Ph.D. Thesis Tolerogenic Dendritic Cells: Immunomodulation of Monocyte-Derived Dendritic Cells with n-Butyrate, NFκB Inhibitor PDTC and JAK3 Inhibitor WHI-P-154 Peter Gyorgy Kelemen, M.D. Semmelweis University, School of Ph.D-Studies Doctoral School: 7. Molecular Medicine Doctoral Program: 7/5. Basic and Clinical Immunology Consultant: Prof. Peter Gergely, M.D. Reviewers: Eva Pocsik, Ph.D. and Zoltan Prohaszka, M.D., Ph.D. Chairman of the Final Examination Committee: Prof. Bela Fekete, M.D. Members: Miklos Benczur, M.D., Ph.D. and Gabriella Sarmay, Ph.D. 2005
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Ph.D. Thesis
Tolerogenic Dendritic Cells: Immunomodulation of Monocyte-Derived Dendritic Cells with
n-Butyrate, NFκB Inhibitor PDTC and JAK3 Inhibitor WHI-P-154
Peter Gyorgy Kelemen, M.D.
Semmelweis University, School of Ph.D-Studies Doctoral School: 7. Molecular Medicine
Doctoral Program: 7/5. Basic and Clinical Immunology Consultant: Prof. Peter Gergely, M.D.
Reviewers: Eva Pocsik, Ph.D. and Zoltan Prohaszka, M.D., Ph.D.
Chairman of the Final Examination Committee: Prof. Bela Fekete, M.D. Members: Miklos Benczur, M.D., Ph.D. and Gabriella Sarmay, Ph.D.
2005
TABLE OF CONTENTS
TABLE OF CONTENTS ................................................................................................. 1
TABLE OF FIGURES ..................................................................................................... 3
A.) „Bacterial Metabolite Interference with Maturation of Human Monocyte-Derived Dendritic Cells”.......................................................................23
1.) Phenotypical and functional impairment of DC maturation by n-butyrate.............23
B.) „Hyporesponsiveness in Alloreactive T-Cells by NFκB Inhibitor-Treated Dendritic Cells: Resistance to Calcineurin Inhibition” ........................................27
1.) Arrest in DC maturation by treatment with the NFκB inhibitor PDTC..................27
2.) PDTC-modulated DC exhibit defective stimulatory capacity for allogeneic
3.) Induction of allogeneic hyporesponsiveness by PDTC-modulated DC..................31
4.) CsA does not counteract the suppres sive state induced by PDTC-modulated DC 32
5.) T-cell hyporesponsiveness with PDTC-modulated DC in T cells from patients
with renal allografts under calcineurin inhibitor-based immunosuppression.........34
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C.) „Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3” .....................................36
1.) Targeting JAK3 disrupts CD40-triggered DC maturation ......................................36
2.) JAK3 inhibition does not interfere with the activation-associated
down-regulation of the DC-antigen uptake machinery ..........................................36
3.) Prevention of T-cell stimulatory capacity and induction of antigen-specific
hyporeactivity by JAK3 inhibited DC....................................................................39
4.) Effect of JAK3 Inhibitor Treatment on Homotypic T-Cell Aggregation................41
SUMMARY OF THE RESULTS....................................................................................42
Ab antibody Ag antigen APC antigen-presenting cells CCR chemokine receptor CD cluster of differentiation CsA Cyclosporine A CTL cytotoxic T lymphocytes DC1 myeloid-related dendritic cells DC2 lymphoid-related dendritic cells DC dendritic cells DC-LAMP DC-lysosome-associated
culture PTK protein tyrosine kinase rh recombinant human SCFA short-chain fatty acid SCF stem cell factor SCID severe combined
immunodeficiency SD standard deviation STAT signal transducers and
activators of transcription T regs regulatory T cells TCR T-cell receptor TGF-β transforming growth
factor-β Th1 T helper 1 Th2 T helper 2 TLR Toll-like-receptor TNF-α tumor necrosis factor-α
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INTRODUCTION
Dendritic Cells
The onset of an immune response requires not only antigen (Ag) and
lymphocytes, but also a third-party cell to present antigens to the lymphocytes. Long
recognized as a requirement for so-called accessory or feeder cells in vitro, the exact cell
type remained elusive until dendritic cells (DC) were first purified and distinguished
from other antigen-presenting cells (APC) in mice, such as B lymphocytes and
macrophages 1,2-4. DC were originally discovered in mice a little more than a quarter
century ago 1,3-4, but only in the past decade has it become feasible to generate either
animal or human DC in sufficiently large numbers and purity for large scale
experimental and clinical investigation.
DC are uniquely specialized among all leukocytes to couple the presentation of
antigen, bound to major histocompatibility complex (MHC) molecules, with all the
adhesive and costimulatory signals, collectively termed accessory molecules, required
to initiate cellular immune responses 5. Unlike B cells that recognize soluble or native
antigen and transform into antibody (Ab)-secreting plasma cells, T-cell receptors
(TCRs) can only recognize peptide fragments of antigen bound to MHC molecules on
APC. MHC molecules are of two types, class I MHC and class II MHC. For the most
part, class I MHC molecules bind intracellular or endogenous antigens that have been
cut into peptides in the cytosol. Class II MHC molecules bind extracellular or
exogenous antigens that have entered the endocytic pathway of the APC. Class I and II
MHC-peptide complexes stimulate CD8+ cytotoxic T lymphocytes (CTL) and CD4+
helper T cells, respectively. DC also have a unique capacity to capture exogenous
antigens from dying cells, apoptotic or necrotic tumor cells, virus-infected cells and
immune complexes, which then access the class I MHC pathway for Ag presentation 6-8. This pathway, called cross-presentation, permits DC to elicit CD8+ as well as CD4+
T-cell responses to exogenous antigens. Cross-presentation is linked to specific DC
antigen uptake receptors, which may be targeted in strategies to load exogenous
antigens onto both MHC I and II. DC also interact directly with B cells and
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lymphocytes of the innate immune system. Activated myeloid DC can directly induce
B-cell proliferation, immunoglobulin isotype switching, and plasma cell differentiation.
Activated lymphoid DC can induce the differentiation of CD40-activated B cells into
plasma cells. DC can also activate and induce the expansion of resting natural killer
cells. Activated natural killer cells can kill immature, but not mature DC and can
stimulate DC to induce protective CD8+ T-cell responses. Lipid and glycolipid antigens
expressed on pathogens or self tissues are presented by DC to T cells on CD1 molecules
(CD1a-d), which are structurally similar to MHC I but specialized to bind lipids instead
of peptides. Processing of lipid antigens onto CD1 molecules is carried out in
specialized intracellular compartments, much like antigen processing onto MHC II.
CD1 molecules present lipid antigens to a variety of lymphocytes, including T cells with
substantial T-cell receptor diversity as well as relatively invariant natural killer T cells.
While DC express abundant MHC molecules of both types, they also constitutively
express large numbers of accessory molecules. The inducible increase in expression of
these adhesive and costimulatory molecules coordinates with maturation and activation
stimuli in culture. This mimics the in vivo capture of Ags by DC in the periphery,
followed by their maturation and migration to secondary lymphoid organs, where DC
complete their activation while stimulating T cells 9-13. It is this pairing of Ag/MHC
presentation with potent accessory function that enables DC to stimulate T cell
immunity without additional adjuvants. DC have therefore often been termed “nature’s
adjuvants” or “professional APC.”
DC could be regarded as a multilineage system of leukocytes with variable
function rather than as a homogenous cell type with predetermined functional properties.
Because the DC systems a whole can present Ags in an immunogenic or tolerogenic
fashion, it is possible that the outcome of an immune response initiated by DC depends
on a combination of several factors, including Ag presentation by a specific type of DC,
the nature of the DC-activating signal, and the stage of DC maturation. Successful
cytokine-driven growth and differentiation of DC from defined precursors has revealed
that DC comprise at least two types, myeloid and plasmacytoid or lymphoid, of which
the myeloid DC comprise at least two subsets (Figure 1) 14. There is also a spectrum of
differentiation within these divisions, from marrow-derived clonogenic progenitors,
to circulating precursors in blood and lymphatics, to immature DC resident in peripheral
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Figure 1: Hematopoietic development of human dendritic cells.
All human DC develop originally from CD34+ hematopoietic progenitor cells (HPC) from either bone marrow, cord blood, or cytokine elicited peripheral blood stem cells. Expansion is supported in vitro by factors like FLT-3-ligand (FL) and c-kit-ligand or stem cell factor (KL/SCF), with additional contributions from either granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3). GM-CSF supports myeloid DC expansion and differentiation, whereas IL-3 supports that of lymphoid-related DC. Tumor necrosis factor-alpha (TNF) exerts pleiotropic effects, recruiting transferrin receptor positive CD34+ HPC into cell cycle, at least partially suppressing granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) receptors early in CD34+ HPC differentiation, and later supporting terminal DC maturation. Additional cytokines like interleukin-4 (IL-4) and transforming growth factor-beta 1 (TGF) suppress CD14+ macrophage differentiation. TGF supports the differentiation of Langerhans cells (LC). DC precursors as represented here are no longer in cell cycle. Activated LC continue to express Langerin, but none of the myeloid DC express this antigen. CD1a is expressed by all myeloid DC types 14.
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tissues, to mature or maturing forms in the thymus and secondary lymphoid organs 15-16.
The two types of myeloid DC are Langerhans cells in epithelial surfaces and dermal or
interstitial DC in the dermis of skin or interstitium of solid organs. C-fms-like tyrosine
kinase ligand (FLT-3 ligand) is a hemopoietic cytokine whose receptor, FLT3, is
expressed on pluripotent stem and progenitor cells. Proceeding from original studies in
mice, FLT-3L also mobilizes both myeloid and lymphoid DC in vivo in humans 17-18.
FLT-3L can increase circulating human myeloid DC by a mean 44-fold and lymphoid
DC by a mean 12-fold, with little toxicity 18. Physical methods can then enrich the DC
from leukopheresis products 19-22. Evidence best supports the direct differentiation of LC
from CD34+ hematopoietic progenitor cells (HPC) 23-27, whereas interstitial or dermal
DC develop from CD34+ HPC via a CD14+ bipotential intermediate 28-31. This CD14+
intermediate is not clonogenic and is termed bipotential because it can alternatively
develop into macrophages instead of DC 29-30. The latter DC type is usually considered
the homologue of the peripheral blood monocyte-derived DC (moDC). CD14+ blood
monocyte cultures require IL-4 in addition to GM-CSF in order to suppress macrophage
differentiation 32 and develop into moDC. LC move into the T cell-rich parafollicular
areas where they are especially likely to encounter naive T cells. In these sites they are
termed interdigitating DC. Although LC have usually been thought of as immature DC,
a new specific marker, Langerin 33-34, still distinguishes some of the mature, activated LC
from the other myeloid interstitial or dermal dendritic cells. They have unique
intracellular organelles called Birbeck granules. Interstitial or dermal DC migrate into
germinal centers where they are termed germinal center DC (GCDC). GCDC present
processed antigen to memory T cells and additionally participate in humoral immunity
by direct interaction with germinal center B cells and indirectly by stimulation of CD4+
T cells that provide cognate help for B cell differentiation 31,35.
In human blood DC are traditionally divided into two populations by staining
with antibodies to CD11c and CD123 (interleukin 3 receptor α [IL-3Rα]). D11c+CD123lo
DC have a monocytoid appearance and are called myeloid DC, whereas CD11c-CD123hi
DC have morphologic features similar to plasma cells and are thus called plasmacytoid
or lymphoid DC. Recently, the terms DC1 and DC2 have been used to distinguish
myeloid DC (DC1) from lymphoid DC (DC2), based on the propensity of each type
to stimulate T helper 1 (Th1) versus T helper 2 (Th2) responses, respectively 36.
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The myeloid DC1 refers to the moDC or its counterpart in the CD34+-derived system,
the dermal or interstitial DC. The term DC1 was not intended to encompass LC, although
LC are also myeloid and meet many of the criteria for DC1. Most investigators are
focusing on the myeloid DC and/or DC1 to stimulate acquired antiviral and antitumoral
immunity, as these DC stimulate Th1 responses that support CTL generation. DC2
precursors (pDC2) are critical effectors of the innate immune system as they rapidly
produce large amounts of interferon-α (IFN-α, type I IFN) in response to enveloped
viruses, bacteria, and tumor cells, and upon terminal differentiation become professional
antigen-presenting type 2 DC for stimulation of Th2 responses 37-39. The tolerizing role
that DC2 may play, either directly or indirectly, is of considerable interest, but is
more pertinent to transplantation and autoimmunity than to the generation of CTL
against viruses and tumors 40-42.
DC are most likely to encounter and capture Ags in the periphery, leading to
their maturation and migration via afferent lymphatics into draining lymphoid organs.
This culminates in the final activation of DC as they stimulate incoming clones of
Ag-specific naive or resting memory T cells, which then exit to function as producers of
helper cytokines or as CTL in the periphery. Antigen capture and processing versus
antigen presentation and T cell stimulation are processes that are dynamically related
to the maturation state of DC 43-44. Mature DC are more stimulatory of Ag-specific
T cells than are immature DC. Chemokine receptors are also regulated in relation to
maturation and support trafficking to secondary lymphoid organs 45-48. Chemokines are
a group of structurally related polypeptides that have been recognized recently to have
critical roles in the selective recruitment of leukocyte subsets to secondary lymphoid
organs and to sites of inflammation. Upon exposure to maturation signals DC undergo
a chemokine receptor switch: DC down-regulate inflammatory chemokine receptors
(CCR1, CCR2, and CCR5) followed by induction of CCR7. Toll-like receptors (TLRs),
which are type I integral membrane glycoproteins, are highly conserved microbial
pattern recognition receptors. TLRs recognize bacteria, viruses and parasites and trigger
DC maturation and secretion of numerous chemokines and cytokines. Cytokine
production by DC might be crucial to induce T-cell immunity. It is well known that
fully mature DC produce large amounts of the proinflammatory cytokines IL-12p40 and
the bioactive form p70, TNF-α, IL-1β and IL-6 and nitric oxide. The production of IL-2
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by DC is required to induce T-cell priming and might represent a switch from tolerance
to immunity. Signals that induce cytokine production by DC and full maturation are
associated with microbial recognition as represented by the recognition of evolutionarily
conserved pathogen-associated molecular patterns (PAMPs) by pattern recognition
receptors (PRRs). Such receptors are represented by the recently discovered Toll-like
receptors (TLRs) on DC or indirect recognition through complement and/or omplement
receptors, or antibody and/or Fc-receptor. IL-12-inducing LPS is recognized by TLR4
and might be a prototype for full DC activation. In humans, myeloid DC express TLRs
1 through 5 and, depending on the subset, TLR 7 and/or 8. Human lymphoid DC
express TLRs 1, 7, and 9. Some TLRs act at the cell surface, whereas others such as
TLRs 3, 7, 8, and 9 are found within endosomes and are presumably activated following
capture and internalization of pathogens or their products. Additional stimuli in vitro
ensure that differentiated DC maintain phenotype and function rather than revert to
immature forms or follow alternative differentiation pathways upon cytokine
withdrawal. These stimuli can be in the form of bacterial or viral components
{such as lipopolysaccharide (LPS), unmethylated cytosine poly-guanine (CpG) motifs,
doublestranded RNA products} and pro-inflammatory cytokines {e.g., IL-1β, IL-6,
TNFα, prostaglandin E2 (PGE2), type I IFN α/β} or interactions with molecules of
the TNF receptor family {i.e. CD40, receptor activator of nuclear factor-κB (NF-κB),
TNF-receptor} on the DC surface with their ligands during cognate T cell–DC
interaction 49,50-53. Cytokines secreted by activated macrophages can also support
terminal and irreversible differentiation of monocyte-derived DC 54. However,
standardization between donors can be difficult, leading some investigators to prefer a
recombinant cytokine cocktail that reproduces the effects of macrophage-conditioned
medium 53. CD40-ligand (CD40L or CD154) is also a powerful stimulus for DC, as
evidenced by an activated phenotype, increased T cell stimulatory capacity, and
secretion of IL-12 9-10,55. The importance of CD40 in T cell activation is suggested by its
localization in the immunological synapse where it is found along with MHC II.
CD40 is different than other costimulatory molecules in that it acts bidirectionally.
CD40 on the DC stimulates T cells through CD40 ligand and, conversely, CD40L on
the T cell provides a maturation signal through CD40 on the DC. Soluble CD40L
binding to CD40, which is upregulated on mature DC, mimics the full activation of DC
10
accomplished by their initial interactions and crosstalk with CD4+ T cells at the onset of
an immune response 11-13. Although, CD40 triggering alone is unable to induce
IL-12p70 production in vivo. Therefore, only the combination of microbial plus CD40
signals might be optimal inducers for full DC maturation, at least for Th1 responses.
Upon successful maturation of myeloid DC, there are important properties that are
always useful to distinguish them from their less mature precursors and from
other leukocytes. The circumferential cytoplasmic veils become more prominent with
maturation in vitro. Mature myeloid DC lack any significant expression of epitopes
specific to macrophages (CD14, CD115) or lymphocytes (CD3, CD4, CD8, CD19,
CD20, CD16, CD56). Mature DC increase expression of CD83 56, which is still the best
available marker of maturation. CD83, a cell surface molecule involved in CD4+ T-cell
development and cell-cell interactions, can also be detected intracellularly as evidence
of commitment to the DC lineage. Mature, myeloid DC also express p55/fascin, which
is an actin bundling protein 57-58, abundant class II MHC and DC-lysosome-associated
membrane glycoprotein (DC-LAMP). In addition, they upregulate surface T-cell
costimulatory molecules CD40, B7-2/CD86 more so than B7-1/CD80, OX40 ligand,
inducible costimulator (ICOS) ligand 59-60 and those intercellular adhesion molecules
(CD54 and CD58) required for both physical interaction with T cells and assembly of
immunological synapse. CD45RO, nominally a marker of memory T cells, is also a
marker of activated LC and moDC. Interleukin-2 receptor (IL-2R/CD25) is also
expressed by certain activated DC. All of these mature DC are potent stimulators of
T cells. Comparable T cell proliferation in vitro, used as a measure of the stimulatory
activity of an APC population, requires at least 10- to 100-fold more APC such as
B cells, macrophages, or bulk peripheral blood mononuclear cells, than is required of
mature DC stimulators 43-44.
Investigators now have the tools to generate DC for the control and manipulation
of immune responses against human disease. Initial clinical trials of human DC vaccines
are generating encouraging preliminary results both in patients with cancer and in normal
volunteers 22,61-63,64. Important considerations in the design of such human trials include
antigen selection, methods for introducing the antigen into MHC class I and II rocessing
pathways, methods for isolating and activating dendritic cells, and route of
administration.
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Tolerogenic Dendritic Cells and Allograft Transplantation
Dendritic cells (DC) are highly specialized antigen-presenting cells (APC),
which are the most potent inducers of primary T-cell responses 65. Beyond this
immunostimulatory function, they play critical roles in central tolerance and in the
maintenance of peripheral tolerance in the normal steady state. Different models have
been proposed to explain the mechanism(s) by which DC may induce/maintain
peripheral T-cell tolerance. It is proposed that under steady-state (normal) conditions,
the uptake of Ags by immature DC expressing low cell-surface levels of MHC and
T-cell costimulatory molecules may induce tolerance to those peptides presented to
Ag-specific T cells. This prediction is based on the fact that binding of the T-cell
receptor (TCR) on naive T cells to MHC–peptide complexes on the APC surface in the
absence of or with low levels of T-cell costimulation leads to anergy/apoptosis of the
Ag-specific T cells or to generation of T cells with regulatory function (regulatory
T cells). In this model of peripheral tolerance, immature DC transport self-antigens
continuously from peripheral tissues to lymph nodes and spleen. The concept of
migratory immature DC as the keepers of peripheral T-cell tolerance disagrees with the
experimental observation that lymph-borne DC (also known as veiled cells), obtained
by cannulation of lymphatic vessels in the steady state, exhibit signs of maturation in all
animal models investigated so far. Lutz and Schuler have coined the term semi-mature
DC for these steady-state migrating DC 66. Semi-mature DC that migrate spontaneously
from the periphery may exhibit certain levels of expression of surface MHC, T-cell
costimulatory and adhesion molecules, low levels of pro-inflammatory cytokines, IL-10
production, and absence of bioactive IL-12p70 synthesis. The detailed mechanism(s) by
which immature or semimature DC induce specific T-cell tolerance to self- or nonself
antigens is not entirely known. The inherent tolerogenicity of DC offers considerable
potential for therapy for allograft rejection. Certain DC subsets have been shown to
induce T-cell tolerance in vitro and in vivo 67. Thus, CD8a+ DEC205+ DC have been
shown to induce antigen-specific T-cell apoptosis 68, intestinal DC to promote Th2 cells 69 or thymic lymphoid DC to induce T-cell anergy in vivo 70. Furthermore, it has been
reported thatimmature DC rather than eliciting a weak T-cell response are able to confer
a state of immunotolerance by the induction of regulatory T cells (T regs) in vitro
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and in vivo 71-73. The concept has recently emerged that dialogue between T reg cells
and DC is crucial for the regulation of alloimmune responses. Using a human
in vitro model system, it has been shown that immature DC exposed to T reg cells can
increase the expression of inhibitory molecules needed for the tolerogenic activity of
DC, and that tolerogenic DC, in turn, can induce anergy in alloreactive CD4+ T cells,
thus establishing an inhibitory feedback loop. Strikingly, subcutaneous administration
of autologous immature DC induced antigen-specific T-cell tolerance in human
volunteers 74. Likewise, myeloid immature DC have been shown to prolong allograft
survival in vivo 75. These data suggest that the maturation state of DC is critical for the
outcome of an immune response and indicate that administration of immature DC could
be a useful approach for the therapy of several immune-mediated diseases 66,72,76-79.
Allograft rejection is a key problem in the field of organ transplantation. The
principal mechanism underlying the acute rejection of allogeneic tissue/organ grafts is
the vigorous adaptive immune response mounted by recipient T lymphocytes against
donor MHC antigens and donor MHC-derived peptides presented by self-MHC
molecules. Tolerance can be defined as the inability of a host to respond to antigens
without the need for immunosuppressive drugs. In particular, peripheral tolerance,
the ultimate goal in organ transplantation, can be achieved by altering immune
reactivity through Th2-skewing, T-cell deletion or T-cell anergy, and the induction of
regulator T-cell responses. The role of DC in organ transplantation is multifaceted,
because of the coexistence of graft-derived DC from the donor and DC from the
recipient. The role of donor hematopoietic cell microchimerism in the outcome of
organ transplantation has been the subject of intense interest and debate since the
observations that microchimerism could be detected in lymphoid and non-lymphoid
tissues of successful human organ allograft recipients up to many years after
transplantation. These findings prompted the hypothesis that microchimerism provided
an essential basis for organ transplant tolerance. The inflammation triggered by
transplant surgery and necrosis due to ischemia/reperfusion injury is enough to initiate
maturation and migration of graft-resident and graft-infiltrating DC, which migrate as
passenger leukocytes out of the graft into the draining lymphoid tissues. Once in the
lymph nodes or spleen of the recipient, these passenger DC activate alloreactive naive
T cells and initiate acute graft rejection. Stimulation of DC through CD40 also may play
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a role in DC mobilization after transplantation. CD40–CD40L interaction regulates DC
migration from peripheral tissue indirectly through induction of TNF-a secretion by DC.
In the transplantation setting, graft-infiltrating platelets, activated T cells, and mast cells
resident in the transplanted tissue may be possible sources of CD40L.
Following cell or organ transplantation, DC present antigen to T cells via the
direct or indirect pathways of allorecognition. The direct recognition pathway is mainly
involved in acute rejection, while the indirect pathway is closely related to chronic
rejection. However, more data showed that indirect recognition might play a more
important role in whole allograft rejection. In the direct pathway of allogeneic antigen
recognition, donor dendritic cells leave the graft and migrate towards recipient
lymphoid organs here they present allogeneic (non-self) MHC molecules to recipient
T cells. The vigorous T-cell proliferation that occurs in primary allogeneic mixed
lymphocyte culture (MLC) is caused mainly by direct allorecognition and is due to the
high comparative frequency of allospecific responder T cells (approx. 1 per 200). In the
indirect pathway, recipient DC internalize and process donor antigens (soluble MHC
molecules, fragments/blebs derived from donor apoptotic or necrotic cells, vesicles
exchanged between living cells). These are then presented to recipient T cells as a
restricted repertoire of immunodominant allogeneic peptides bound to self-MHC
molecules on the dendritic cell surface. Most allogeneic peptides derive from
hypervariable regions of donor MHC class II molecules, and to a lesser extent MHC
class I or minor MHC antigens. The frequency of recipient T cells specific for
allopeptides bound to self-MHC molecules is similar to the frequency of T cells
responding to nominal antigen peptides (<1 out of 10 000), and significantly lower than
that of T cells engaged in direct recognition.
There are many experimental models with different grafts and approaches to
therapy of allograft rejection by targeting donor or recipient DC (Table 1.). Various
attempts have been made to convert DC into tolerogenic APC such as treatment of DC
with interleukin-10 (IL-10) 80, transforming growth factor-β (TGF-β) or with low doses
of granulocyte–macrophage-colony-stimulating factor (GM-CSF) 81-83. These treatment
modalities have in common the ability to interfere with the proper maturation of DC.
Several studies indicate that the maturational state of DC is primarily controlled
by NFκB, which regulates a multitude of immunomodulatory genes (e.g., MHC class II,
14
DC SOURCE
APPROACH
TYPE OF DC
TRANSPLANT MODEL
Intravenous administration of DC generated in vitro from bone marrow precursors under specific culture conditions
(mouse) Intravenous administration of DC generated in vitro from
bone marrow precursors by pharmacological treatment (i.e. vitamin D3)
Immature myeloid DC and/or DC unable to produce IL-12p70
Skin (mouse) Pancreatic islets (mouse)
Intravenous, intraperitoneal, or intraportal administration Of DC generated from bone marrow precursors (mice) or peripheral monocytes (humans) and genetically engineered
Myeloid DC incubated with oligodeoxyribonucleotides (ODNs) or encoding a
Heart (mouse) Kidney (mouse) Human skin
(i.e. FasL, CTLA4-Ig, IL-10, and NFkB decoy ODN) in vitro
transgenic protein (SCID mouse)
Intravenous administration of specific DC subpopulation CD8α+(lymphoid-related) DC isolated from spleen of Flt3L-treated mice
Heart (mouse)
Total DC (CD8α- andCD8α+) isolated from spleen of Flt3L-treated mice
Aorta (mouse)
Plasmacytoid DC from blood of patients treated with G-CSF or isolated from spleen of Flt3L-treated mice
GVHD(human) Heart (mouse)
Intrathymic or intravenous injection of DC pulsed with donor MHC I peptide
Thymic or myeloid DC
Heart (rat) Pancreatic islets (rat)
Recipient
Pharmacologic manipulation (in vivo administration of deoxyspergualin; intravenous injection of recipient-type DC
Lymph node DC (primates) or myeloid DC
Kidney (nonhuman primates)
generated in vitro under IL-10, TGFβ, LPS) GVHD (mouse) Intravenous injection of DC generated in vitro from
recipient-type transgenic mice encoding donor MHC I allele Myeloid DC
Heart (mouse)
or infected with a rAd encoding a donor MHC I allele Intravenous administration of donor MHC+ apoptotic cells
Splenic DC (CD8α- and CD8α+)
Bone marrow(mouse) Heart (mouse)
Table 1: Approaches to therapy of allograft rejection by targeting donor or recipient DC
CD80, CD86and CD40) 84-85. The inhibition of NFκB activation not only hampers
the expression of costimulatory, maturational and major histocompatibility complex
(MHC) molecules 85-86, but is also involved in the induction of T-cell hyporeactivity.
Thus, DC treated with vitamin-D3, corticosteroids or proteasome inhibitors, which are
known to prevent nuclear translocation of NFκB, have been reported to induce a state of
T-cell tolerance both in vitro and in vivo 87-89. Likewise, significant prolongation of
organ allograft survival was achieved when allogeneic DC harboring NFκB decoy
oligonucleotides were administered 90-92. The inhibition of NFκB activity might then
disable also full maturation of DC, as alloreactive T cells are not activated by such DC,
which leads to an arrest of DC at an immature stage with low costimulatory molecule
expression, down-regulated stimulatory capacity and suppressed cytokine production
because of a lack of helper signal provision by T cells 77.
15
A major step towards the realization of allograft tolerance may represent the generation
of defined allogeneic APC facilitating graft acceptance and maintenance of T-cell
hyporeactivity. Several studies indicate that tolerizing APC share particularfeatures such
as low/absent IL-12 production, defective stimulatory capacity and high endocytosis
rate 93. Considering the in vivo application of designer DC defined and reproducible
conditions for their generation are required. Furthermore, administered DC should not
mature upon activating stimuli and the establishment of tolerance should not be
abrogated by conventional immunosuppressive protocols thus enabling their
incorporation into current therapies used to treat allograft rejection.
AIMS OF THE STUDY
Our aim was to generate tolerogenic monocyte-derived dendritic cells by
activating immature DC in the presence of different inhibitory substances in order to
obtain DC that exhibit phenotypical features and cytokine production of immature DC,
as well as defective stimulatory capacity for allogeneic T-cell responses. We aimed to
characterize these in vitro modulated-DC and to analyze the allogeneic tolerance
induced by these APC.
In this study three independent attempts had been made to convert DC into
tolerogenic APC such as treatment of DC with n-butyrate, NFκB inhibitor pyrrolidine
dithiocarbamate (PDTC) and Janus Kinase 3 inhibitor WHI-P-154. These treatments
have in common the ability to interfere with the proper maturation of DC. At first we
planned to use the bacterial metabolite n-butyrate, which occurs physiologically in high
concentrations in the gastrointestinal tract and has well-known anti-inflammatory
effects. Several studies indicate that the maturational state of DC is primarily controlled
by NFκB, which regulates a multitude of immunomodulatory genes. n-Butyrate
profoundly inhibits translocation of NFκB to the nucleus in LPS-stimulated DC,
suggesting that the impairment of nuclear translocation of this transcription factor by
16
n-butyrate may account for most features of the altered DC phenotype. To mimic this
physiologically occurring immunomodulatory phenomenon by n-butyrate we chose the
NFκB inhibitor pyrrolidine dithiocarbamate (PDTC) for the next experimental setting.
These two substances have similar features with regard to their immunomodulatory
capacity of DC via similar mechanism interfering the translocation of NFκB. Our aim
was to study the established T-cell tolerance and the influence of calcineurin inhibition
with Cyclosporine A (CsA) in the allogeneic hyporesponsiveness induced by PDTC-
modulated DC. For further investigation of this T-cell hyporesponsiveness we
established an ex vivo model in T cells from patients with renal allografts under CsA-
based immunosuppression in order to assess the ability of in vitro modulated-DC to
incorporate into a conventional immunosuppressive protocol without abrogation of the
tolerance by the clinical therapy. The third inhibitory substance, which was used in our
experiments is engaged in the CD40 signaling pathway in addition to its involvement in
common-gamma chain (cγ) signaling of cytokine receptors. Our next aim was to assess
the consequences of Janus Kinase 3 inhibition with WHI-P-154 during CD40-induced
maturation of monocyte-derived DC and to test the impact thereof on the induction of
T-cell hyporesponsiveness. At the end we aimed to test the influence of this inhibitory
agent on T cells stimulated by TCR triggering with CD3 and CD28 mAbs in the
presence of the Janus Kinase 3 inhibitor WHI-P-154.
In the current work we aimed to study the followings:
A.) „Bacterial Metabolite Interference with Maturation of Human Monocyte-Derived Dendritic Cells” 94
1.) To investigate how monocyte-derived dendritic cells respond to the bacterial
stimulus (lipopolysaccharide) when applied in the presence of n-butyrate with
regard to phenotypical and morphological changes, T-cell stimulatory capacity
and analysis of the DC antigen-uptake machinery.
17
B.) “Hyporesponsiveness in Alloreactive T-Cells by NFκB Inhibitor-Treated Dendritic Cells: Resistance to Calcineurin Inhibition” 95
1.) To generate tolerogenic monocyte-derived dendritic cells by activating
immature DC in the presence of NFκB inhibitor pyrrolidine dithiocarbamate
(PDTC) in order to obtain DC that exhibit phenotypical features and cytokine
production of immature DC.
2.) To characterize allostimulatory potential of PDTC-modulated dendritic cells in
primary mixed lymphocyte culture, as well as the expression of activation
markers and cytokine production of allogeneic T-cells.
3.) To induce allogeneic hyporesponsiveness by PDTC-modulated dendritic cells
and to analyze this state of allogeneic tolerance after restimulation in secondary
mixed lymphocyte culture.
4.) To study the influence of calcineurin inhibition with Cyclosporine A (CsA) in
functional T-cell responses elicited by different DC pretreatment protocols with
PDTC in primary and secondary mixed lymphocyte culture.
5.) To establish T-cell hyporesponsiveness with PDTC-modulated dendritic cells
in T cells from patients with renal allografts under Cyclosporine A (CsA)-based
immunosuppression.
C.) “Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3” 96-98
1.) To explore the impact of Janus Kinase 3 (JAK3) inhibition with WHI-P-154
on the phenotype of monocyte-derived dendritic cells activated through CD40
engagement.
2.) To evaluate receptor-driven endocytosis, macropinocytosis and mannose
receptor expression of JAK3 inhibitor-treated dendritic cells.
3.) To investigate T-cell stimulatory potential of JAK3 inhibitor-treated dendritic
cells (DC) in primary mixed lymphocyte culture, to induce hyporesponsiveness
after restimulation in secondary mixed lymphocyte culture and to assess the
specificity and reversibility of this hyporesponsive state.
4.) To evaluate homotypical cell clustering of T cells activated with CD3 and CD28
mAbs in the presence of Janus Kinase 3 inhibitor WHI-P-154.
18
MATERIALS AND METHODS
Media and reagents
RPMI-1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 2 mM
(FN50), anti-HLA-DR (IgG2a, L243), anti-CD83 (IgG1, clone HB15e) and anti-CD14
(IgG2b, clone MOP9) were from Becton Dickinson. FITC-labeled anti-CD32 (IgG2b,
clone IV.3) was obtained from Medarex (Annandale, NJ). The following PE-labeled
mAbs (obtained from Becton Dickinson) were used: anti-CD80 (IgG1, L307.4),
anti-CD86 (IgG2b, clone IT2.2) and anti-mannose receptor (IgG1, clone 19).
For measurement of cytokines, supernatants were harvested at the indicated time-points.
IL-2, IFN-γ, TNF-α and IL-12p40 were measured by sandwich ELISA using matched
pair antibodies. Capture as well as detection antibodies were obtained from R and D
Systems. Antibodies to human TNF-α were from PharMingen (San Diego, CA, USA).
Standards consisted of human recombinant material from R and D Systems.
Endocytosis assay
To determine mannose receptor (MR)-mediated endocytosis, 1x106 cells/ml were
incubated in medium with FITC-labeled dextran [molecular weight (Mr) 40,000; Sigma
Chemie GmbH Co.] at a concentration of 1 mg/ml. After an incubation period of 60 min
at 37°C or on ice as a control, cells were washed extensively with ice-cold phosphate-
buffered saline (PBS) and analyzed on a FACScalibur. Fluid-phase endocytosis was
measured via cellular uptake of lucifer yellow (LY; Sigma Chemie GmbH Co.) and was
analyzed by flow cytometry.
Allogeneic mixed lymphocyte culture (MLC) and tolerance assays
Stimulator cells as indicated were irradiated (3000 rad, 137Cs source) and added
at the indicated cell numbers to 1x105 allogeneic T cells in 96-well culture plates in
RPMI-1640 medium supplemented with 10% FCS (total volume 200 µL/well). After 4
days, cells were pulsed with 1 µCi [3H]-thymidine (ICN Pharmaceuticals, Irvine, CA).
After another 18 h, the cells were harvested on glass-fiber filters (Packard, Topcount,
Meriden, CI, USA) and DNA-associated radioactivity was determined using a
microplate scintillation counter (Packard). DNA synthesis was expressed as mean cpm
of triplicate cultures. To assess cytokine production in MLC, DC were cocultured with
1x106 purified T cells in 24-well plates. For secondary MLC, first 1x106 purified T cells
were mixed with 1x105 DC subjected to the various treatment protocols (culture volume
21
1 mL in 24-well plates). After washing at day 7, cells (5x104/well) were added to
irradiated DC (5x103/well) from the original donor or unrelated third party donors as a
third-party control in round-bottom 96-well tissue culture plates. Dendritic cells for
restimulation cultures were generated from cryopreserved monocytes and matured by
100 ng/mL LPS for 48 h. For assessment of potential suppressor cells, restimulation
cultures using T cells initially primed with mDC were cocultured with activated DC
from the same donor in the presence/absence of T cells exposed to PDTC-treated DC
stimulated with LPS. For comparison, coculture experiments were performed with
T cells that were incubated with immature DC, immature DC-treated with PDTC or
LPS-treated DC. To evaluate the inhibitory capacity of PDTC-treated DC, PMLC with
T cells and mDC at different ratios were set up in the presence or absence of one of the
respective APC populations. For secondary MLC, 1x106 purified T cells or PBMC
(from renal allografts recipients) were mixed with 1x105 DC subjected to the various
treatment protocols (culture volume 1 mL). After washing at day 7, the cells
(5x104/well) were re-plated in 200 µL culture medium with irradiated cells (5x103/well)
from the original donor or unrelated third party donors as a third party control. DC for
restimulation cultures were generated from monocytes that were cryopreserved
immediately after monocyte isolation. Examination of IL-2 responsiveness of
hyporesponsive T cells was performed in two ways. First, in the respective MLC, 20
U/mL rhIL-2 was added simultaneously with the donor APC to secondary cell cultures.
Second, cells were washed 7 days after initiation of primary MLC, exposed to 20 U/mL
rhIL-2 for 3 days, washed again and then restimulated with the respective APC. DNA
synthesis in secondary MLC was assessed after 3 days. Global T-cell reactivity in
secondary MLC was assayed by stimulation with 10-7 M PMA plus 1 µg/mL CD3 mAb.
Morphological cell analysis
Immature DC were stimulated with LPS in the presence or absence of n-butyrate.
After 4 h, the cells were analyzed by light microscopy on a Leitz Aristoplan microscope
(Wetzlar, Germany). To perform scanning electron microscopy, cells were fixed onto 24
multiwell plates using 2 ml 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for
90 min at 4°C. After rinsing in cacodylate buffer, cells were postfixed with 1% OsO4 for
15 min. The samples were dried in a gradual series of ethanol, transferred to tertiary
22
butanol (Merck, Darmstadt, Germany) for 60 min, and freeze-dried. The samples were
examined in a Stereoscan S90 scanning electron microscope (Cambridge Instruments,
Cambridge, UK). After CD3 and CD28 stimulation (8h) in RPMI 1640 (supplemented
with penicillin (100U/ml), streptomycin (100 µg/ml), glutamine (2mM), and 10% fetal
calf serum) purified T cells were analyzed by light microscopy on a Leitz Aristoplan
microscope (Wetzlar, Germany).
Statistics
Comparisons were performed using the Student’s t-test. A p-value < 0.05 was
considered statistically significant.
RESULTS
A.) “Bacterial Metabolite Interference with Maturation of Human Monocyte Derived Dendritic Cells” 94
1.) Phenotypical and functional impairment of DC maturation by n-butyrate
Because terminal DC maturation critically determines the outcome of
antimicrobial immune responses, we evaluated the impact of n-butyrate on this stage of
the DC life cycle. We first investigated the phenotypical changes in immature DC
exposed to LPS under the influence of this bacterial metabolite. As shown in Figure 2,
addition of LPS to immature DC resulted in the neoexpression of the maturation
markers CD83 and CD25, the α-subunit of the IL-2 receptor, and the upregulation of
major histocompatibility complex (MHC) class I and II and costimulatory molecules.
However, concomitant treatment with n-butyrate yielded DC with a markedly
altered phenotype (Figures 2 and 3). Expression of CD25 and CD83 and of critical
23
costimulatory molecules, i.e., CD40, CD80, and CD86, was reduced substantially.
Furthermore, a clear suppression of the up-regulation of MHC class I and II antigens by
n-butyrate was observed.
Figure 2: n-Butyrate prevents DC maturation.
Monocytes were cultured for 7 days with GM-CSF (50 ng/ml) plus IL-4 (10 ng/ml). Subsequently, these immature DC (5x105/ml) were activated with LPS (100 ng/ml) with or without 1 mM n-butyrate for 48 h. Open profiles (fine line) in the upper panel represent a staining pattern with an isotype control, and open profiles (bold line) represent staining with mAb of the indicated specificity in immature DC before LPS activation. In the lower panel, surface expression results from cultures containing LPS (open profiles with bold line) or LPS plus 1 mM n-butyrate (solid profiles) are depicted. Open profiles (fine line) represent the staining pattern with an isotope control. Data are representative of three independent experiments.
A striking feature of activated DC is the occurrence of large cell clusters arising a few
hours after addition of LPS (Figure 3). In comparison, little or no cell clustering occurs
in cultures activated in the presence of n-butyrate (Figure 3). Moreover, the majority of
cells in the n-butyrate-treated cultures imposed with widespread cytoplasmic
projections. In addition to the reduced expression of DC maturation markers, we found
a significant dose-dependent reduction of the allostimulatory capacity of immature DC
treated with LPS and n-butyrate (Figure 4). A series of experiments confirmed that n-
butyrate does not impede the viability of DC; i.e., we found identical numbers of cells in
cultures treated with or without n-butyrate for 48 h. More importantly, FACS analysis
measuring propidium-iodide uptake confirmed the same number of viable cells in
n-butyrate and nontreated cultures (unpublished results). Thus, the effects of this
24
compound resulted from specific action rather than from unspecific effects, such as cell
death.
Figure 3: Effect of n-butyrate on maturation-associated clustering of DC.
Immature DC were stimulated with LPS (100 ng/ml) in the absence (A, C) or presence (B, D) of 1 mM n-butyrate. After 4 h of cultivation, cells were analyzed by photomicrographs using light microscopy (A, B) or scanning electron microscopy (C, D). The insert in photomicrograph D shows a bulb-like ending of a cytoplasmic projection in n-butyrate-treated cells. Similar results were obtained in four different experiments.
n-Butyrate inhibited the expression of the antigen-uptake molecules CD32 and mannose
receptor (MR). Assessment of macropinocytosis, a special type of actin-dependent
fluid-phase uptake, revealed impaired internalization of lucifer yellow (LY) but
a less pronounced inhibition of receptor-mediated endocytosis of dextran molecules
(Figure 5).
25
Figure 4: Reduced T-cell stimulatory capacity of DC matured in the presence of n-butyrate.
Immature DC were stimulated with LPS (100 ng/ml) with or without n-butyrate at the indicated concentrations. After 48 h, the cells were washed extensively, irradiated, and cocultured with 1x105 allogeneic T cells at the indicated ratios. DNA synthesis was assessed at day 5. Shown are the means ± SE of six to nine experiments. *, P < 0.01.
Figure 5: Analysis of the DC antigen-uptake machinery of DC differentiated in the presence of n-butyrate. Monocytes were cultured for 7 days with GM-CSF (50 ng/ml) plus IL-4 (10 ng/ml) in the absence (Ctrl-DC) or presence (nB-DC) of 1 mM n-butyrate. For assessment of CD32 or MR expression, cells were stained with the respective antibodies and analyzed by flow cytometry. For functional endocytosis assays, cells were pulsed with 1 mg/ml FITC-dextran (DEX) or 1 mg/ml LY for 60 min. Open profiles (fine line) show the background uptake on ice; open profiles with bold line (Ctrl-DC) and solid profiles (nB-DC) indicate antigen uptake of cells at 37°C.
26
B.) “Hyporesponsiveness in Alloreactive T-Cells by NFκB Inhibitor- Treated Dendritic Cells: Resistance to Calcineurin Inhibition” 95
1.) Arrest in DC maturation by treatment with the NFκB inhibitor PDTC
Immature DC and maturation-resistant DC have recently been shown to be
effective inducers of T-cell tolerance in vitro and in vivo 74,83,99. In order to obtain
potentially tolerogenic DC, we aimed to generate DC that exhibit phenotypical features
of immature DC and that are resistant to further activation stimuli. Since triggering
of the DC maturation program represents an irreversible step of DC development,
we employed immature DC that were treated with LPS in the presence of an effective
agent interfering with this activation signal. Because transition of immature DC to the
mature stage is primarily controlled by activation of the transcription factor NFκB 84-
85,100-101 we have chosen the dithiocarbamate NFκB inhibitor PDTC. Dithiocarbamates
such as PDTC have been shown to block NFκB-dependent cytokine production in
human myeloid cells such as monocytes 102-103. As seen in Figure 6A, activation of
immature DC by LPS leads to neoexpression of the DC maturation marker CD83,
upregulation of major histocompatibility complex (MHC) class II antigens and
costimulatory molecules. In contrast, concomitant treatment with PDTC yielded DC
with a markedly altered phenotype. Thus expression of CD83 and of critical
costimulatory molecules such as CD40, CD80 and CD86 was reduced substantially.
Likewise, up-regulation of MHC class II antigens was found to be clearly suppressed by
PDTC. Decreased survival as assessed by propidium-iodide staining was seen only at
doses ≥50 µM (data not shown). Analyzing cytokine production during DC maturation,
we observed a clear inhibition of the production of the immunostimulatory cytokines
IL-12 and TNF-α by PDTC (Figure 6B), which demonstrates the ability of
dithiocarbamate based NFκB inhibition to effectively interfere with the LPS-triggered
process of DC maturation. We further analyzed the ability of PDTC to block other
activation signals. As seen in Figure 7A, PDTC was also effectively abrogating
phenotypical DC maturation initiated by exposure to cross-linked CD40L,
peptidoglycan and TNF-α. Furthermore, also upon other activation stimuli involving
CD40, toll-like-receptor-2 (TLR)-2 and pro-inflammatory cytokine signaling,
27
Figure 6: Treatment with the NFκB inhibitor pyrrolidine dithiocarbamate (PDTC) prevents the phenotypic changes associated with dendritic cell (DC) maturation.
(A) Monocytes were cultured for 7 d with granulocyte–macrophage-colony-stimulating factor (GM-CSF) (50 ng/mL) and interleukin-4 (IL-4) (10 ng/mL). Subsequently, these immature DC (iDC, 5x105/mL) were activated with LPS (100 ng/mL) for 48 h after preincubation with or without 10 µM PDTC for 2 h. Open profiles (fine line) in the upper panel represent the staining pattern with an isotype control and open profiles (bold line) represent staining with mAb of the indicated specificity in immature DC before LPS stimulation. In the lower panel, surface expression results from cultures containing LPS (open profile with bold line) or LPS plus 10 µM PDTC (solid profiles) are depicted. Open profiles (fine line) represent the staining pattern with an isotype control. Data are representative of three independent experiments. (B) DC were cultured as described above and cell-free supernatants were collected 48 h after endotoxin stimulation and analyzed by ELISA. Mean cytokine levels ±SEM obtained from three independent experiments are depicted.*level of significance, p < 0.05 for comparison to cultures in the absence of PDTC.
28
we observed a significant inhibition of cytokine production (Figure 7B). These findings
indicate maturation resistance of such modulated DC, which prompted us to further
evaluate their tolerogenic properties.
Figure 7: PDTC blocks DC activation to various maturation stimuli.
(A) After differentiation to iDC, cells were activated with LPS (100 ng/mL), peptidoglycan (Pg, 10 µg/mL), tumor necrosis factor-α (TNF-α) (20 ng/mL) or CD40L (200 ng/mL) followed by antiflag crosslinking (4.4 µg/mL), for 48 h after pre-incubation with or without 10 µM PDTC for 2 h. Open profiles (fine line) represent the staining pattern with an isotype control and open profiles (bold line) represent staining with mAb of the indicated specificity. Data are representative of three independent experiments. (B) DC were cultured as described above and cell-free supernatants were collected 48 h after respective stimulation and analyzed by ELISA. Mean cytokine levels + SEM obtained from three to five independent experiments are depicted. *level of significance, p<0.05 for comparison to cultures in the absence of PDTC; NA: not applicable because of exogenous TNF-α addition.
29
2.) PDTC-modulated DC exhibit defective stimulatory capacity for allogeneic T-cell responses
To get a first hint towards the immunomodulating properties of PDTC-
modulated DC, we tested their allostimulatory capacity in MLC. As depicted
in Figure 8A, pretreatment of DC with PDTC induced a DC population with a low
stimulatory capacity. Analyzing the production of distinct T cell-derived cytokines, we
found a vigorous production of IL-2 and IFN-γ from T cells stimulated with mature DC.
In contrast, T cells challenged with modulated DC exhibited a dose-dependent
suppression of IL-2 and IFN-γ production (Figure 8B). To get further insight into the
Figure 8: PDTC-modulated DC fail to support full allogeneic T-cell activation.
Immature DC (iDC), LPS-treated DC (mDC) and PDTCmodulated DC stimulated with LPS (PDTC-DC) were generated as described in Materials and Methods and after 48 h the cells were washed and harvested, irradiated, and cocultured with allogeneic T cells (5x105) at the indicated ratios. (A) T-cell proliferation was assessed by DNA synthesis at day 5 and (B) T-cell cytokines were harvested on day 3 (IFN-γ) and 4 (IL-2) after initiation of the MLC and analyzed by ELISA. Results are representative of at least three independent experiments. (C) Analysis of activation marker expression on allogeneic T cells was determined by flow cytometry 48 h after initiation of the MLC. Data are represented as dot blots and percentages in the upper right quadrants indicate percent positivity of the respective markers in CD3-positive lymphocytes. Similar results were obtained in three other independent MLC.
30
activation profile of the responding allogeneic T-cell population, we analyzed surface
molecules indicative of proper T-cell activation such as CD69 and CD25. As shown in
Figure 8C, T-cells challenged with modified DC did not express CD69, similarly
expression of CD25 was profoundly impaired in the respective cell cultures.
To investigate whether PDTC-modulated DC induce T-cell apoptosis, we stained
allogeneic T cells with Annexin V and propidium iodide. After incubation of allogeneic
T cells for 3 and 6 d after initiation of the MLC no increase in cell death as compared
with control MLC was observed (data not shown).
3.) Induction of allogeneic hyporesponsiveness by PDTC-modulated DC
To further determine the impact of the observed lack of allogeneic T-cell
proliferation in primary MLC with PDTC-modulated DC, we analyzed the functional
outcome of T cells challenged with this subset of DC compared with T cells stimulated
with mature and immature DC after restimulation. Therefore, allogeneic T cells were
cocultured with the respective DC for 7 d, washed and incubated with medium alone for
another 24–48 h before they were restimulated. While allogeneic T cells pre-cultured
with immature or mature DC readily responded to antigenspecific restimulation
(Figure 9A), T cells exposed to PDTC-modulated DC did not proliferate upon challenge
with immature or even fully mature DC from both original and third party donor DC
(Figure 9A). In contrast, APC-independent T-cell stimulation with PMA plus OKT-3
was unaffected (Figure 9A). To assess the possibility to revert the hyporesponsive state
exerted by PDTC-modulated DC, rIL-2 was added to secondary MLC. As depicted in
Figure 9B, rIL-2 partially restored proliferative responses towards allogeneic DC in
MLC containing PDTC-modulated DC while it induced an unspecific increase in
secondary proliferative responses. In the absence of T-cell receptor (TCR)-mediated
activation, rIL-2 alone induced a low grade of proliferation in tolerant T cells.
In order to demonstrate specificity for maturation-resistant DC to induce T-cell
hyporesponsiveness, we exposed immature DC to PDTC and then used these cells to
stimulate allogeneic T cells. Interestingly, while such cells induced a T-cell response
comparable to immature DC or maturation-resistant DC in primary MLC (Figure 9D),
secondary stimulation revealed an absence of hyporesponsiveness induction by
31
PDTC-treated immature DC (Figure 9C). To evaluate the possibility of suppressor cells
mediating the observed hyporesponsiveness exerted by PDTC-modulated DC, the
impact of T cells recovered from hyporesponsive T-cell cultures on MLC with the
specific donor cells was analyzed. As shown in Figure 9C, hyporesponsive T cells were
unable to significantly suppress allogeneic activation of syngeneic T cells and allowed
T-cell responsiveness comparable to T cells previously exposed to immature DC or
immature DC treated with PDTC. For the assessment of the inhibitory capacity of
PDTC-treated DC, allogeneic stimulation of T cells with mature DC was performed in
the presence or absence of PDTC-modulated DC. As depicted in Figure 9D such cells
were unable to suppress alloresponsiveness to mature DC from the same donor. In this
respect they behaved similar to immature DC or immature DC exposed to PDTC.
As expected, addition of mature DC resulted in increased stimulation in cultures
set up at a stimulator/responder ratio of 1:40.
4.) CsA does not counteract the suppressive state induced by PDTC-modulated DC
While CsA is the current immunosuppressant for the prevention of renal
allograft rejection, many studies have demonstrated that calcineurin inhibition prevents
the induction of stable tolerance in several experimental models of organ transplantation 104-106. The finding of a profound state of T-cell hyporeactivity by maturation-resistant
DC prompted us to study whether calcineurin inhibition with CsA influences the
functional T-cell responses elicited by different DC pretreatment protocols. Addition of
CsA to primary MLC led to an inhibition of the allogeneic T-cell proliferation
regardless of the mode of APC pretreatment (Figure 10A). However, as shown in Figure
10B,C, the presence of CsA had no influence on the proliferative response of T cells in
secondary cultures when they were stimulated with immature or mature DC in primary
MLC. Interestingly, secondary MLC revealed that the presence of CsA during
alloantigenic priming did not influence the induction of the hyporesponsive state by
PDTC-modulated DC.
32
Figure 9: Induction of allogeneic T-cell hyporesponsiveness by PDTC-modulated DC. Primary MLCs were set up by incubating immature DC (iDC), immature DC plus PDTC (iDC + PDTC), LPS-treated DC (mDC) and PDTC-modulated DC stimulated with LPS (PDTC-DC) with allogeneic T cells as indicated. Seven days after initiation of the primary MLC the cells were washed and restimulated (5x104/well) with irradiated cells (5x103/mL) from the original donor or unrelated third party donors or phorbol myristate acetate (PMA) (1 µM) plus anti-CD3 mAb OKT-3(1 µg/mL). T-cell proliferation was measured after 3d by [3H]-thymidine incorporation for the last 18 h of the culture period. Results are representative of at least three independent experiments. Restimulation cultures were performed in the absence (A) or presence (B) of rIL-2 (20 U/mL). (C) To test for the presence of potential T regs, restimulation cultures using T cells initially primed with mDC (5x104/mL) were cocultured with mDC (5x103/mL) from the same donor in the presence or absence of T cells (5x104/mL) preincubated with one of the four different APC populations. (D) To evaluate the inhibitory capacity of PDTC-treated DC pMLC with T cells (5x104/mL) and mDC were set up in the presence or absence of one of the four different APC populations (5x103/mL).
33
Figure 10: Effect of Cyclosporine A (CsA) treatment on allogeneic hyporesponsiveness induced by PDTC-modulated DC.
Immature DC (iDC), LPS-treated DC (mDC) and PDTC-modulated DC stimulated with LPS (PDTC-DC) were generated as described in Materials and Methods and after 48 h the cells were washed and harvested, irradiated, and cocultured with 5x105/mL allogeneic T cells at an APC/T cell ratio of 1:10 in the absence or presence of 100 ng/mL CsA. Subsequently secondary MLC were set up as described in Figure 8. T-cell proliferation was assessed by DNA synthesis on day 5 in primary MLC and on day 3 in secondary MLC. Results of pMLC (A) and restimulation cultures using cells from pMLC in the absence (B) or presence (C) of CsA are shown. Results are representative of at least three independent experiments.
5.) T-cell hyporesponsiveness with PDTC-modulated DC in T cells from patients with renal allografts under calcineurin inhibitor-based immunosuppression
To further examine the possibility that a state of T-cell hyporesponsiveness can
be established with PDTC-modulated DC in T cells from patients with renal allografts
under CsA-based immunosuppression, freshly isolated T cells were challenged with
various DC subgroups generated from frozen spleen cells of the respective kidney
donors. While the primary MLC response was profoundly reduced using PDTC-
modulated DC (data not shown), a conspicuous state of T-cell hyporesponsiveness was
observed in restimulation cultures supplemented with allogeneic stimulator cells
34
(Figure 11). In contrast, T-cell responsiveness was unimpaired when various polyclonal
stimuli were employed (Figure 11). Similar results were obtained when T cells from
patients with FK506 instead of CsA-based immunosuppression were exposed to in vitro
modulated DC (data not shown). Hence, it is concluded that induction of T-cell
hyporeactivity towards alloantigens by maturation-resistant DC is not dependent on
calcineurin activation and furthermore is feasible in T cells exposed to CsA in vivo.
Figure 11: Feasibility of modulating alloresponsiveness in T cells from patients with renal allografts by altered DC.
Secondary cultures were set up as described in Figure 8. For the generation of DC from kidney donors, spleen cells frozen at the time of organ harvesting were differentiated into the respective DC populations as described in Materials and Methods before they were cocultured with allogeneic T cells. T-cell proliferation was assessed by DNA synthesis 3 d after initiation of the secondary culture. Data represent mean ±SD from triplicate wells. Depicted is one representative experiment out of four different experiments performed: mDC, LPS-treated DC; PDTC-DC, PDTC-modulated DC stimulated with LPS.
35
C.) “Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3” 96-98
1.) Targeting JAK3 disrupts CD40-triggered DC maturation
We explored the impact of JAK3 inhibition on the phenotype of DC activated
through CD40 engagement. Such activated DC demonstrated a strong up-regulation of
the costimulatory molecules CD80, CD86 and CD40, the MHC class I and II molecules
as well as of the specific DC maturation marker CD83, when compared with immature
DC. In contrast, DC activated through CD40 in the presence of the JAK3 inhibitor
maintained an immature phenotype (Figure 12A,B). Importantly, JAK3 inhibitor-treated
DC did not revert to a monocyte/macrophage stage as demonstrated by the lack of
expression of CD14 (data not shown).
2.) JAK3 inhibition does not interfere with the activation-associated down-regulation of the DC-antigen uptake machinery
Immature DC are known to effectively internalize antigens through
macropinocytosis and receptor-mediated endocytosis 107-108. When DC mature they
down-regulate their antigen-capturing capability and simultaneously increase their
antigen-presenting and costimulatory efficiency. In the next set of experiments we
analyzed whether JAK3 targeting might affect down-regulation of the DC-antigen
capture machinery upon CD40-ligation. As shown in Figure 13A receptor-driven
endocytosis and macropinocytosis, which were evaluated by incorporation of FITC-
labeled dextran or of lucifer yellow (LY), respectively, were more pronounced in
immature DC than in CD40-activated DC. Interestingly, endocytosis and macro-
pinocytosis was similar in JAK3 inhibitor-treated DC and mature DC (Figure 13B).
Furthermore, CD40 ligation led to a reduction in mannose receptor expression in DC
(Figure 13B). As observed for antigen uptake, JAK3 inhibitor-treated DC behaved
similar to mature DC with regard to mannose receptor expression (Figure 13B).
36
(A)
(B)
Figure 12: JAK3 targeting prevents the phenotypic alterations induced by CD40 ligation.
(A) Monocytes were cultured for 7 days with granulocytemonocyte colony stimulating factor (GM-CSF) (50 ng/mL) plus IL-4 (10 ng/mL). Subsequently these immature DC (5x105/mL) were activated with CD40L (200 ng/mL) with or without the JAK3 inhibitor (10 µg/mL) for 48 h. Open profiles (fine line) in the upper panel represent staining pattern with an isotype control, and open profiles with bold line staining with mAbs of the indicated specificity in immature DC before CD40 activation. In the lower panel surface expression in cultures containing CD40L (open profiles with bold line) or CD40L plus 10 µg/mL of JAK3 inhibitor (black profiles) is depicted. Data are representative of six independent experiments. (B) Percent responses ± SEM calculated from median fluorescence intensities (MFI) from six experiments are shown. Expression of the respective surface markers in mature and JAK3 inhibitor-treated DC was related to expression in immature DC. Median fluorescence intensities of immature DC in the absence of the JAK3 inhibitor were as follows: isotype control-FITC: 5.9 ± 1.8, CD40-FITC: 202 ± 63, CD83-FITC: 12 ± 3, MHCI-FITC: 457 ± 85, MHC II-FITC: 808 ± 272, isotype control-PE: 4.4 ± 1.2, CD86-PE: 79 ± 20, CD80-PE: 92 ± 23. Open bars, immature DC; hatched bars, DC activated by CD40 ligation in the absence of the JAK3 inhibitor; solid bars, DC activated by CD40 ligation in the presence of the JAK3 inhibitor.
37
Figure 13: Dendritic cells (DC) activated by CD40 ligation down-regulate their antigenuptake machinery in the presence of Janus kinase 3 (JAK3) inhibition.
(A) Immature DC or DC activated via CD40 in the absence or presence of the JAK3 inhibitor (10 µg/mL) were pulsed with 1 mg/mL of FITC-dextran (DEX) or 1 mg/mL of Lucifer yellow (LY) for 60 min. Open pro-files with fine line show the background uptake on ice and open profiles with bold line indicate antigen-uptake at 37 °C. Data are representative of four independent experiments. (B) Summary of four experiments ± SD indicating the behavior of immature DC or of DC stimulated via CD40 in the presence or absence of 10 µg/mL of JAK3 inhibitor. MR, mannose receptor expression.
38
3.) Prevention of T-cell stimulatory capacity and induction of antigen-specific hyporeactivity by JAK3 inhibited DC
Mature DC are known to be the strongest stimulators of MHC-mismatched
peripheral blood T-lymphocytes. The finding that the response of DC to CD40
ligation was profoundly modified in the presence of JAK3 inhibition prompted us to
investigate the T-cell stimulatory potential of these cells. CD40 triggering of DC
induced a vigorous T-cell stimulating capacity compared with immature DC (Figure
14A). Treatment of DC with the JAK3-inhibitor during CD40 triggering resulted in an
allostimulatory capacity similar to immature DC (Figure 14A). Regarding cytokine
production lower levels of IL-2 and IFN-γ were detected in cultures stimulated with
JAK3 inhibitor-treated DC than in cultures stimulated with mature DC (data not
shown). To address the functional outcome of defective immunostimulation by JAK3-
inhibited DC, T cells initially primed with allogeneic immature DC or DC that had been
activated by CD40-ligation in the presence or absence of JAK3 inhibition were
restimulated with LPS-matured DC from the original donor and from unrelated third-
party donors. Figure 14B shows that preculturing allogeneic T cells with CD40-
triggered DC led to a strong secondary T-cell alloresponse. In contrast, preculture with
CD40-triggered DC exposed to the JAK3 inhibitor resulted in a dramatically reduced
responsiveness of allogeneic T cells upon restimulation. Priming of allogeneic T cells
with immature DC resulted in a comparably low extent of secondary T-cell reactivity
(Figure 14B). Restimulation with allogeneic mature DC from unrelated third-party
donors was also associated with defective T-cell proliferation in JAK3-treated DC or
immature DC-primed T cells, indicating nonspecific T-cell hyporesponsiveness induced
by both APC populations (Figure 14C). For the assessment of the reversibility of the
hyporesponsive state of allogeneic T cells challenged with JAK3 inhibitor-pretreated
DC, rIL-2 was added to secondary MLC. As shown in Figure 15, addition of IL-2 led to
a complete restoration of the defective T-cell proliferation of T cells coincubated with
JAK3 inhibitor-pretreated DC in primary MLC.
39
Figure 14: Reduced stimulatory activity of dendritic cells (DC) matured in the presence of Janus kinase 3 (JAK3) inhibition and induction of a state of T-cell hyporeactivity. For primary MLC, (A) immature DC (5x105/mL) were activated with rh-CD40L and anti-FLAG mAbs in the presence or absence of the JAK3 inhibitor (10 µg/mL). After 48 h, the cells were washed extensively, irradiated, and cocultured with 1x105 purified allogeneic T cells at the indicated ratios. DNA synthesis was assessed at day 5. Shown are the means ± SEM of six to nine independent experiments. *p < 0.01 for comparison of alloresponses induced by DC activated by CD40L in the presence or absence of the JAK3 inhibitor. For restimulation cultures, purified T cells precultured with immature DC, activated DC or JAK3 inhibitor-treated DC were harvested after 5 days. Then the T cells were restimulated with mature DC from (B) the original donor or (C) third-party donors as described in Materials and Methods. DNA synthesis was assessed at day 3. Data are expressed as mean of five different experiments ± SEM. Open bars, immature DC; hatched bars, DC activated by CD40 ligation in the absenceof the JAK3 inhibitor; solid bars, DC activated by CD40 ligation in the presence of the JAK3 inhibitor.
Figure 15: Reversal of T-cell hypo-responsiveness by exogenous IL-2. T cells were challenged with immature dendritic cells (DC) (white bars), CD40-triggered DC (hatched bars), or DC activated by CD40 ligation in the presence of the Janus kinase 3 (JAK3) inhibitor (black bars) and restimulated with monocytes from the original donor with IL-2 (20 U/mL) or without.
40
4.) Effect of JAK3 Inhibitor Treatment on Homotypic T-Cell Aggregation
We evaluated homotypical cell clustering, which is a typical hallmark of
activated T cells and depends on TCR-induced induction of integrin clustering and
integrin avidity changes 109. Although T cells activated with CD3 and CD28 mAb
exhibited prominent cell clusters 8 hr after initiation of the culture, JAK3 inhibitor-
supplemented cultures were completely devoid of any T-cell clusters (Figure 16).
Figure 16: JAK3 targeting blocks phenotypical hallmarks of T-cell activation.
Effect of JAK3 targeting on the activation-associated homoaggregation of human T cells. Purified T cells were left unstimulated in the absence (upperleft) or presence of 10 µg/mL WHI-P-154 (upper right) or stimulated with CD3 (1 µg/mL) and CD28 (2.5 µg/mL) mAb in the absence (lower left) or presence of 10 µg/mL WHI-154 (lower right) for 8 hr. Data are representative of four different donors.
41
SUMMARY OF THE RESULTS
A.) “Bacterial Metabolite Interference with Maturation of Human
Monocyte-Derived Dendritic Cells” 94
1.) Phenotypical and functional impairment of DC maturation by n-butyrate Expression of CD25 and CD83 and of critical costimulatory molecules, i.e.,
CD40, CD80, and CD86, was reduced substantially. A clear suppression of the
up-regulation of MHC class I and II antigens was observed. Little or no cell
clustering occured in cultures, moreover, the majority of cells imposed with
widespread cytoplasmic projections. We found a significant dose-dependent
reduction of the allostimulatory capacity. The expression of the antigen-uptake
molecules CD32 and mannose receptor was inhibited. Assessment of
macropinocytosis revealed impaired internalization of lucifer yellow but a less
pronounced inhibition of receptor-mediated endocytosis of dextran molecules.
B.) “Hyporesponsiveness in Alloreactive T-Cells by NFκB Inhibitor-Treated Dendritic Cells: Resistance to Calcineurin Inhibition” 95
1.) Arrest in DC maturation by treatment with the NFκB inhibitor PDTC Expression of CD83 and of critical costimulatory molecules such as CD40,
CD80 and CD86 was reduced substantially. Up-regulation of MHC class II
antigens was found to be clearly suppressed. We observed a clear inhibition of
the production of the immunostimulatory cytokines IL-12 and TNF-α.
2.) PDTC-modulated DC exhibit defective stimulatory capacity for allogeneic T-cell responses
Pretreatment with PDTC induced a DC population with a low stimulatory
capacity. T-cells challenged with modified DC did not express CD69, similarly
expression of CD25 was profoundly impaired. We found a dose-dependent
suppression of IL-2 and IFN-γ production.
42
3.) Induction of allogeneic hyporesponsiveness by PDTC-modulated DC
T cells exposed to PDTC-modulated DC did not proliferate upon challenge with
immature or even fully mature DC from both original and third party donor DC.
In contrast, APC-independent T-cell stimulation with PMA plus OKT-3 was
unaffected. Recombinans human IL-2 partially restored proliferative responses
towards allogeneic DC while it induced an unspecific increase in secondary
proliferative responses. In the absence of T-cell receptor (TCR)-mediated
activation, IL-2 alone induced a low grade of proliferation in tolerant T cells.
Hyporesponsive T cells were unable to significantly suppress allogeneic
activation of syngeneic T cells and allowed T-cell responsiveness comparable to
T cells previously exposed to immature DC or immature DC treated with
PDTC. PDTC-modulated DC were unable to suppress alloresponsiveness to
mature DC from the same donor.
4.) CsA does not counteract the suppressive state induced by PDTC-treated DC Addition of CsA to primary MLC led to an inhibition of the allogeneic T-cell
proliferation regardless of the mode of APC pretreatment. The presence of CsA
had no influence on the proliferative response of T cells in secondary cultures
when they were stimulated with immature or mature DC in primary MLC.
Interestingly, secondary MLC revealed that the presence of CsA during
alloantigenic priming did not influence the induction of the hyporesponsive
state by PDTC-modulated DC.
5.) T-cell hyporesponsiveness with PDTC-modulated DC in T cells from patients with renal allografts under calcineurin inhibitor-based immunosuppression While the primary MLC response was profoundly reduced using PDTC-
modulated DC, a conspicuous state of T-cell hyporesponsiveness was observed
in restimulation cultures supplemented with allogeneic stimulator cells. In
contrast, T-cell responsiveness was unimpaired when various polyclonal stimuli
were employed. It is concluded that induction of T-cell hyporeactivity towards
alloantigens by maturation-resistant DC is not dependent on calcineurin
activation and furthermore is feasible in T cells exposed to CsA in vivo.
43
C.) “Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3” 96-98
1.) Targeting JAK3 disrupts CD40-triggered DC maturation
Up-regulation of the costimulatory molecules CD80, CD86 and CD40,
the MHC class I and II molecules as well as of the specific DC maturation
marker CD83 was inhibited. JAK3 inhibitor-treated DC did not revert to a
monocyte/ macrophage stage.
2.) JAK3 inhibition does not interfere with the activation-associated down-regulation of the DC-antigen uptake machinery
Endocytosis and macropinocytosis, which were evaluated by incorporation of
FITC-labeled dextran or of lucifer yellow (LY), respectively, were more
pronounced in immature DC than in CD40-activated DC. Interestingly,
endocytosis and macropinocytosis was similar in JAK3 inhibitor-treated DC and
mature DC. Furthermore, CD40 ligation led to a reduction in mannose receptor
expression in DC. As observed for antigen uptake, JAK3 inhibitor-treated DC
behaved similar to mature DC with regard to mannose receptor expression.
3.) Prevention of T-cell stimulatory capacity and induction of antigen-specific hyporeactivity by JAK3 inhibited DC
CD40 triggering of DC induced a vigorous T-cell stimulating capacity compared
with immature DC. Treatment of DC with the JAK3-inhibitor during CD40
triggering resulted in an allostimulatory capacity similar to immature DC.
Preculturing allogeneic T cells with CD40-triggered DC led to a strong
secondary T-cell alloresponse. In contrast, preculture with CD40-triggered DC
exposed to the JAK3 inhibitor resulted in a dramatically reduced responsiveness
of allogeneic T cells upon restimulation. Restimulation with allogeneic mature
DC from unrelated third-party donors was also associated with defective T-cell
proliferation in JAK3-treated DC or immature DC-primed T cells, indicating
nonspecific T-cell hyporesponsiveness induced by both APC populations.
Addition of IL-2 led to a complete restoration of the defective T-cell
44
proliferation of T cells coincubated with JAK3 inhibitor-pretreated DC in
primary MLC.
4.) Effect of JAK3 Inhibitor Treatment on Homotypic T-Cell Aggregation
T cells activated with CD3 and CD28 mAbs exhibited prominent cell clusters
8 hr after initiation of the culture, JAK3 inhibitor-supplemented cultures were
completely devoid of any T-cell clusters.
DISCUSSION
Several lines of evidence indicate that bacteria and viruses have evolved
strategies to evade immune surveillance and in particular to block DC function 110-119. It
has been shown that n-butyrate, a physiologically occurring short-chain fatty acid
(SCFA) derived from breakdown of carbohydrates by the intestinal microflora, exerts
profound anti-inflammatory effects in vitro 120-128. It was demonstrated that this
bacterial metabolite severely hampers interferon-γ (IFN-γ) production as a result of
defective IL-12 production and IL-12-receptor β1/2-chain expression 121. Mounting
evidence suggests that apart from its physiological function as the essential energy
source for colonocytes, n-butyrate exerts strong anti-inflammatory activity in several
states of mucosal inflammation 129-132. The intestinal lumen has been shown to contain
n-butyrate concentrations from 0.1 mM to 16 mM in the small intestine and 40 mM
in the colon depending on quality and quantity of daily food intake 133. Because
n-butyrate occurs in the portal blood at 0.04 mM 133, it is conceivable that the
concentrations used in our study actually occur in the mucosa, where contact between
butyrate and DC can be expected. Finally, in vitro and in vivo experiments have
demonstrated that n-butyrate is able to induce a state of T-cell anergy 134-136. We
investigated how DC respond to the bacterial DC stimulus lipopolysaccharide (LPS)
45
when applied in the presence of n-butyrate, both of which occur at high concentrations
in the mucosal immune system. We described a novel way of bacterial interference
with DC maturation and provided phenotypical, morphological, and functional
evidence that n-butyrate profoundly suppresses the development of monocyte-derived
DC in vitro. The bacterial metabolite n-butyrate inhibited the development of mature
DC, resulting in impaired DC function and defective cytokine production. n-Butyrate
did not promote a classical differentiation program toward macrophages as has been
demonstrated for corticosteroids, IL-10 and IL-6 80,137-138. Furthermore, n-butyrate
substantially impaired the upregulation of MHC antigens, costimulatory molecules, and
DC-specific maturation markers, correlating with reduced antigen-specific T-cell
proliferation. Inhibition of costimulatory molecule expression on DC by n-butyrate was
also striking given their critical role in T-cell stimulation 139-141. DC exposed to n-
butyrate during maturation by LPS induced a state of hyporeactivity in alloreactive
T cells in secondary cultures (unpublished data not shown). In particular, n-butyrate
prevented homotypic DC clustering, inhibited IL-12 while sparing IL-10 production,
and at the molecular level, blocked NFκB translocation 95,142. It is tempting to speculate
that the intestinal microflora in the gut uses this mechanism to escape immune
surveillance. Mice deficient in functional NFκB have no mature DC and present with
impaired cellular immunity 143-145. Likewise, selective inhibition of NFκB activity has
also been shown to impair DC maturation 146-149. Impairment of nuclear translocation of
this transcription factor by n-butyrate may account for most features of the altered DC
phenotype 150 as well as for the sensitive inhibition of cytokine production by n-
butyrate, because the human IL-12, but not the IL-10 promoter, contains crucial NFκB
binding sites within its promoter 151-152. Endotoxin-stimulated PBMC also showed
inhibited NFκB activity in the presence of n-butyrate, which caused a decreased
production of proinflammatory cytokines 122. Finally, in animal models of colitis
mucosal, NFκB-DNA binding activity was reduced markedly when high intestinal n-
butyrate concentrations were achieved by SCFA enemas or dietary means 122,129-130.
We described conditions for the establishment of a novel form of tolerogenic
dendritic cells. This population of DC was generated from peripheral blood monocytes
applying a pharmacological NFκB-inhibitor (PDTC) in combination with a well-known
maturation stimulus. PDTC-pretreatment of DC leads to an arrest in maturation as
46
reflected by down-regulated major histocompatibility complex (MHC) antigens and
costimulatory molecules, suppressed immunostimulatory cytokines and an impaired
capability to support allogeneic T-cell activation. Tolerization was accompanied by
blunted cytokine production and a failure to express distinct activation molecules such
as CD69 and CD25 in primary MLC. The inhibition of NF-κB activation is also
involved in the induction of a deep state of T-cell hyporeactivity in secondary MLC that
cannot even be reverted by mature allogeneic DC. Allogeneic T cells challenged with
PDTC-modulated DC are refractory upon restimulation with alloantigens but not to
polyclonal stimuli. While DC-based tolerance protocols may be an attractive therapeutic
modality if reproducible in the clinical environment, it is presently not clear whether the
immunotolerizing properties of maturation-resistant DC are affected by the presence of
immunosuppressive drugs currently employed in clinical transplantation. Calcineurin
inhibitors such as CsA or FK506 are presently the basic standard immunosuppressive
agents in allogeneic transplantation. They inhibit the transactivation of the transcription
factor NF-AT thus preventing the induction of a variety of genes such as cytokine
genes. There are conflicting results from studies employing diverse strategies to induce
alloantigen-specific tolerance in combination with CsA. Thus CsA prevents the
induction of anergy when the TCR is engaged in the absence of costimulation in vitro.
Likewise, CsA has been shown to abrogate permanent allograft acceptance induced by
B7 plus CD154 blockade 104-105. Conversely, administration of CsA did not prevent the
establishment of regulatory cells induced by perioperative CD4 mAb therapy 153 or
tolerance employing a protocol with mixed chimerism using post-transplant total
lymphoid irradiation 154. The functional consequences of calcineurin inhibition with
Cyclosporine A were studied in T cells exposed to PDTC-modulated DC. For further
investigation of this T-cell hyporesponsiveness we established an ex vivo model in
T cells from patients with renal allografts under CsA-based immunosuppression in order
to assess the ability of in vitro modulated-DC to incorporate into a conventional
immunosuppressive protocol without abrogation of the tolerance by the clinical therapy.
Interestingly, the successful establishment of alloantigenic hyporesponsiveness is not
prevented by concomitant calcineurin inhibition in vitro as well as in T cells from
patients under CsA-based immunosuppression ex vivo. Our results convincingly
demonstrate that CsA, while synergistically suppressing allogeneic T-cell
47
responsiveness in primary MLC, does not prevent allogeneic T-cell hyporeactivity
induced by PDTC-modulated DC. Importantly, similar results were obtained in T cells
from renal allograft recipients indicating that the effects of DC-based regulation of
allogeneic immune responsiveness is conceivable, when modulated DC are incorporated
into a conventional clinical immunosuppressive protocol. These data may have
important implications for the design of clinical regimens for the establishment of
antidonor hyporeactivity in organ transplantation using in vitro modulated DC.
CD40 ligation by CD154 on activated T cells enables DC for sustained
stimulation of T cells leading to clonal T-cell expansion 155. Impaired DC maturation
may be a pivotal mechanism for the success of the CD154–CD40 blockade 156-162.
Premature decline of antigen-specific T-cell responses was observed in conjunction with
decreased DC persistence when CD40–CD154 interactions were absent 163. Similarly,
the efficacy of the CD40–CD154 blockade as immune intervention strategy for the
treatment of allogeneic rejection is well established 164-167. It has also been demonstrated
that engraftment of immature donor-derived DC in conjunction with anti-CD154 mAb
therapy promotes the induction of transplant tolerance 156-157,168. Recently,
pharmacological JAK3 inhibition has successfully been used to suppress T-cell
reactivity in experimental acute GVHD as well as in allogeneic transplantation 169-173.
Therapeutic targeting of this protein tyrosine kinase (PTK), which is a member of the
Janus family PTK, might be of particular advantage because of its association with the
common-gamma chain (cγ) of cytokine receptors that serves for multiple T-cell
cytokines such as IL-2, IL-4, IL-7, IL-9 and IL-15 174-175. Upon ligand-binding,
receptor associated JAKs are recruited, leading to their phosphorylation and subsequent
activation of STAT (signal transducers and activators of transcription) proteins that
dimerize and transactivate the transcription of specific target genes 176-177. Janus kinase
3 deficiency disables phosphorylation of the transcription factors STAT5a/b 178 and
manifests as severe combined immunodeficiency (SCID) 179-180. This disease is
associated with a profound defect in the immune system with significant alterations in
lymphocyte development, a functional incompetence and increased susceptibility for
apoptosis in peripheral T cells 181. Recent evidence suggests that in addition to its
involvement in common-gamma chain (cγ) signaling of cytokine receptors 182-183, JAK3
is also engaged in the CD40 signaling pathway of peripheral blood monocytes 184-185. In
48
this study, we assessed the consequences of JAK3 inhibition during CD40-induced
maturation of myeloid dendritic cells (DC), and tested the impact thereof on the
induction of T-cell alloreactivity. CD40-triggering of monocyte-derived DC in the
presence of selective JAK3 inhibitors resulted in the generation of APC with low
costimulatory molecule expression, defective cytokine production and impaired
allostimulatory capacity, which even confered a state of hyporeactivity to allogeneic
T cells that was reversible upon exogenous IL-2 supplementation to secondary cultures.
These results suggest that immunosuppressive therapies targeting the tyrosine kinase
JAK3 may also affect the function of myeloid cells. This property of JAK3 inhibitors
therefore represents a further level of interference, which together with the well-
established suppression of common-gamma chain signaling could be responsible for
their clinical efficacy. Current data indicate a critical role for JAK3 in signaling linked
to the T-cell antigen receptor 186-188, therefore, we investigated whether targeting JAK3
with a rationally designed inhibitor affects early T-cell activation events. T-cells were
stimulated by CD3 and CD28 cross-linking. We found that JAK3 inhibitor treatment