation of cytotoxic effectors which could substitute for the mutated cytotoxic effector pathway. Specifically, we addressed the question whether IL-2 signaling may reconstitute CD95-Ligand and TRAIL mediated killing. Methods: Cellular cytotoxicity was tested in 8% endotoxin-free FCS against 51chromium labeled K562 targets, cultured hemophagocytic DC and Mo cells, and EBV-positive allogeneic and autologous B cells. Quantification of sCD25 in patients’ plasma was performed by a chemiluminescence assay (DBC Biermann, Bad Nauheim, Germany). Surface expression of CD25 and TRAIL signaling receptors DR4 and DR5 was measured by flow cytometry (FACScalibur, BD). To activate cytotoxic effectors by IL-2, high dose IL-2 culture (103 IE/ml rh-IL-2) was performed for 10 to 28 days. Results: Following a lag phase of 4-6 days, non-adherent lymphocytes of patients with HLH started to proliferate in IL-2 containing medium. Control lymphocytes proliferated without delay. IL-2 driven cell lines were generated from heterozygous and homozygous nonsense mutations in the perforin gene, the Syntaxin 11 and Rab27a gene. Eight out of ten IL-2 driven cell lines mediated calcium dependent cytolysis of allogeneic cultured hemophagocytic cells in 4 hours. In addition, four IL-2 cell lines recognized spontaneously transformed B cell lines, established from EBV-positive patients with HLH. The cytolysis was perforin independent but could be blocked by TRAIL-antibody (RIK-2). Thus IL-2 activation could indeed induce TRAIL specific cytolysis of hemophagocytic cells and EBV-virus infected B cells. Target hemophagocytic Mo and DC expressed the TRAIL receptor DR4, and EBV+ B cell targets were DR5 positive in addition. Conclusion: Tumor necrosis factor related apoptosis inducing ligand is a key effector molecule expressed by natural killer (NK) cells and has been





shown to control tumor immunity and leukocyte subpopulations. Here we demonstrate that TRAIL is a dominant cytotoxic effector molecule expressed by IL-2 driven lymphocyte cell lines generated from patients with HLH. Reconstitution of IL-2 responsiveness and TRAIL effector pathways may be a novel aspect for treating HLH.





B055A subset of immature NK cells in the spleen and bone marrow expresses surface Lymphotoxin- Kirsten Schneider, Chris A. Benedict and Carl F. Ware. Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA USA.

We are interested in the role that Lymphotoxin (LT) and LIGHT, members of the TNF superfamily of cytokines, play in the innate and adaptive immune response to Cytomegalovirus (CMV) infection. LT is crucial for the development and homeostasis of secondary lymphoid tissues but also contributes to the differentiation of NK, NK-T and dendritic cell populations. LT also takes part in the initiation of the innate immune response contributing to the early type I Interferon response to mouse CMV (MCMV). In the C57BL/6 strain of mice NK cells mediate an important early host defense against MCMV.Due to the fact that NK cells and LT both have crucial roles in the immune response to MCMV we were interested in defining the role that LT plays in the differentiation and effector function of NK cells during MCMV infection. The percentage of DX5+CD3- NK cells were reduced by 2 fold in the spleen of LT, LT and LTR deficient mice, but not in LIGHT or HVEM deficient mice. However, the percentage of DX5+ NK cells in the bone marrow of the LT-deficient mice were normal suggesting LT may be involved in NK cell differentiation. Previous studies have shown that LTR expressed on bone marrow stromal cells is required for NK cell development, but it remained unclear weather the NK cells themselves or another cell type expressed surface LT required to activate the LTR signal transduction pathway needed for NK cell differentiation. Flow cytometric analysis revealed that LT was expressed on the surface of a small percentage (3-5%) of splenic NK cells and NK cells of the BM. The LT expressing NK cells were DX5 positive, but did not express members of the Ly49 family of NK cell receptors, and also lacked Mac-1, which is up-regulated in mature NK cells. These results suggest that NK cells with an immature phenotype express LT on their surface and may be the cells responsible for initiating the signaling of the LTR in stromal cells required for NK cell differentiation.





B056Memory cytotoxic T cell induction by the poliovirus receptor family ligands of the DNAM-1 (CD226) receptorShort Title: Memory T cell induction by DNAM-1 ligandsAkira Shibuya, Satoko Tahara-Hanaoka, Kazuko Shibuya

Department of Immunology, Institute of Basic Medical Sciences and Center for TARA, Graduate School of Comprehensive Human Sciences, and Center for TARA, University of Tsukuba, Ibaraki 305-8575, Japan. (e-mail; ashibuya @md.tsukuba.ac.jp)

The poliovirus receptor CD155 and its family member CD112 (nectin-2) are the ligands for the activating cell surface receptor DNAM-1 on CD8+ T cells and NK cells. Here, we demonstrated that, while the RMA tumor grew in syngeneic mice, DNAM-1 ligands-transduced RMA was rejected, in which CD8+ T cells and NK cells played an essential role. Importantly, CD8+ memory cytotoxic T cells to parental RMA were generated in these mice. We found that DNAM-1 was also expressed on CD8+, rather than CD8-, dendritic cells. Cross-linking DNAM-1 induced maturation of CD8+ dendritic cells. Antigen presentation by these stimulated dendritic cells drived Th1 cells. Moreover, the rejection of DNAM-1 ligands-transduced RMA was canceled in CD4+ T cell-depleted and MHC class II deficient mice. Taken together, these results suggest that DNAM-1 ligands stimulate innate immunity by CD8+ dendritic cells as well as NK cells, which efficiently prime cell-mediated tumor specific immunity.





B057Crystal structure and efficient Leukocyte Ig-like receptor signaling of a disulfide-linked dimer of non-classical MHC molecule, HLA-GMitsunori Shiroishia, Kimiko Kurokia, Toyoyuki Osea, Linda Rasubalaa, Ikuo Shiratorib, Hisashi Araseb, Daisuke Kohdaa, Katsumi Maenaka a

aMedical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. bResearch Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871 Japan.

HLA-G is a non-classical MHC class I molecule (MHCI) and confers immunological tolerance in the maternal-fetal interface by binding to Leukocyte Ig-like receptors (LILRs, also called as LIRs, ILTs and CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Dimer formations have also been observed for MHCIs expressed on the activated T cells and HLA-B27, associated with spondyloarthropathies. However, no three-dimensional structure of a disulfide-linked MHCI dimer has been elucidated, and the LILR and CD8 binding characteristics of the HLA-G dimer remain unknown. Here we determined the crystal structure of the disulfide-linked wild-type dimer of HLA-G with the intermolecular Cys42-Cys42 disulfide bond. The dimer formation rendered LILR/CD8 binding sites exposed to solvents, providing the plausible 1:2 (dimer : receptors) complex model. Furthermore, the LILRB1/2 and CD8 binding studies of the HLA-G dimer showed that the dimer exhibited higher overall affinity to LILRB1/2 and CD8 than the monomer by significant avidity effect, consistent with the dimer structure. These results give new insight into the dominant role of HLA-G dimers on LILR- and CD8-mediated signaling and furthermore provide the structural and functional implications of other MHCI dimers.





B058

Role of KARAP/DAP12 signaling in Ly49D receptor modulation and specific clustering in the NK immune synapse upon ligand interaction Hanna Sjölin 1 , Esther Nolte-'t Hoen2, Sungjin Kim3, Katja Andersson1, Maria Johansson1, Wayne Yokoyama3 , Petter Höglund1, Daniel Davis2 and Klas Kärre1.1 Microbiology and Tumor Biology Center, Karolinska Institute, S 171 77 Stockholm, Sweden; 2 Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College, London SW7 2AZ, United Kingdom; 3Howard Hughes Medical Institute, Rheumatology Division, Washington University School of Medicine, St Louis, MO 63110, USA;

Recent studies have addressed the redistribution of inhibiting and activating receptors and their ligands in the NK cell immune synapse. The role of receptor signaling in these processes is however less clear. Ly49D is an activating receptor on mouse natural killer (NK) cells, and its surface expression as well as signaling is dependent on association with the transmembrane region of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing adaptor protein DAP12. Crosslinking of the Ly49D/DAP12 receptor complex with a hamster MHC class I like molecule Hm1-C4, expressed on Chinese hamster ovary (CHO) cells triggers NK cell mediated cytotoxicity. In order to study Ly49D receptor modulation and synapse formation, we have established a system based on in vitro co-incubation with resting or IL-2 activated mouse NK cells and ligand expressing target cells. To determine the role of DAP12 signaling we are using mutant mice bearing DAP12 molecules with non-functional ITAMs. While Ly49D staining intensity on C57BL/6 NK cells was largely decreased upon co-culture with CHO cells, there were only marginal effects on the NK cells from DAP12 mutant mice. These data suggest that DAP12 signaling is crucial for ligand induced down modulation of at least one of its associated receptors. In addition, we have preliminary results indicating that Ly49D specifically clusters in the NK cell immune synapse upon ligand interaction, and we are currently investigating whether receptor clustering and conjugate formation requires DAP12 signaling.





B059Tumour immune surveillance by NK cells – the role of NKG2D.Mark J. Smyth, Nadeen Zerafa, Erika Cretney, Jeremy Swann, and Yoshihiro HayakawaCancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, 3002, Victoria, Australia

There is emerging evidence that the innate arm of the immune system mediates and regulates tumour immunity. Importantly, coordination of innate and adaptive responses is very much controlled by interactions between NK cells and DC and the regulatory role of specialized T cell populations such as NKT cells and CD4+CD25+ regulatory T cells. NK cells are regulated by both cells and cytokines in their immediate environment and when interacting with tumour directly, a delicate balance between inhibitory signals mediated by MHC class I molecules and activating signals triggered by specific ligands. The activation NKG2D receptor has been shown to play an important role in the control of experimental tumour growth and metastases expressing ligands for NKG2D, however a function for this recognition pathway in host protection from de novo tumourigenesis has never been demonstrated. We have now shown that neutralization of NKG2D enhances the sensitivity of wild-type C57BL/6 and BALB/c mice to methylcholanthrene (MCA)-induced fibrosarcoma. The importance of the NKG2D pathway was additionally illustrated in mice deficient for either IFN- or TRAIL, while mice depleted of NK cells, T cells, or deficient for perforin did not display any detectable NKG2D phenotype. Furthermore, IL-12 therapy preventing MCA-induced sarcoma formation was also largely dependent upon the NKG2D pathway. This was consistent with our observation that some cytokines mediate much of their anti-tumour activity via NKG2D activation when the tumour expresses the appropriate ligands. While NKG2D ligand expression was variable or absent on sarcomas emerging in wild-type mice, sarcomas derived from perforin-deficient mice or those mice neutralized for NKG2D were universally Rae-1+ and immunogenic when transferred into wild-type syngeneic mice. These findings suggest an important early role for the NKG2D in controlling and shaping tumour formation. Furthermore, we have recently illustrated an important role for CD4+CD25+ regulatory T cells in controlling NKG2D-mediated suppression of tumour growth and metastases by NK cells. Using the knowledge we have gained from using cytokines, activating NKT cells or depleting regulatory





T cells, we have begun to rationally design combined therapies that optimize NK cell-mediated tumour destruction.





B060Multiple sclerosis, the innate immunity and the “hygiene hypothesis”.Sotgiu S#, Angius A*, Fois ML#, Arru G#, Pugliatti M#, Rosati G# and Musumeci S*°.# Institute of Clinical Neurology, University of Sassari, *Institute of Population Genetics, CNR, Alghero and °Department of Pharmacology, Gynecology-Obstetrics, Pediatrics, University of Sassari, Italy

BACKGROUND. MS geographical distribution describes areas at high (northern Europe), medium (Mediterranean basin) and low prevalence rate (Africa). MS frequency in Sardinia has dramatically increased in the last 40 years resulting among the highest MS rates in the world despite its Mediterranean location. Epidemiological and genetic evidences link past malaria endemy with MS in Sardinia. Malaria is associated with an innate immune response including a high activity of chitotriosidase (Chit), a macrophage enzyme. A Chit gene mutation (CHIT1) causing Chit deficiency is found at variable frequency in areas of past malaria and absent in African areas presently endemic for malaria. Since macrophages represent the pathological basis of MS lesions, we tested whether CHIT1 frequency relates to Chit production in individuals from Africa, Sicily and Sardinia and to past malaria endemism in Sicily, Sardinia and Taiwan, which allow to propose a “malaria hypothesis” in connection with the MS frequency of the above regions.METHODS. Plasma from 46 healthy Sardinians, 149 healthy Africans from Burkina Faso and 331 healthy Sicilians were studied. Determination of Chit plasma activity and CHIT1 mutation have been already described.RESULTS. The different MS frequencies in Sardinia (high), Sicily (moderate) and Taiwan (low) inversely correlates with the CHIT1 mutation frequency, whilst both CHIT1 polymorphism and MS frequency were very low amongst Africans. The average Chit level in plasma of African donors was 15.6 nmol/ml/h, whereas that of Sardinian and Sicilian donors was 9.3 and 4.6 nmol/ml/h, respectively (T test p <0.001)CONCLUSION. The finding fits very well with the working hygiene hypothesis that MS incidence is increasing amongst Sardinians because, having not completely “dismissed” malaria-related genes, their infiltrating macrophages continue to secrete macrophage-derived products, including chitotriosidase, in response to no existing parasites, contributing to autoimmunity. On the





contrary, the presence of malaria amongst Africans prevent their immune system to attack other innocuous or “self” antigens.





B061Expression and role of phosphatidylcholine-specific phospholipase C (PC-PLC) in human NK cells and its relationship with CD16 receptor. Francesca Spadaro & Serena Cecchetti, Franca Podo and Carlo Ramoni. Istituto Superiore di Sanità, viale Regina Elena 299, 00161, Rome Italy.

Our previous studies showed that PC-PLC is involved in the functional role of NK-mediated cell killing and in the lytic granules exocytosis process. We identified and characterized different subpopulations of human peripheral blood lymphocytes (PBL) in relation to PC-PLC enzyme surface expression. All lymphoid subsets exibited a large intracellular amount of PC-PLC. According to our previous observations on NK cells, in CD8+ T lymphocytes this enzyme translocated to the synapsis region with PFN molecules, suggesting a common pathway for the PFN-dependent cell-mediated lytic process. On the other hand, PC-PLC was detected at high intensity only on NK membrane surface, was present at low level on B lymphocytes, on some T /+ and on rare CD8+ cells, and was totally absent on CD4+ T lymphocytes. Moreover, in NK cells we could distinguish different subpopulations: CD56dim PC-PLCbright and CD56bright PC-PLClow/- cells, corresponding to two distinct NK subsets with cytolytic and immunoregolatory functions respectively. Interestingly, PC-PLC expression on NK membrane surface was directly proportional to that of CD16, suggesting a possible correlation between the enzyme expression and NK cell maturation. Pre-incubation of NK cells with the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) determined a dramatic decrease of PC-PLC fraction expressed on NK plasma membrane. Surprisingly, D609 also down-modulated the membrane expression of CD16 receptor. Among phenotype markers of PBL subsets analyzed after D609 treatment, only CD16 was deeply down-modulated, thus suggesting a possible specific role of PC-PLC enzyme in regulating CD16 receptor expression. In order to better understand the relationship between the two molecules, we analyzed their trafficking from plasma membrane to cytoplasmic regions. PC-PLC and CD16 were internalized upon antibody engagement, degraded and newly synthetized, moreover, they needed an integral and functional actin cytoskeleton during their trafficking.Collectively, our data suggest that among PBLs an intense PC-PLC expression on the cell surface is a peculiarity of NK cytolytic cells, in which the enzyme plays an important role on sensitive target cells killing, and that PC-PLC may be





involved in CD16 signal transduction cascade and/or in the regulation of CD16 membrane surface expression.





B062Ly49 and CD94/NKG2 receptor acquisition by NK cells does not require lymphotoxin- receptor expression. Frederik Stevenaert, Katrien Van Beneden, Veerle De Colvenaer, Ann Sophie Franki, Veronique Debacker, Tom Boterberg, Dieter Deforce, Klaus Pfeffer, Jean Plum, Dirk Elewaut, and Georges Leclercq. Ghent University, 4 Klok A, DePintelas A85, Ghent B-9000 Belgium, and University of Dusseldorf, Dusseldorf, Germany

A crucial step in murine NK cell development, mediated by bone marrow stromal cells, is the induction of Ly49 and CD94/NKG2 receptor expression. The signals that regulate Ly49 receptor expression are still largely undetermined. It has been shown that interaction between LT12 and LTR, expressed on lymphoid progenitor cells and non-lymphoid bone marrow stromal cells respectively, is important for both quantitative and functional NK cell development. Therefore, we have investigated the role of LT-LTR-mediated signaling in Ly49 and CD94/NKG2 receptor acquisition. We show that the NK receptor repertoire of LTR-/- mice can only be partially analysed because of the residual 129/Ola mouse genetic background, due to a physical linkage of the LTR locus and the loci encoding the Ly49 and CD94/NKG2 receptors. Therefore, we transferred wild type B6 lymphoid-committed progenitor cells into LTR-/- mice, which differentiated into NK cells with a normal NK cell receptor repertoire. Also, administration of LTR-Ig, which acts as a soluble receptor for LT12, resulted in reduced NK cell percentages but did not influence the Ly49 and CD94/NKG2 receptor acquisition on remaining NK cells. These results indicate that LTR-mediated signals are not required for Ly49 and CD94/NKG2 receptor acquisition.





B063Recognition of peptide-MHC class I complexes by activating Killer Immunoglobulin-like ReceptorsC. Andrew Stewart1, Fanny Laugier-Anfossi1, Frédéric Vély1,2, François Romagné3, Alessandro Moretta4,5, Peter D. Sun6, Sophie Ugolini1, Eric Vivier1

1Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS-Univ. Méditerranée, Marseille, France2Current Address: Faculté de Pharmacie, INSERM U608, Marseille, France3Innate-Pharma SA, Marseille, France4Dipartimento di Medicina Sperimentale, and 5Centro di Eccellenza per le Ricerche Biomediche, Università degli Studi di Genova, Genova, Italy6National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA

Inhibitory receptors for Major Histocompatibility Complex (MHC) class I molecules increase the threshold of lymphocyte activation. Natural Killer (NK) cells express a large number of such inhibitory receptors including the human Killer Immunoglobulin-like Receptors (KIR). However, activating members of the KIR family have poorly defined ligands and functions. Here we describe the use of activating KIR tetramer reagents as probes to detect their ligands. Infection of cells with Epstein-Barr virus (EBV) leads to expression of a detectable ligand for the activating receptor KIR2DS1. In this case, KIR2DS1 interacts with up-regulated peptide-MHC class I complexes on EBV-infected cells in a transporter associated with antigen processing (TAP)-dependent manner. In tetramer based cellular assays and direct affinity measurements, this interaction with MHC class I is facilitated by a broad spectrum of peptides. KIR2DS1 and its inhibitory homologue, KIR2DL1, share sensitivity to peptide sequence alterations at positions 7 and 8. These results fit a model in which activating and inhibitory receptors recognize the same sets of self MHC class I molecules, differing only in their binding affinities. Importantly, KIR2DS1 is not always sufficient to trigger NK effector responses when faced with cognate ligand, consistent with fine control during NK activation. We will discuss how our results for KIR2DS1 and parallel studies on KIR2DS2 relate to the association between activating KIR genes and susceptibility to autoimmune disorders.





B064Prognostic Significance of Very Low CD57 Natural Killer Cells in Patients with Chronic Lyme Disease. Raphael B. Stricker,1 Edward E. Winger.2 1California Pacific Medical Center, San Francisco, CA, and 2Laboratory Corporation of America, San Leandro, CA.Background: Lyme disease is caused by infection with the spirochete Borrelia burgdorferi and has become the most common tickborne illness in the world today. Previously we identified a decrease in the CD57 natural killer (NK) subset in patients with chronic Lyme disease (Stricker & Winger, Immunol Lett 2001;76:43). We have now examined the clinical features of a subset of Lyme disease patients with very low CD57 NK levels. Methods: Eighty-nine adult patients with chronic Lyme disease were prospectively evaluated over a period of at least one year. All patients had clinical and serologic evidence of chronic infection with Borrelia burgdorferi. Clinical symptoms of Lyme disease were assessed using a four-point Lyme Disease Symptom Scale (0=none, 1=mild, 2=moderate, 3=severe). CD3(-) CD57(+) lymphocyte levels were measured by flow cytometry prior to the start of antibiotic therapy and at bimonthly intervals during treatment. Results: The 89 patients were divided into two groups: Group 1 consisted of 76 patients (85%) who had CD57 NK levels >10 cells/ul (normal range 60-360 cells/ul), while Group 2 consisted of 13 patients (15%) who had CD57 NK levels between 0-10 cells/ul. The mean time from presumed infection to Lyme disease diagnosis was 36 ± 28 months (range, 3-124 months) in Group 1 compared to 78 ± 98 months (range, 9-300 months) in Group 2 (P=0.0019). Coinfection with Babesia, Anaplasma or Bartonella species was detected in 13 of 76 patients (17%) in Group 1 compared to 11 of 13 patients (85%) in Group 2 (P<0.0001). In Group 1, 55 of 76 patients (72%) had predominant musculoskeletal symptoms, while in Group 2, 10 of 13 patients (77%) had predominant neurologic symptoms. The mean initial symptom score was 1.96 ± 0.58 (moderate) in Group 1 compared to 2.92 ± 0.27 (severe) in Group 2 (P<0.0001). With antibiotic treatment (mean 17 ± 5 months, range 8-25 months) the mean symptom score decreased to 1.22 ± 0.58 (mild) in Group 1 compared to 2.08 ± 0.86 (moderate) in Group 2 (P<0.0001). With treatment, the mean CD57 NK level in Group 1 increased from 51 ± 35 cells/ul (range, 11-198 cells/ul) to 61 ± 44 cells/ul (range, 15-252 cells/ul) (P=0.0044). In Group 2 the mean CD57 NK level increased from 3 ± 5 cells/ul (range, 0-10 cells/ul) to 18 ± 12 cells/ul (range, 0-51 cells/ul) (P=0.002). By the end of the treatment period, normal CD57 NK levels were found in 28 of 76 patients in Group 1 compared to 0





of 13 patients in Group 2 (P<0.0001). Conclusions: Patients with chronic Lyme disease and very low CD57 NK levels have significantly more coinfections, delayed diagnosis, more neurologic disease and persistent immunologic defects compared to patients with higher CD57 NK levels. A very low level of CD57 NK cells appears to be an important prognostic indicator in chronic Lyme disease.





B065GlcNAc8-dendrimer as NKR-P1A ligand in effector-target cell interaction and G-protein signaling. Jan Svoboda1, Marketa Kuldova1, Katarina Hulikova1, Richard G. Miller2 and Anna Fiserova1. 1Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, Prague, Czech Republic. 2University of Toronto and the Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada.

N-acetyl--D-glucosamine-coated octadendrimers (GlcNAc8) have been shown in our previous research as a high affinity ligands of recombinant rat NKR-P1A molecule and were found to have beneficial effect on anticancer immunity in vivo.

To define their role in effector-target cell interaction we followed the NKR-P1A expression in cytotoxic cells and GTP-binding proteins-mediated signaling in syngeneic C6 glioma model in Wistar rats in both lymphoid and tumor cell compartments. The specificity of GlcNAc8 for NKR-P1A was studied on YTS NK cell line, transfected with either A or B isoforms of rat NKR-P1 receptor. Cholate membrane extracts were prepared, and measured by competitive sandwich ELISA using polyclonal anti-G-protein subunit antibodies (Gq/11, Gi1,2, Gs, G0). NKR-P1A expression was assessed by flow cytometry using surface and intracellular staining.

Results have shown that either in vivo GlcNAc8 treatment or in vitro anti-NKR-P1A MAb addition increased the expression of Gq/11-protein in splenocytes to the same extent. However, the previous in vivo glycodendrimer treatment attenuated the anti-NKR-P1A MAb-mediated Gq/11-protein stimulation. FACS analysis of rat splenocytes demonstrated overall decrease in surface expression of NKR-P1A after in vivo GlcNAc8 exposure. Detailed study revealed an increased intracellular receptor occurrence at the expense of surface NKR-P1A levels. Evidence of GlcNAc8 specificity to the A isoform was provided by FACS analysis of trasfected YTS cell lines, which revealed changes in NKR-P1A, but not NKR-P1B surface vs intracellular receptor distribution. This disproportion could be connected to the reduction of anti-NKR-P1A MAb-induced Gq/11-protein expression after in vivo glycodendrimer treatment. These events possibly account for the receptor internalisation, described on other GPCRs as well.

Furthermore, we focused our interest on possible effect of glycodendrimer treatment on C6 gliomas. In ex vivo isolated C6 cells GlcNAc8 down modulated the Gs-protein expression, without influencing Gq/11 -subunit, thus affecting glioma cells through distinct downstream effectors (PLC vs adenylyl cyclase). However, we have not observed any direct in vitro effect of glycodendrimer on C6 cell line, thus GlcNAc8 could elicit these changes by modulating the effector functions of NKR-P1A positive cells (cell-cell interaction, cytokine production).





Taken together, GlcNAc8 was proven to be a candidate ligand of rat NKR-P1A molecule, it is involved in G-protein signaling and subsequent changes in tumor microenvironment.This work was supported by grants 524/04/0102, IAA5020403, IAA400200503 and Institutional Research Concept AV0Z50200510.





B066Anticancer effect of fucoidan based on biological and clinical study. Tachikawa D 1 ,2 M.D.Ph.D , Maemura M1, Fujii M1,3 Ph.D1NPO Fucoidan Research Lab, 1-6-14 shirogane chuou-ku Fukuoka city, Japan2Ordermadeclinic shirogane, 2-15-4 shirogane chuou-ku Fukuoka city, Japan3Faculty of Agriculture, Kagoshima University, 1-21-24 korimoto Kagoshima city, Japan

Health maintenance effects of foods, particularly prevention of cancers, have been attracting high social attention in recent years. We have been focusing on the functions of an algal sulfated polysaccharide, fucoidan. Fucoidan was one of water soluble dietary fiber and was reported anticancer effect caused by immunoenhancement, induction of apoptosis and suppression of angiogenesis. We have been investigated the immunoenhancement effect of fucoidan by NK cell activity. Now we have been examined the antitumor effect of fucoidan in various human cancer cells by MTT assay. As for clinical application in human, the food was administered to patients with cancer, and the preventive effect was evaluated.Sarcoma 180(2 105/0.2 ml) was subcutaneously transplanted in 5-week-old BALB/c mice, and the mice were fed fucoidan-mixed food product for 20 days. After completion of the feeding experiment, tumors formed under the skin were excised and weighed. In addition, the splenic NK cell activity was measured using YAC-1 as the target, and the cytotoxicity rate was obtained. Human stomach cancer cell(KATO-III), human colon and rectum cancer cell(HCT-15, LoVo), human hepatic cancer cell(HepG2), human breast cancer cell(MCF-7), and neonatal epitheolicyte of mouse(JB6C141) were cultivated at 37℃ in 5% CO2 incubator for 24 hours and then samples were added in each 96well plates, respectively. After incubation for 48 hours, MTT test reagent were added. Furthermore incubation for 4 hours, we measured microplate-reader(595nm) with Mosmann method. When cancer-transplanted mice ingested the fucoidan-mixed food product, the tumor weight was significantly decreased compared to that in the control mice, and the splenic NK cell activity was high, suggesting that increased NK cell activity inhibited the cancer cell growth. Each fucoidan suppressed increase in almost all cancer cells on concentration-dependence, especially Mozuku fucoidan showed strong effect of suppression for increase in LoVo and HepG2 cells. But the sample of Agaricus blazei Murrill could not show the same effect. More over each fucoidan could not suppressed increase in JB6C141 cells in spite of concentration. So, it was suggested that the suppression of increase in cell line of each fucoidan were peculiar for cancer cells, and its effect was different from the kind of cancer cells or the kind of fucoidan. In clinical study, after administration of fucoidan, it is





recognized that the reduction of tumor size, improvement of tumor marker and decrease of side effects by chemotherapy. In conclusion, it is suggested that the fucoidan therapy appears to be effective for carcinoma is useful and improves QOL of patients.





B067An MHC Class I Independent Mechanism for NK Self Tolerance Ruth T. Taniguchi, Megan E. McNerney, Dustin Guzior, and Vinay Kumar.Department of Pathology, Committee on Immunology, University of Chicago, 5841 South Maryland Avenue, S-315 MC3083, Chicago, IL 60637, USA

According to the missing self hypothesis, NK cells are rendered self tolerant by inhibitory receptors for self MHC molecules. Recent data extends this hypothesis by demonstrating that developing NK cells must interact with MHC class I molecules for them to become mature effector cells. Such “licensing” by MHC class I may prevent auto reactivity in the absence of class I MHC-mediated inhibition. However, while resting NK cells from class I-deficient mice are anergic, upon activation with IL-2 or poly(I:C), they rapidly acquire cytolytic and secretory functions. Such activation likely occurs during infections in vivo and indeed 2m-/- mice clear MCMV infections as well as do wild type mice. In addition, 2m-/- mice do not show increased killing of self, compared to wild type mice, despite the absence of self MHC. We propose that NK cells in class I-deficient mice and the Ly49-negative subset of NK cells in WT mice are rendered self tolerant by non-MHC dependent inhibitory mechanisms during activation, as exemplified by the receptor ligand pair 2B4-CD48. In support of this, we find that NK cells from 2m-/- and wild type mice are inhibited by 2B4 in vitro and in vivo. Furthermore, NK cells are prevented from killing syngeneic NK cells and DCs by inhibitory signals mediated by 2B4. Blocking 2B4-CD48 interactions with anti-2B4, or anti-CD48 antibodies in wild type NK cells or by use of NK cells from 2B4/CD48 knockout mice causes NK cells to lyse other NK cells, leading to suboptimal NK functions. Such fratricide does not occur in perforin-/- mice even if 2B4-CD48 interaction is blocked, indicating that NK-NK killing is mediated by perforin-dependent mechanisms. These findings suggest that NK cell self tolerance is maintained by several distinct and non-redundant MHC dependent and independent mechanisms.





B068Phenotype and functionality of transplanted NK cell populations in haploidentical bone marrow and stem cell transplantation. Dominik ter Meer1, Iris Bigalke1, Barbara Mosetter1, Barbara Simm1, Monika Braun1, Dusan Prevalsek2, Christoph Schmid2, Hans-Jochem Kolb2 and Christine S. Falk1. 1 Institute for Molecular Immunology, GSF Research Center for Environment and Health, Munich; 2 III. Medical Clinic Großhadern, LMU Munich, Germany.Introduction: Recent evidence from Velardi, Ruggeri et al. suggests that alloreactive NK cells in haploidentical BMT/SCT may provide graft-versus-leukemia (GVL) properties but do not lead to graft-versus host (GVHD) disease. The biological basis for this may be the expression of HLA-C-specific killer-Ig-like Receptors (KIR) on specific NK subsets and the genetic constellation of KIR+ NK cells in absence of inhibitory HLA-C ligands. Several clinical protocols currently attempt to benefit from this potential NK activity by selecting special KIR-HLA-C incompatible NK-cells.Materials and Methods: Since 1996, more than 76 patients suffering from different hematopoietic malignancies have been transplanted with haploidentical bone marrow at day 0 at the III. Medical Clinic, followed by transplantation of G-CSF-mobilized T cell–depleted peripheral blood stem cells (PBSC) at day 6 under immunosuppressive regiment (ATG, MTX, CyA). Fresh PBSC derived from 20 donors were analyzed for phenotypes of NK and contaminating T cell subpopulations prior to and after T cell depletion. Furthermore, the functionalities of NK-populations were determined by studying cell-proliferation and cytotoxicity against K562, in some cases additionally against haploidentical recipient cells, as well as determining cytokine secretion by multiplex protein array technique. In addition, high resolution HLA-C and KIR typing was performed using Luminex typing methods. Results and Conclusions: Although complete bone marrow was transplanted at day 0 according to the published protocol, no enhancement of GVHD was observed in the above mentioned 76 patients, when compared with HLA identical BMT. For all patients, 100% chimerism was detected post transplantation. Moreover, the T cell-depleted PBSC contained more NK cells than CD34+ stem cells, clearly demonstrating, that transplantation of up to 65 x 106 NK cells / kg body weight does not result in enhanced risk for GVHD. Extended phenotypical and functional analyses of the NK subpopulations within the PBSC prior to and after T cell-depletion of 20 samples revealed i) differences in cytokine production among particular donor/recipient constellations, ii) weak cytotoxic activity against





K562 and iii) reduced proliferative capacity in response to K562 of T cell-depleted PBSC samples compared to PBSC prior to depletion. In conclusion, haploidentical BMT in combination with T cell-depleted-PBSC may represent a promising treatment protocol for patients with advanced leukaemia due to improved engraftment and chimerism with simultaneous achievement of immunological tolerance.





B069Gliadin drives, through NK-DC crosstalk, the specific T response towards a Th1 polarization in celiac disease. Terrazzano G 1, Sica M 1, Gianfrani C 2, Mazzarella G 2, Maurano F 2, Giulio B 2, de Siant-Mezard S 3 , Zanzi D 4, Jabri B 3, Troncone R 4, Auricchio S 4, Zappacosta S 1

and Carbone E 1,5,6

1Dept Biol Pat Cell Mol, University of Naples Federico II, Italy, 2Institute of Food Science and Technology, CNR, Italy, 3Dept of Pathology, University of Chicago, USA,4Dept of Pediatrics and European Laboratory for the Investigation of Food-Induced Diseases, University Federico II, Italy, 5Dept of Exp. Medicine University of Catanzaro “Magna Graecia”, Italy, 6Strategic Research Center IRIS, Karolinska Institutet, S-17177 Stockholm, Sweden.

A key role in modulating immune diseases course has been demonstrated for NK cells in several studies. So far there are no direct prove that NK cells – DC crosstalk could be part of autoimmune pathogenesis. Celiac Disease (CD) has been described as an immune mediated disease elicited in some individual by gluten ingestion. We analysed the NK cells functional interactions with gliadin presenting DC in relation with the gliadin specific CD4+ T cell responses from intestinal mucosa of CD patients. Our data demonstrate that the interplay between autologous NK cells with gliadin presenting iDC results in a powerful increase of the IFN- production during the gliadin restricted CD4+T cell line response.The gliadin pre-treatment, but not with other dietary proteins studied, prevented the iDC elimination by NK cells. Furthermore the gliadin-dependent inhibition of NK mediated cytotoxicity by gliadin did not impair the IFN- production by autologous NK cell population and could be reversed by CD94/NKG2A receptor blocking. A strict correlation between the gliadin induced, iDC resistance to NK-mediated lysis and HLA-E expression on iDC was also observed.The most common gliadin subunits (lpha, amma and omega fractions) were analysed for the presence of amminoacidic (aa) sequences consensus motifs able to bind the HLA-E groove. Several gliadin peptides with different degrees of homology with the HLA-E specific peptide VMAPRTLLL were found in

lphaandomega sequences. Accordingly, fraction lpha and omega were the most efficient in conferring to iDC the protection to NK-mediated lysis and the increased expression of HLA-E. One peptide derived from gliadin lpha





,SQQPYLQLQ, increased HLA-E cell membrane levels on RMA-S HLA-E transfected lines. Analysis of HLA-E expression on either the small intestinal mucosa of celiac patients on gluten-containing diet confirmed the in vitro data.





B070Adoptive immunotherapy of cancer with ex vivo expanded, autologous NK cells. Hiroshi Terunuma 1,2 , Xuewen Deng1, Md. Zahidunnabi Dewan3,4, Naoki Yamamoto3,4, and Hiroyuki Abe2. 1Biotherapy Institute of Japan, 2-4-8 Edagawa, Koutou-ku, Tokyo 135-0051, Japan; 2Kudan Clinic, 1-9-5 Kudankita, Chiyoda-ku, Tokyo 102-0073, Japan; 3Department of Molecular Virology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; 4AIDS Research Center, National institute of Infectious Disease, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

Background: Natural killer (NK) cells are an important component of the innate immune response against malignant tumors without restriction of major histocompatibility complex. However, only few clinical studies of adoptive immunotherapy of cancer using autologous activated NK cells have been reported. In the present study, we examined the phenotypic and functional characteristics of an ex vivo expanded NK cell, and efficacy and safety of the autologous activated NK cell infusion into cancer patients.Materials and methods : Informed consent regarding the immunotherapy with autologous activated NK cells was obtained from cancer patients and healthy donors after we explained this therapy to them. Blood was taken from the patients and the donors. NK activity, flow cytometric analysis, ex vivo cultivation of NK cells from blood under Good Manufacturing Process conditions, and intravenous infusion of expanded NK cells was performed.Results: Activity of NK cells was significantly lower in cancer patients as compared with healthy donors. Several hundred to 2,500-fold NK cells were expanded ex vivo from PBMCs for 2 weeks cultivation. Cytotoxic activity of NK cells, CD69 expression level on the surface of NK cells, and perforin and granzyme expression level in the NK cells increased after 2 weeks activation of PBMCs. No contaminated pathogen was detected with an endotoxin test and agar plate cultures. After intravenous transfusion of autologous activated NK cells in volunteers, NK activity and the number of NK, B and T cells significantly increased in their blood. Expanded autologous NK cells-rich effector cells were repeatedly infused into more than 600 cancer





patients and no side effect was observed except fever at the night of NK cells infusion in a few patients. When patients received autologous NK cells expanded by ex vivo more than several times, their NK activity in the blood increased. Improvement of QOL, the decrease or stabilizing in tumor size and tumor markers was observed in more than half cases.Conclusion: We successfully expanded activated NK-rich effector cells by ex vivo. NK immunotherapy using such expanded cells was safe and useful in cancer patients.





B071KLRG1 interacts with a protein moiety expressed on the cell surface that is conserved among mammalian species. Marlowe S. Tessmer1, Frederick Stevenaert2, Olga V. Naidenko3, George Leclercq2, and Laurent Brossay1.1 Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912; 2 Department of Clinical Chemistry, Microbiology & Immunology; University of Ghent, Ghent, Belgium; 3Department of Pathology and Immunology, Washington University School of Medicine, 660 S Euclid, St. Louis, MO 63110.

The killer cell lectin-like receptor G1 (KLRG1) is a C-type lectin inhibitory receptor expressed on mouse NK cells and subsets of T cells. Inhibitory receptors oppose stimulatory signals via an immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic domains. Infection with MCMV and other pathogens, as well as indirect stimulation, induces a dramatic increase in the percentage of NK cells that are KLRG1+ and KLRG1 crosslinking inhibits NK cell effector functions. These findings suggest a role for KLRG1 in the negative regulation of NK cell function. KLRG1 signaling is presumably initiated upon ligand engagement, however, the ligand for KLRG1 remains unknown. While many of the receptors within this family bind to MHC class I like molecules, an interaction between KLRG1 and MHC class I has not been demonstrated. In order to identify candidates we generated a KLRG1 tetramer to detect cell lines that express binding moieties through flow cytometric analysis. Cells that stain positively with the tetramer are indicative of potential KLRG1 ligand expression. In order to confirm the specificity of this interaction, reporter cells were generated. A chimeric construct consisting of the KLRG1 extracellular domain in frame with the transmembrane and cytoplasmic domains of the Ly49H activating receptor was generated and expressed along with the adaptor protein DAP12. Engagement of the KLRG1/Ly49H fusion protein activates the reporter cells and induces the expression of -galactosidase, allowing for visual identification of ligand binding. Using these and other strategies, we demonstrate KLRG1 ligand expression at the surface of several cell lines and that this expression is evolutionarily conserved among various species. Successful detection of the KLRG1 ligand will provide important information about the role of this molecule in immune system regulation. Supported by NIH Grant AI58181.





B072Search for potential NKG2D ligands in human, mouse and viral genomes. Kathryn Trandem, Sai Durisetti, Sergei Radaev and Peter D. Sun. Laboratory of Immunogenetics, National Institutes of Allergy and Infectious Diseases. National Institutes of Health. 12441 Parklawn Dr., Rockville, MD 20852, USA

NK cell activating receptor NKG2D recognizes diverse ligands with low sequence identity. Crystal structures of several NKG2D-ligand complexes revealed common pattern in their hydrogen bonds and hydrophobic interactions. However, general principles of NKG2D ligand recognition and most importantly prediction of its new potential ligands still remain challenging. A structure-based ligand recognition algorithm was developed to evaluate hydrogen bonding and hydrophobic interactions for potential NKG2D ligands. This algorithm was applied for search on up-to-date human, mouse and viral genomes. Studies to evaluate the potential candidates are currently underway.





B073Signalling by inhibitory Killer Ig-like Receptors occurs in discrete domains of human NK cell immunological synapses. Bebhinn Treanor, Peter M.P. Lanigan, Chris Dunsby, Ian Munro, Björn Önfelt, Deborah N. Burshtyn*, Paul M.W. French, and Daniel M. Davis. Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, SW7 2AZ, UK. *Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.

NK cell activation is regulated by the balance between activating and inhibitory receptor signals. Inhibition of NK cell cytotoxicity by killer Ig-like receptors (KIR) depends on tyrosine phosphorylation at immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic domain. KIR, and their cognate MHC class I ligand, have been shown to cluster at the immunological synapse (IS) between NK cells and target cells. Here, we aim to determine where and when KIR signalling occurs at the NK cell IS and whether or not the spatio-temporal organisation of KIR phosphorylation is important in disrupting activating signals. For this, we present a methodology to image KIR phosphorylation at the NK cell IS using fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET). Using the human NK line YTS expressing GFP-tagged KIR2DL1 (YTS-KIR2DL1-GFP) and a Cy3-tagged generic anti-phosphotyrosine antibody, KIR phosphorylation was detected as a reduction in the fluorescence lifetime of KIR2DL1-GFP caused by FRET between GFP and Cy3. At the IS between YTS-KIR2DL1-GFP and a target cell expressing a cognate MHC ligand, HLA-Cw6, KIR phosphorylation is evident at the IS and not elsewhere at the cell surface. As a biological control, FRET was not detected in cells expressing a mutant of KIR2DL1 which lacks the ITIMs in the cytoplasmic tail. In addition, pre-treatment of NK cells with pharmacological inhibitors of the Src family kinases completely abrogated KIR phosphorylation. After 5 minutes of co-incubation of cells, the extent of KIR phosphorylation at the IS is variable, often with phosphorylation being restricted to small patches within the cluster of KIR. By 10 minutes, KIR phosphorylation is clear at most synapses and generally across the whole cluster of KIR. After 20 minutes KIR phosphorylation is reduced and again occurs within discrete domains of the cluster of KIR. Thus, there is a supramolecular organisation of KIR phosphorylation that is distinct from the organisation of KIR per se. We now aim to compare the extent and organisation of KIR





phosphorylation at synapses where the balance of activating and inhibitory signals varies.





B074Receptor binding of a Grb2-Vav1 intermediate is required for human NK cell NKG2D-initiated cytotoxicity. Jadee L. Upshaw, Laura N. Arneson, Renee A. Schoon, Christopher J. Dick, Daniel D. Billadeau, Paul J. Leibson. Mayo Clinic College of Medicine, 200 First Street SW, Rochester MN USA.

The human NK cell activating receptor NKG2D utilizes the transmembrane adaptor DAP10 for intracellular signaling. However, DAP10 does not have an ITAM and does not utilize Syk or LAT when triggering cytotoxicity. Instead it contains a YINM motif that binds to the PI3K regulatory subunit p85, which has traditionally been associated with co-stimulation. Although other potential DAP10 binding partners have been described (e.g. Grb2), their functional significance remains unknown. For example, it remains to be determined how DAP10 couples to needed downstream signaling elements (e.g. PLC-2, SLP-76, and Vav1) during the cytotoxic response. In this study we show that in contrast to the traditional assumptions, p85 binding to DAP10 is by itself insufficient to trigger NKG2D-initiated cell-mediated cytotoxicity. We specifically evaluated the functional significance of a DAP10-Grb2 interaction in the development of a cytotoxic response. A mutant DAP10-containing chimeric receptor was designed that disrupts Grb2 binding but not p85 binding. We found that the DAP10-Grb2 interaction is required for DAP10-mediated phosphorylation of PLC-2, SLP-76, and Vav1, as well as calcium flux and cytotoxicity. Furthermore, we utilized a Vav1 mutant that abrogates the Vav1-Grb2 interaction to show that DAP10-mediated phosphorylation of PLC-2, SLP-76, and Vav1, calcium flux, and cytotoxicity are all dependent on a Grb2-Vav1 interaction. These data suggest that upon NKG2D stimulation, a DAP10-Grb2-Vav1 complex forms that couples the receptor to downstream signaling elements required for calcium flux and NK cell-mediated cytotoxicity. Our findings identify a previously unknown mechanism by which receptor complexes that lack ITAM motifs can trigger lymphocyte activation.





B075Phenotypic and functional characterisation of CD4 T cells expressing KIR. Jeroen van Bergen, Allan Thompson, Yvonne Kooy and Frits Koning. Dept. of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Albinusdreef 2, 2333 ZA Leiden, the Netherlands.

Killer Ig-like Receptors (KIR) are commonly found on human NK cells, T cells and CD8 T cells. Although KIR+ CD4 T cells are found in certain patients, their prevalence in healthy donors is controversial. We now provide definitive proof that such cells are present in most individuals, and report on their frequency, surface phenotype, cytokine profile and antigen specificity. The number of KIR+ CD4 T cells detected in peripheral blood increased with age. In contrast with regular KIR- CD4 T cells, the majority of KIR+ CD4 T cells lacked surface expression of CD27, CD28, CCR4, and CCR7, but did express CD57 and 2B4. What's more, KIR were detected on approximately one tenth of CD28- and CD57+ memory CD4 T cells. In line with the absence of the Th2 marker CCR4, the KIR+ CD4 cells produced mainly IFN and little IL-4 or IL-10 upon TCR triggering. Furthermore, the KIR+ CD4 population was enriched for cells that respond to CMV antigens in an HLA class II-restricted fashion. Together, our data indicate that KIR expressing CD4 T cells are predominantly HLA class II-restricted effector memory Th1 cells, and that a significant, previously unrecognised fraction of effector memory Th1 cells expresses KIR. Current experiments aim to elucidate the role of KIR-HLA class I interactions in the specific recognition of CMV infected cells by KIR+ CD4 T cell clones.





B076Expression of rearranged TCR genes in natural killer cells suggests a thymus-dependent pathway of lineage commitment. Linnea L. Veinotte, Chelsea P. Greenwood, Nastaran Mohammadi, Christine A. Parachoniak and Fumio Takei

Terry Fox Laboratory, British Columbia Cancer Agency andDepartment of Pathology and Laboratory Medicine, University of British Columbia675 West 10th Avenue, Vancouver, British Columbia, Canada, V5Z 1L3

NK cells are thought to develop from common lymphoid progenitors in the bone marrow. However, multiple progenitors with NK cell potential have been described including bipotent T/NK progenitors in the fetal blood, spleen, liver, and thymus and immature thymocytes in both the fetal and adult mouse. The NK cell lineage commitment pathway is unclear and currently, the contribution of the thymus-dependent pathway in normal steady-state NK cell development is unknown. Through microarray analysis of neonatal and adult NK cells, we detected expression of the T-cell receptor gamma gene. Genomic and RT-PCR analysis show that the TCR gene expression represents true V(D)J genomic rearrangement and the percentage of productive rearrangements are close to the expected rate for random recombination. TCR genes are rearranged in approximately 5% of neonatal and 1% of adult mouse splenic NK cells and similar levels are detected in NK cells from TCR,-double knockout mice, excluding the possibility of T cell contamination. Also, the TCR rearrangement is not detected in NK cells from Nude mice which lack the thymus, suggesting that the NK cells with rearranged TCR are thymus-dependant. Very low levels of TCR delta rearrangements are present in NK cells but TCR beta rearrangement is absent. In addition, B cells lack TCR rearrangement suggesting that the subpopulation of NK cells develop from thymic T/NK bipotential progenitors that have rearranged TCR genes and have lost B cell potential. This study has revealed a thymic pathway of NK cell lineage commitment that includes TCR rearrangement and shows that NK cells follow at least two separate pathways of lineage commitment, one in the thymus shared with developing T cells and another pathway in the BM. It is important to know that the BM pathway is not representative of all NK cells in steady-state NK cell development.





B077NK-cell reconstitution after pediatric stem cell transplantation: correlation of adenoviral infections with NK-cell number and phenotype. Dirk H.J. Verhoeven, Monique M. ten Dam, Arjan C. Lankester, Marco W. Schilham and Maarten J.D. van Tol. Pediatric Bone Marrow Transplantation Unit, Department of Pediatrics, Leiden University Medical Center, PObox 9600, 2300 RC Leiden, The Netherlands.NK-cells are generally the first lymphocytes to reconstitute after successful allogeneic stem cell transplantation (SCT), but their functionality and their contribution to clearance of viral infections early after SCT is largely uncharacterized.In order to correlate NK-cell numbers and phenotype with viral infections, phenotypical analysis of several activating and inhibitory NK-cell receptors was investigated weekly for the first 8 weeks and in regular intervals thereafter after SCT in 25 patients. Furthermore, quantitative viral loads and/or cultures of Epstein Barr Virus (EBV), Cytomegalovirus (CMV), Herpes Simples Virus-1 (HSV-1) and Adenovirus (AdV) were determined weekly.NK-cells appear early after SCT and reach normal numbers (>90 NK-cells/ul) within 4-5 weeks after SCT, depending on the type of donor. There is a marked decrease in the percentage of NKG2A positive NK-cells in the first months after SCT: 4 weeks after SCT, generally >80-90% of NK-cells expresses NKG2A, whereas this decreases to 40-60% of NK-cells 6 months after SCT.A marked correlation was observed between increasing NK-cell numbers and increasing expression of NKG2A, NKG2D, NKp46, NKp30 and CD244 and increasing absolute numbers of NKp44 positive NK-cells in 3/6 patients with disseminated Adenoviral infection early after SCT. Furthermore, in all of these patients a clearance of disseminated Adenovirus was detected by quantitative PCR within 1- 3 weeks after NK-cell peak. These 3 patients did not have substantial numbers (<50/ul) of peripheral T-cells present at time of infection or in the weeks after, making it unlikely that T-cell reactivity, as demonstrated earlier by our group, has contributed to control Adenovirus in vivo in these patients. In contrast, the other 3 patients had either no NK-cells or also a substantial amount of recovering NK- and T- cells at time of adenoviral infection.No correlation between changes in NK-cell numbers or phenotype was observed during EBV, CMV and HSV-1 infection.In conclusion, disseminated adenoviral infection can be cleared early post SCT with peaking NK-cell numbers but without peripheral T-cells present. Further





experiments need to establish a functional correlation between changes in NK-cell number or phenotype and clearance of adenoviral infection early post SCT.

This research is supported by grants of The Netherlands Organization for Health Research and Development (ZonMw), Foundation KiKa and the Leiden University Medical Center.





B078Effect of alcohol consumption by C57BL/6 mice on natural killer cell activation after systemic or pulmonary infection by murine cytomegalovirus. Debbie Vidlak, Jennifer Strachota, and Thomas R. Jerrells. University of Nebraska Medical Center, 986495 Nebraska Medical Center, Omaha, NE USA.The immune response of C57BL/6 mice to murine cytomegalovirus (MCMV) is critically dependent on activation of natural killer (NK) cells, as well as some of the early pathogenic effects in the liver. Consumption of alcohol by C57BL/6 mice has been shown by us to impair clearance of MCMV and result in a prolonged hepatitis. Alcohol abuse has been associated with increased susceptibility to infections of human beings. Studies have been conducted to evaluate the effects of alcohol consumption on spontaneous cytolytic activity of NK cells and lymphokine-activated killer cells, but no consistent changes have been shown in human beings. The experiments described in this abstract were designed to determine the effects of alcohol consumption by C57BL/6 mice on NK cell IFN- production associated with MCMV infection, which is suggested by us to be the more relevant parameter of NK cell biology than spontaneous cytotoxicity. Female C57BL/6 mice were provided ethanol in either a liquid diet (short-term consumption, 7–10 days) or as a 20% v/v solution in drinking water (chronic consumption, 4–6 weeks). Infection with MCMV was induced intraperitoneally or intranasally. At various times after infection (ranging from 24 h to 7 days, depending on the experiment), mononuclear cells were isolated from the peritoneal cavity, spleen, liver, and lungs, and the proportion of IFN- NK cells (NK1.1+) was determined with the Interferon-Secretion Assay (Miltenyi Biotec) without further purification and immediately after cells were obtained. Short-term alcohol consumption resulted in decreased NK1.1+ cells in cells from the peritoneal cavity obtained 72 h after infection, but the number of IFN-+ NK cells was not affected. Alcohol consumption had little effect on either NK cell percentages or NK IFN- secretion in the spleen, but it had significant effects on the NK1.1–/IFN-+ cells in the spleen. In contrast, 72 h after infection the total number of NK cells in the liver was significantly higher, and the NK1.1–/IFN- cells were decreased. Pulmonary infection of mice provided ethanol in drinking water for 6 weeks was associated with a decreased number of NK1.1 cells and NK1.1 cells secreting IFN- in the spleen 48 and 72 h after infection. A trend toward lower total NK1.1 cell numbers and NK1.1/IFN-+ cells was seen in cells isolated from the lungs, but this did not reach statistical significance. Together,





these preliminary findings support the suggestion that short-term or chronic alcohol consumption results in changes in NK cell activation induced by MCMV infection, but these changes were transient and differed in kinetics and magnitude of change, depending on the organ evaluated. More profound effects of alcohol on IFN- production were seen in NK1.1– cells.





B079Involvement of Notch signaling in NK cell maturation. Haruka WADA, Kiyokazu KAKUGAWA, Hiroshi KAWAMOTO. Laboratory for Lymphocyte Development, Research Center for Allergy and Immunology, RIKEN, N609A 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

The developmental stages of NK cells from progenitors to mature NK cells have been well characterized. Along with differentiation, NK precursor sequentially acquires various molecules including NKR-P1, NKG2, Ly49, DX5, Mac-1, and then they functionally mature. Some cytokine/receptor signals, such as IL-15/IL-15R and LT/LTR, have been shown to be required for full NK cell development. However, environmental factors involved in NK cell development other than cytokine/receptor signals have been poorly understood. Present study aims to clarify whether Notch signal is involved in the development and/or maturation of NK cells, because the Notch signaling plays a critical role in the development of various organs, including hematopoiesis and T cell development.

As a source of NK cell lineage committed progenitors (pNK), we used lineage marker (CD3, CD4, CD8, TER, B220, Mac-1, Gr-1)-NK1.1-DX5-CD122+ cells (CD122+cells) from 14 dpc (days post coitus) murine fetal thymuses. CD122+ cells were co-cultured with control fibroblastic stromal cells (TSt-4) or those enforced to over-express Delta like-1 (TSt-4/DLL-1), which is one of the Notch ligand, in the presence of IL-7, SCF and IL-15. After 12 days, we flow cytometrically analyzed generated cells. In both culture conditions, large proportions of generated cells expressed NKR-P1, CD94, NKG2A/C/E and NKG2D, but they did not express Ly49A, Ly49D or Ly49G2. Strikingly, whereas cells generated on control stromal cells were DX5-/low, most of cells generated on TSt-4/DLL-1 highly expressed DX5. We then isolated NK1.1+DX5-/low cells from adult liver, which have been proposed to serve as a precursor pool for mature DX5hi NK cells, and were co-cultured with TSt-4/DLL-1 for 7 days. While cells remained DX5-/low in control culture, they came to be DX5hi by co-culturing with TSt-4/DLL-1. The high expression of DX5 was cancelled by blocking the Notch signal using -secretase inhibitors, confirming that differentiation from DX5-/low to DX5hi on stromal cells is induced by Notch signaling. These results suggest that Notch signaling is involved in the maturation step of NK cells.

Since the transition step from DX5low to DX5hi is known to coincide with the acquisition of lytic activity, we are currently studying whether Notch signal is involved in functional maturation of NK cells.





B080Kinome of a killer. Joseph A. Wahle 1 , Sander Diks2, Amy L. Hazen1, Maikel Peppelenbosch2, William G. Kerr1. 1H. Lee Moffitt Cancer Center and Research Institute and the University of South Florida, 12902 Magnolia Dr. Tampa, FL USA. 2University Medical Center of Groningen, 9713 AV Groningen, The Netherlands.

Natural killer cells are lymphocytic cells that play a role in both the innate and adaptive immune responses through direct cytolysis of target cells and through release of cytokines and chemokines. NK cells develop along a distinct lineage, delineated by the acquisition of the various surface receptors involved in NK function and self-tolerance. The functional maturation of these cells is, in part, regulated through signaling pathways that involve protein phosphorylation. The kinases that perform these phosphorylations play a major role in regulating signaling cascades that determine cell cycle entry, survival and the maturation of cells in the mammalian body, including the hematopoietic system. In this study we have performed a massively parallel and unbiased determination of the different kinase activities present in mature and immature NK cells. To this end we hope to better elucidate the molecular processes that are involved in the regulation of NK development and function. We have developed peptide array technology where 1176 different peptide substrates are arrayed in duplicate on a single chip. Whole cell lysates applied to these chips lead to the phosphorylation of specific peptides. After determining the phosphorylation levels for each of the 1176 kinase substrates a phosphorylation signature or kinome is generated. As expected our initial comparison of the kinome profiles of mature and immature NK cells reveals a significant degree of overlap in their respective kinomes. However, these comparisons also reveal a significant number of differentially regulated kinase activities in these two cell types. In total 234 peptides were shown to be differentially regulated between these 2 cell types, of these, 84 were found to be higher in mature NK cells and 149 were found to be higher in immature NK cells. Within these differentially phosphorylated peptides some trends have emerged such as the differential phosphorylation of proteins involved in cell cycle, chromatin packaging, and Lyn signaling. In addition to the comparison of these two cell types we have performed comparisons with the mature NK cell to other mature cell types, namely mature B cells and myeloid cells. In these comparisons we once again find a high degree of overlap with over 900 peptides having a similar kinome signature; however, there are also





significant differences that yield a specific kinome for the NK lineage. These kinome studies have the potential to further elucidate, as well as identify, new signaling pathways that are crucial to the maturation and function of the NK cell.





B081The Unexpected and Differential Effect of Cyclosporin A on CD56bright and CD56dim NK cell Expansion, Phenotype, Function and Development From Progenitor Cells. Hongbo Wang, Alisa Lee, Valarie McCullar, Jeffery Miller and Michael Verneris. Division of Bone Marrow Transplantation, University of Minnesota, MMC 266/420 Delaware Street, SE, Minneapolis, MN 55455 USA

Following allogeneic hematopoietic cell transplantation, NK cells play important roles in hematopoietic cell engraftment, anti-viral responses, graft vs. host disease (GVHD) and graft vs. leukemia (GVL) reactions. Calcineurin inhibitors, such as cyclosporine A (CsA), are frequently administered to prevent or treat GVHD. It is generally considered that these immune suppressive agents inhibit GVL reactions. To date, little is known about the impact of these immune suppressants on NK cell function. To investigate this, NK cells were isolated from normal donors by negative selection and cultured with IL-2 (100 U/ml), IL-15 (10 ng/ml) and either physiological levels of CsA (1µg/ml) or vehicle control. Under these conditions, we consistently found that CsA treated cultures showed a reduction in NK cell expansion at one week (4.88 vs. 1.87 fold expansion, n=10). The phenotype of CsA treated NK cells was markedly different than controls. More specifically, after 7-10 days of culture with CsA there were significantly more CD56brightCD16- cells and significantly less CD56dimCD16+ cells compared to controls (p<0.001 for both). Accordingly, the percentage of KIR receptor (CD158a/h, CD158b/j and CD158e) expressing cells was significantly less in CsA treated cultures. No consistent changes were detected in the expression of NKG2D, NKp30, NKp44, or NKp46 on CsA treated NK cells relative to controls. To further investigate the influence of CsA on NK cell subset expansion, freshly isolated NK cells were stained with CFSE then cultured with CsA or vehicle control for 1 week. Using FACS we monitored CD56dimCD16+ and CD56brightCD16- cell division. There was no difference in the number of CD56birghtCD16- cell divisions comparing CsA treated cultures to controls. In contrast, the CsA treated CD56dimCD16+ cells had fewer cell divisions, demonstrating that CsA selectively inhibits CD56dimCD16+ cell proliferation. These results were confirmed with freshly isolated, FACS sorted CD56dimCD16+ and CD56birghtCD16- populations cultured with CsA or vehicle control. To investigate the functional activity of CsA treated NK cells, we performed standard killing assays using K562 and Raji cells as targets. Surprisingly, we consistently found higher cytotoxicity in CsA treated cells compared to controls (K562, p <0.05 and Raji, p <0.05). One of the main functions of CsA is to inhibit cytokine secretion by preventing cytoplasmic NFAT translocation into the nucleus upon cell activation. Since CsA treated NK cell cultures have higher percentages of CD56brightCD16- cells, we investigated the intracellular IFN- secretion following IL-12/IL18 stimulation. In 5 consecutive experiments the percentage of IFN- secreting cells was higher in CsA treated NK cells compared to vehicle controls (44% vs. 24%, p<0.05). Lastly, we investigated the effect of CsA on NK cell differentiation from progenitor cells (CD34+Lin-CD38-) using an in vitro differentiation system. Briefly, progenitor cells were cultured on a murine feeder cell line (AFT-024) for 42 days in the presence of IL-3, IL-7, IL-15, SCF and FLT3L +/- CsA (or vehicle control). We





found that CsA treated cultures had less KIR expressing cells compared to controls. Taken together these results suggest that CsA inhibits CD56dimCD16+ cell growth, resulting in a population of NK cells that have less KIR receptor expression and higher cytokine secretion. These findings may have important implications for both GVHD and GVL following hematopoietic marrow transplantation.





B082Pre-activation of T lymphocytes by low dose of concanavalin A aggravates toll-like receptor-3 ligand-induced NK cell-mediated liver injury. Jing Wang *, Rui Sun*†, Haiming Wei*, Zhongjun Dong*, Zhigang Tian†. *School of Life Sciences, University of Science and Technology of China, Hefei 230027, China; †School of Pharmacy, Shandong Univeristy, Jinan 250002, China

Poly I:C, a well-known potent activator for NK cells, can stimulate surface TLR-3 and trigger immune responses resembling viral infection owing to its double-stranded viral RNA-like structure. Our previous study has demonstrated that injection of poly I:C into mice could preferentially recruit and activate NK cells in liver with moderate elevation of serum transaminase levels and mild liver inflammation, which is verified to be NK cell-dependent. Con A-induced fulminant liver injury is well described to be mediated by activated T cells, or NKT cells as recently reported. Several inflammatory cytokines such as TNF- and IFN- are also considered to play an important role in mediating the development of massive hepatocellular necrosis. In this study, we find that hepatic T cells, when pre-activated by a non-hepatotoxic low dose of Con A, can positively affect NK cells and then aggravates the liver injury induced by poly I:C-activated NK cells, indicating that interaction between T cells and NK cells participates in liver damage.

In order to evaluate the effect of non-hepatotoxic Con A on hepatic NK cells, pretreatment of mice with low dose of Con A was performed on polyinosinic-polycytidylic acid (poly I:C) [toll-like receptor-3 (TLR-3) ligand]-induced NK cell-mediated hepatitis. The role of the Con A-pre-activated T cells in NK cell function was then determined. The results demonstrated that Con A pretreatment aggravated poly I:C-induced liver injury with elevation of serum transaminases, hepatic necrosis and serum IFN- level. In parallel to the enhanced accumulation of activated NK cells into liver, the natural cytotoxicity and IFN- production by hepatic NK cells were significantly augmented. Moreover, depletion of T cells suppressed the aggravating effect of Con A pretreatment and downregulated NK cell activation. Collectively, the aggravation of poly I:C-induced hepatitis by Con A pretreatment may be due to the generation of more vigorous NK cells which is dependent on the presence of T cells. This investigation will help to explain the synergistic effects of NK and T cells in liver inflammatory injury.





B083Poly I:C Prevents T Cell-mediated Hepatitis via an NK-dependent Mechanism. Jing Wang, Haiming Wei, Rui Sun, Zhongjun Dong, Zhigang Tian. *School of Life Sciences, University of Science and Technology of China, Hefei 230027, China; In contrast to T cells, NKT cells, and Kupffer cells that are essential for T cell-mediated hepatitis, NK cells have been shown to play a minor role in Con A-induced T cell-mediated hepatitis, as depletion of NK cells did not significantly affect Con A-induced liver injury. However, activation of NK cells induced by poly I:C was able to cause mild liver injury in a TNF related apoptosis inducing ligand (TRAIL)-dependent mechanism and negatively regulate liver regeneration in an IFN--dependent mechanism. Thus, we hypothesize that activation of NK cells by poly I:C may also modulate Con A-induced T cell-mediated hepatitis.

In this study, pretreatment with poly I:C 12 h prior to Con A injection completely prevented Con A-induced liver injury. Such protective effect was further confirmed by pathology. Furthermore, poly I:C pretreatment protected mice from the lethality associated with lethal dose of Con A. Con A administration caused significant increases in serum TNF-, IFN-, and IL-4 levels. Poly I:C pretreatment profoundly suppresses the elevation of serum IL-4 and IFN- levels, and to a lesser degree, TNF-. Using flow cytometry, we observed IL-4 was mainly produced by liver NKT cells, which was largely suppressed by poly I:C pretreatment. Depletion of NK cells did not affect Con A-induced serum ALT elevation, pretreatment with poly I:C caused a 90% reduction of serum ALT levels in mice containing NK cells, but only caused a 40% reduction in NK-depleted mice. These findings suggest that NK cells contribute to the inhibitory effect of poly I:C on Con A-induced hepatitis. In NK-depleted mice receiving an intrahepatic transfer of hepatic MNCs or purified NK cells, poly I:C pretreatment reduced serum ALT level by 80% to 90%, significantly lower than about 40% reduction in non-transferred group. Collectively, our data suggest that adoptive transfer of NK cells largely restores the protective effect of poly I:C on Con A-induced liver injury in NK-depleted mice.

Since NKT and T cells are essential for Con A-induced hepatitis, we wondered whether the protective effect of poly I:C is mediated via modulation of these effector cells. A marked reduction of T and NKT cells was detected 3-24 h post poly I:C injection with peak decline at 12 h post injection. In contrast, NK cells strikingly accumulated in liver during this period. In NK-depleted mice, poly I:C treatment did not markedly affect NKT cell number, and did not decrease rather significantly increased T cells, suggesting that poly I:C treatment decreases NKT and T cells via an NK-dependent mechanism. Expression of Annexin V on NKT and T cells was rapidly upregulated after poly I:C injection, while the expression on NK cells was not significantly changed. Meanwhile, expression of Fas protein on T and NKT cells was also elevated, while expression of Fas L was enhanced on NK cells. To confirm the pivotal role of Fas/Fas L, we selected Fas L-/- or Fas-/- mice. Poly I:C downregulation of NKT cells in wild-type mice was diminished significantly in these mice 12 h post poly I:C treatment. Depletion of NKT cells was also markedly suppressed in Fas L-/- mice at 24 h compared to wild-type mice, but was only slightly diminished in Fas-/- mice.





[This work was supported by Natural Science Foundation of China (#30125038, #30230340, #30328022), 973 Program by Ministry of Science and Technology of China (#2001CB510009; #2003515501) and Foundation of Chinese Academy of Science (#KSCX2-2-08)].





B084Naïve CD8 T cells expressing inhibitory NK receptors in cord blood. Hilary S. Warren1,2 , Purna M. Rana1, Duncan T. Rieger2, Jane E. Dahlstrom3,5 and Alison L. Kent.4,5 1Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia, 2 Cancer Research Unit, 3Department of Anatomical Pathology, 4Department of Neonatology, The Canberra Hospital, Canberra, ACT, Australia, and 5ANU Medical School, Australian National University, Canberra, ACT, Australia. [email protected]

Inhibitory NK receptors (iNKR), the Killer Ig-like Receptors (KIR) and CD94/NKG2A receptor, are expressed on a small proportion of CD8 T cells in adult blood. CD8 T cells expressing iNKR are reportedly not present in umbilical cord blood or thymus. The proportion of CD8 T cells expressing iNKR in adult blood increases with age, consistent with an hypothesis that iNKR are induced on CD8 T cells as a consequence of chronic antigen or autoantigen stimulation. In this study we show that a minor subset of CD8 T cells in cord blood do express iNKR. The proportion of CD8 T cells expressing iNKR is similar for cord blood from healthy term babies and preterm (25-30 weeks gestation) babies and also in cases where placental inflammation was found on histology. The proportion of CD8 T cells expressing KIR in cord blood from healthy term babies (n = 27) was on average 1.67% (range 0.8% to 4.92%), and the proportion expressing NKG2A was on average 0.74% (range 0.04% to 3.52%). As expected adult blood (age 20-40 years) contained significantly (p<0.0001) higher proportions of CD8 T cells expressing KIR (mean 5.08%, range 1.8% to 14.1%, n = 15) and NKG2A (mean 4.16%, range 1.45% to 11.44%, n = 24). Nevertheless, more than half of the values recorded for cord blood are within the same range as those for normal donors. To compare the differentiation state of CD8 T cells expressing iNKR in cord blood compared to adult blood we analysed these cells for expression of CD27 and granzyme B. iNKR+ CD8 T cells in cord blood (n = 9) are entirely CD27+, and almost all lack expression of granzyme B, indicative of a naïve status. By contrast iNKR+ CD8 T cells in adult blood (n = 11) showed considerably heterogeneity in expression of CD27 and granzyme B, with a majority expressing the differentiated CD27- granzyme B+ phenotype. This study demonstrates a hitherto unappreciated complexity for iNKR+ CD8 T cells. That cord blood from healthy term babies contains a naïve population of iNKR+ CD8 T cells suggests that iNKR are constitutively expressed by these cells. The





pool of iNKR+ CD8 T cells in adult blood is clearly heterogeneous with both naïve and differentiated cells present. The possibility that iNKT+ CD8 T cells in adult blood arise at least in part from neonatal iNKR+ CD8 T cells needs to be considered.





B085IL-21 differently affects CD56dim and CD56bright NK cells. Katy Wendt, Esther Wilk, Sabine Buyny, Sebastian Bremer, Reinhold E. Schmidt, Roland Jacobs. Department of Clinical Immunology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

Interleukin (IL)-21 is known to play an important role in activation and differentiation of human lymphocytes. The receptor (IL-21R) shares the common -chain for signal transduction with IL-2R, IL-15R and IL-7R. As determined by gene chip and FACS analyses we found IL-21R equally expressed by CD56dim and CD56bright NK cells. To investigate the role of IL-21 in both NK cell subsets human PBMC or sorted NK cells were stimulated with IL-21 and/or suboptimal doses of IL-2. FACS analyses after 24 hours revealed a differential regulation of activation markers CD69 and CD25. While CD69 was preferentially up-regulated by CD56dim NK cells after stimulation with IL-21 alone or in combination with IL-2, CD25 expression was mainly regulated by the CD56bright NK subset. A significant decrease of this marker was measured after stimulation with IL-2 or both mediators but CD25 was slightly enhanced after activation with IL-21 alone. Preliminary data show that other NK cell receptors like CD158a, CD158b, NKp44, CD16 and IL-21R are not affected by IL-21.To study the functional impact of IL-21 on CD56dim and CD56bright NK cells we performed 72 hours 3H-thymidin and cytotoxicity assays. Proliferation of both subsets increased by treatment with cytokines alone or in combination with stronger responses detected in CD56bright NK cells. Cytotoxicity against K562 was mainly enhanced in the CD56dim subset.Since expression of IL-21R is not altered by the treatment used in this study we assume other mechanisms responsible for the synergistic effect of IL-2 and IL-21 on NK cell subsets.Our data indicate that IL-21 modulates immune responses and functions in CD56dim and CD56bright NK cells differently.Supported by DFG Priority Program SPP1110, project Ja1058 and Graduate Program of Lower Saxony





B086The Role of V14i NK T Cells during MCMV Infection. Johnna D. Wesley, Marlowe S. Tessmer, Stephanie C. Terrizzi, and Laurent Brossay. Department of Molecular Microbiology & Immunology, Brown University Providence, RI 02912.

Anti-viral immunity requires both innate and adaptive responses to control infection and promote clearance. V14i natural killer T (NK T) cells are a unique population of lymphocytes that rapidly activate natural killer (NK) and CD8+ T cells. Here we investigate the role of NK T cells in the intiation and propagation of the immune response using the well-characterized infection model murine cytomegalovirus (MCMV). We find that very early in the course of MCMV infection NK T cells display characteristics of activation, specifically TCR and NK1.1 down-regulation, followed by IFN- release in the liver and spleen of infected animals. Interestingly, the activation of NK T cells occurs prior to that of NK cells, follows similar kinetics, and requires similar cytokine signals. This demonstrates that, in addition to NK cells, NK T cells act as early sensors of infection. Also, the MCMV-dependent effect on the NK T cell population is maintained throughout the adaptive immune response and resolution of the infection. Surprisingly, the absence of the NK T cell population during infection leads to dysregulation of the cellular immune response, resulting in fewer virus-induced activated CD8+ T cells and enhanced MCMV-mediated liver damage. Additionally, we find that the absence of the NK T cell subset results in the generation of functionally impaired CD8+ T cells. Collectively, the data provide evidence that NK T cells influence the quality of the anti-viral immune response. Supported by NIH research grant AI46709.





B087Regulation of experimental autoimmunity in vivo by depletion of NK cells: alteration of primary adaptive responses. Robin T. Winkler-Pickett, James Cherry, John W. Diehl, William E. Bere, Anna T. Mason, and John R. Ortaldo. Lab. Exp. Immunol, & SAIC, NCI-Frederick, P.O. Box B, Frederick, MD 21701

Innate immune responses provide the body with its first line of defense against infections. Signals generated by a subset of lymphocytes, including natural killer (NK) cells, natural killer T (NKT) cells, and antigen presenting cells during the early host response determine the nature of downstream adaptive immune responses. Recent studies in autoimmune models have implied early regulation of the adaptive interface by innate cells. In the present study, we have extended these observations in the MOG peptide induced EAE model. Our studies have shown that NK cells can affect the outcome of these adaptive immune responses as NK cells, but not NKT cells, were found to regulate the degree of clinical and immune adaptive responses to MOG (35-55). The requirement for NK cells was reflected by the reduced helper T cell response to MOG peptide and suppression of clinical disease was seen in mice treated with anti- NK1.1 , anti- asGM1, and selected Ly49 subtype-depleted mice. Further analysis of TcR V-beta usage indicated not only a diminished T cell response, but an alteration in the TcR V-beta usage at the site of disease, e.g. the brain. These findings establish a previously unexplored link between NK cells and the development of autoreactive T cells and may imply a similar process for autoreactive B cells.





B088Allelic differences in membrane expression and secretion of KIR2DL4. Campbell S Witt1, Jodie Goodridge2, F Christiansen1,2

1Dept Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Wellington St, Perth, West Australia2Dept Surgery and Pathology, University of Western Australia, Nedlands, West Australia.

KIR2DL4 is unique among the KIR receptors in that it 1) is not part of a stable phenotype but appears transiently during NK cell activation 2) has structural features suggesting both activating and inhibitory function 3) may be a receptor for a non-polymorphic ligand, HLA-G. HLA-G is expressed on invading foetal trophoblast but is also upregulated in a variety of inflammatory situations. Stimulation of NK cells via KIR2DL4 results in IFNg secretion. Secretion of IFNg by KIR2DL4 interaction with HLA-G in inflamed tissue therefore has potential to modify ongoing immune responses. We have previously described a common allele (gene frequency = 0.5) of KIR2DL4 (9A allele) that differs from the alternative allele (10A) by a single nucleotide deletion in the transmembrane region. This deletion causes splicing out of the transmembrane region resulting in a receptor which cannot be expressed on the membrane but is potentially secreted. CD56bright NK cells from most subjects with the 10A allele express KIR2DL4 whereas those who are homozygous for the 9A allele do not. We now report that some subjects with a 10A allele whose CD56bright NK cells do not express KIR2DL4, have an allele that can be distinguished by nucleotide substitutions in intron 6 and the Ig-domains. Although not expressed on resting CD56bright NK cells, this new allele (termed 10A-B to distinguish it from the other 10A allele, 10A-A), which has a gene frequency of 20%, is expressed on activated NK cells. In contrast to the 10A-A allele, 50% of the 10A-B transcripts are missing one of the 2 Ig-domains. Longitudinal studies of KIR2DL4 mRNA production and surface expression reveal that mRNA production of all 3 alleles (9A, 10A-A, 10A-B) is strongly upregulated during activation. In activated NK cells that have become quiescent again, KIR2DL4 mRNA remains maximal in all 3 genotypes but surface expression is lost suggesting the existence of a mechanism that suppresses membrane expression of KIR2DL4. In addition, we now show that the 9A allele results in copious secretion of soluble KIR2DL4 whereas the 10A alleles do not. These data indicate that genetic selection is maintaining three different functional alleles of KIR2DL4 that differ in their





membrane expression and secretion properties. Given its potential to interact with HLA-G and stimulate IFNg in inflamed tissues, the genetic polymorphism may be a mechanism for diversifying the nature of immune responses.





B089Perforin mediated anti-cryptococcal activity requires PI3 kinase and ERK1/2. Jeremy C Wiseman 1 , Kaleb Marr1 and Christopher H. Mody1, 2. Departments of 1Medical Science and 2Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta.

Natural killer cells are important in the innate defense against malignancy, viral infections and intracellular bacterial pathogens. They are also appreciated for their ability to mediate anti-fungal activity. NK cells have been observed to interact directly with and kill the opportunistic fungal pathogen C. neoformans through a granule mediated perforin dependent mechanism. The signaling initiated upon contact and the receptor responsible for adhesion remain elusive. Signaling of perforin dependent natural cytotoxicity against tumor targets by NK cells relies on PI3 kinase and ERK1/2 dependent pathways. In this report we sought to determine if such pathways are activated and required during the interaction between C. neoformans and NK cells. Using the NK-like cell line YT, we demonstrate cryptococcal killing by YT cells, that contact between YT cells and C. neoformans results in the activation of PI3 kinase as indicated by immunoblot for Akt phosphorylation, and that pharmacological inhibition of PI3 kinase or siRNA mediated gene silencing of the 85 kDa subunit of PI3 kinase significantly inhibited observed killing of C. neoformans. Downstream of PI3 kinase ERK1/2 is activated following contact with C. neoformans, as indicated by immunoblot for dual tyrosine/threonine phosphorylation. ERK 1/2 is also required for cryptococcal killing, as inhibition of ERK1/2 activity with the pharmacological inhibitor U0126 resulted in significantly inhibited killing. Furthermore, we demonstrate that perforin release is stimulated by 4h following contact of YT cells with C. neoformans and that release of perforin is blocked by pharacological inhibition of both PI3 kinase and ERK. Taken together these data would suggest that NK cells signal perforin dependent killing of C. neoformans through a PI3 kinase - ERK1/2 dependent pathway.





B090Synthetic TLR agonists potently activate cell-mediated immunity in cutaneous T cell lymphoma patients. Maria Wysocka, Sarah Newton, Bernice M. Benoit and Alain H.Rook. University of Pennsylvania, Department of Dermatology, 245 CRB, 415 Curie Boulevard, Philadelphia, PA 19104, USACutaneous T cell lymphomas (CTCL) are a heterogenous group of lymphoproliferative disorders caused by clonally derived, skin-invasive T cells. The advanced form of CTCL, Sezary syndrome (SS), represents the leukemic form of the disease with malignant T cells disseminated into the bloodstream. Progression to SS is associated with a decrease in cell-mediated immunity, including a decrease in the production of TH1 type cytokines such as IFN- or IL-2 and with increased production of TH2 type cytokines such as IL-4 and IL-5. Clinical data has consistently shown that CTCL patients benefit much more from therapies involving biologic response modifiers than from conventional treatment such as chemotherapy or radiotherapy, suggesting that the host immune system plays an integral role in controlling and containing the disease. In recent years, the list of potent immune response modifiers has been systematically expanded as compounds targeting Toll-like receptors (TLR) become known and available. Stimulation of TLRs, present on cells such as DC or monocytes, trigger through their ligands signal transduction pathways eventually responsible for the initiation of innate immune responses and further development of antigen-specific acquired immunity. We investigated the biological effects of TLR7, TLR8 and TLR9 synthetic agonists on in vitro responses of PBMC from CTCL patients. TLR7- and TLR8-dependent responses were stimulated by synthetic imidazoquinolines while TLR9 was activated by synthetic oligodeoxynucleotides with CpG motifs (CpG ODN). TLR7 ligands significantly induced high levels of IFN-, while the TLR 8 ligands induced IL-12, IL-15 and IFN-. These cytokine-inducing effects were associated with marked activation of NK and CD8 T cells shown by CD69 upregulation and cytolytic activity. Stimulation of patients’ PBMC with CpG-A, a TLR9 agonist, also resulted in marked induction of IFN- release. Consequently, significant activation of NK cells and CD8 T cells occurred as assessed by up-regulation of CD69 expression and by natural killer cytolytic activity. Nevertheless, the PBMC of patients exhibited blunted responses to CpG-A compared to normal volunteers. In such cases, IL-15 was capable of inducing levels of NK activation that were superior to CpG-A, while the combined effects of CpG-A plus IL-15 induced maximal activation of NK cells and further enhanced activation of CD8 T cells. Such pre-clinical findings have typically been





associated with significant clinical improvement when put into clinical practice and therefore have important implications for the potential enhancement of anti-tumor immunity among patients with advanced CTCL.





B091How diverse human KIR phenotype repertoires are modulated by HLA class I interactions: comparison with analogous system of murine Ly49 receptors. Makoto Yawata 1 , Nobuyo Yawata1, Fotini Partheniou2, Ann-Margaret Little2,3 and Peter Parham1. 1Stanford University, School of Medicine, Dept of Structural Biology, Stanford, CA 94304 USA, 2Histocompatibility Laboratories, The Anthony Nolan Trust, London, UK, 3Dept of Haematology, The Royal Free Hospital, London, UK.

KIR phenotypes are highly diverse between human individuals. Expression of KIR on the CD56+CD3- lymphocyte subset was studied in 120 healthy, unrelated donors by flow cytometry, utilizing four anti-KIR antibodies EB6, DX27, DX9 and DX31.Median fluorescence intensity [MFI] and frequency of expression within the NK cell subset [%NK] of KIR in each individual were analyzed in conjunction with HLA class I and KIR alleles. Detailed analyses revealed many analogies between human KIR and murine Ly49 expression.First, allele specific expression was clearly observed for KIR3DL1, where alleles of 3DL1 defined distinct ranges in expression level (MFI) and expression frequency (%NK). KIR3DL2 also displayed allele dependent phenotypes in MFI. Second, independent expression of KIR alleles created distinct bimodal peaks in anti-3DL1 antibody staining on NK cells in heterozygotes carrying combinations of high and low binding alleles of 3DL1. Third, presence of the cognate ligand modulated KIR expression. KIR2DL1 was expressed at higher frequencies within the NK cell subset in the presence of group 2 HLA-C. Limited to the subset of donors with two copies of high expression alleles of 3DL1, a combination with strong inhibitory potential, expression frequencies of 3DL1 were significantly higher in donors with HLA-Bw4 alleles, the cognate ligand. Fourth, the presence of more class I ligands in addition to the cognate ligand, affected KIR phenotypes as well. This was evident in the cumulative decrease in expression frequency which was most prominent for 3DL1 and 2DL3, again limited to the subset of donors homozygous for high expression alleles of 3DL1. That these latter two phenomena were limited to the subset of donors carrying KIR combinations with strong inhibitory potential, implies that human KIR repertoires are likely formed through modes analogous to the receptor acquisition model described for Ly49 during murine NK cell development, where self-inhibitory receptors are sequentially or stochastically expressed on the NK cell surface until cumulative inhibition reaches a threshold whereupon further receptor acquisition is terminated. As KIR and HLA genes reside on different chromosomes, they are inherited independently, in cases giving rise to receptor-ligand combinations resulting in unfavorable levels of inhibition in an individual, thus necessitating mechanisms





allowing for adjustment in levels of NK cell inhibition. Our study has revealed presence of these mechanisms for the KIR system in human, which had previously been difficult to establish confounded by extensive allelic diversification in both KIR and HLA.





B092HLA class I polymorphisms drive selection of KIR distribution in human populations. Nobuyo Yawata 1 , Makoto Yawata1, Monia Draghi1, Fotini Partheniou2, Ann-Margaret Little2,3 and Peter Parham1. 1Stanford University, School of Medicine, Dept. of Structural Biology, Stanford, CA 94305 USA, 2Histocompatibility Laboratories, The Anthony Nolan Trust, London, UK, 3Dept. of Haematology, The Royal Free Hospital, London, UK.Polymorphic HLA class I molecules regulate NK functions. KIR, especially those with inhibitory function, play a major role in the recognition of HLA in this context where they must maintain self-tolerance while avoiding immune-suppression. The extensive population diversity of HLA alleles poses a challenge in this framework, as not all HLA alleles are recognized by human KIR. We investigated whether and how HLA polymorphisms in the world populations affect KIR distribution. KIR alleles were defined by sequencing KIR transcripts and SSP-PCR typing in a panel of 120 Japanese donors. Locus and allele frequencies were analyzed in conjunction with HLA class I alleles. Results were compared with KIR/HLA distributions in Caucasian and other populations. HLA allele distributions in Japanese and Caucasians are contrasting in the context of KIR recognition where Japanese tend to inherit less ligands for KIR. Group 1 HLA-C (HLA-C1) and HLA-B alleles with Bw4 motifs are the mainstay for KIR-mediated inhibition in Japanese whereas Caucasian individuals tend to have more ligands for KIR, namely group 2 HLA-C (HLA-C2) and HLA-A3/-A11. This pattern was reflected in KIR distributions favoring gene content encoding stronger inhibition in Japanese: the group A haplotype characterized by high content of inhibitory receptors, KIR alleles with higher levels of expression and stronger inhibitory potential. Of special note were distinct correlations between HLA-C2 and KIR. Donors with HLA-C2 alleles in this panel tended to associate with truncated variants of 2DL4, a striking relationship that was extrapolated to Caucasoid populations as well.In a comparison among world populations, incidence of HLA-C2 alleles correlated with KIR haplotypes lacking the KIR2DL1 locus. As human KIR haplotype structures infer that essentially all individuals carry inhibitory receptors for HLA-C1 (2DL2/3), the presence of HLA-C2 alleles potentiates an additional level of NK cell inhibition, likely driving selection avoiding KIR haplotypes carrying its cognate receptor KIR2DL1. This paradigm apparently extends to other KIR loci, such as 2DL4, in these individuals.





Thus, KIR-HLA correlations imply ongoing selection in the human populations: increased inhibitory KIR with stronger functions prevalent in populations with less HLA ligands, and vice versa. The associations observed for HLA-C and -G, both KIR ligands expressed in the placenta, suggest the importance of adequately regulating the activity of uterine NK cells at the feto-maternal interface for successful reproduction: under-inhibition of uterine NK cells causing miscarriage and over-inhibition resulting in pre-eclampsia.





B093Stage-dependent Gene Expression Profiles during Natural Killer Cell Development. Suk-Ran Yoon1, Hyung-Sik Kang2, Mira Chung1, Eun-Mi Kim2 and Inpyo Choi1. 1Laboratory of Immunology, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Taejeon 305-333, Republic of Korea. 2School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea

Natural killer (NK) cells develop from hematopoietic stem cells (HSCs) in the bone marrow (BM). During development, NK cells acquire a set of surface molecules, which are transiently regulated in a stage-specific manner. Although recent progresses have been made to decipher the molecular events in NK cell development, but NK cell ontogeny is not completely understood. To understand molecular regulation of NK cell development, serial analysis of gene expression (SAGE) was applied to HSCs, NK precursor (pNK) and mature NK cells (mNK) cultured without (-OP9) or with (+OP9) stromal cells, OP9. From 170,464 total individual tags from four SAGE libraries, 59657unique transcripts and 35385 unique genes were identified. Among 59,657 distinct transcripts, 77.9% were single copy, 16.8% were between two and four copies, 3.2% were between five and nine copies, 1.9% were between ten and ninety nine copies, and only 0.2% of tags were present in more than 100 copies. The genes were clustered into four groups according to expression profiles as HSCs were differentiated into mNK cells. A set of genes was expressed in a stage-specific manner; 15 genes in HSC, 30 genes in pNK, and 27 genes in mNK cells. Among them, lipoprotein lipase induces NK maturation and cytotoxicity activity. Identification of genome-wide profiles of gene expression in different stages of NK cell development affords us a fundamental basis for defining the molecular network during NK cell development.





B094Mouse dendritic cells mediated NK cell activation. Ivan Zanoni, Maria Foti, Paola Ricciardi-Castagnoli and Francesca Granucci. Department of Biotechnology and Bioscience, University of Milano-Bicocca, P.zza della Scienza 2, 20126, Milan, Italy

Dendritic cells (DCs) have an important role in the activation of natural killer (NK) cells that exert a direct anti-tumor and anti-microbial effect and can influence the development of adaptive T cell responses. We have identified the molecular mechanisms responsible of DC-mediated NK cell activation during a bacterial infection. Two soluble molecules produced by bacterially activated myeloid DC are required for optimal priming of NK cells. Type I interferons (IFNs) promote the cytotoxic functions of NK cells and IL-2 is necessary, both in vitro and in vivo, for the efficient production of IFN, which, in turn, has an important anti-bacterial function.Furthermore, we have investigated the nature of the stimuli that, other than bacterial stimuli, confer to DCs NK cell activating capacity. After exposure of DCs to Toll like Receptor (TLR)-dependent and independent microbial stimuli and to non-microbial stimuli we have evaluated the ability of activated DCs to elicit IFN production from NK cells in vitro and to promote NK cell activation in vivo. We demonstrated that only TLR-dependent microbial stimuli typically associated with Th1 responses enable DCs to activate NK cells in an IL-2-dependent manner, while stimuli associated with Th2 responses do not show this property.





B095Chimeric NK receptor-bearing T cells mediate anti-tumor immunotherapy. Tong Zhang, Bethany A. Lemoi, and Charles L. Sentman. Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH, 03756 USA

NKG2D is an activating cell surface receptor expressed on natural killer (NK) cells and some T cell subsets. Its ligands are primarily expressed on tumor cells. The aim of this study was to determine whether chimeric NK receptor bearing T cells would directly kill tumor cells and lead to induction of host immunity against tumors. Chimeric NK receptors were produced by linking NKG2D or Dap10 to the cytoplasmic portion of the CD3 chain. Our results showed that chNKG2D-bearing T cells responded to NKG2D ligand-bearing tumor cells (RMA/Rae-1, EG7) but not to wildtype tumor cells (RMA). This response was dependent upon ligand expression on the target cells but not on expression of MHC molecules, and the response could be blocked by anti-NKG2D antibodies. These T cells produced large amounts of Th1 cytokines and proinflamatory chemokines and killed ligand-expressing tumor cells. Adoptive transfer of chNKG2D-bearing T cells inhibited RMA/Rae-1 tumor growth in vivo. Moreover, mice that had remained tumor-free were resistant to subsequent challenge with the wildtype RMA tumor cells, suggesting the generation of immunity against other tumor antigens. Taken together, our findings indicate that modification of T cells with chimeric NKG2D receptors represents a promising approach for immunotherapy against cancer.

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