Persistence T Cell Function during Viral of CD8 Memory ...

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2007;179;141-153 J. Immunol. Ravneberg, Janet L. Weslow-Schmidt and Emilio Flaño Stephanie S. Cush, Kathleen M. Anderson, David H. 

Persistence T Cell Function during Viral+of CD8

Memory Generation and Maintenance

http://www.jimmunol.org/cgi/content/full/179/1/141

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Memory Generation and Maintenance of CD8� T CellFunction during Viral Persistence1

Stephanie S. Cush,* Kathleen M. Anderson,* David H. Ravneberg,* Janet L. Weslow-Schmidt,*and Emilio Flano2*†

During infection with viruses that establish latency, the immune system needs to maintain lifelong control of the infectious agentin the presence of persistent Ag. By using a �-herpesvirus (�HV) infection model, we demonstrate that a small number ofvirus-specific central-memory CD8� T cells develop early during infection, and that virus-specific CD8� T cells maintain func-tional and protective capacities during chronic infection despite low-level Ag persistence. During the primary immune response,we show generation of CD8� memory T cell precursors expressing lymphoid homing molecules (CCR7, L-selectin) and homeo-static cytokine receptors (IL-7�, IL-2/IL-15�). During long-term persistent infection, central-memory cells constitute 20–50% ofthe virus-specific CD8� T cell population and maintain the expression of L-selectin, CCR7, and IL-7R molecules. Functionalanalyses demonstrate that during viral persistence: 1) CD8� T cells maintain TCR affinity for peptide/MHC complexes, 2) thefunctional avidity of CD8� T cells measured as the capacity to produce IFN-� is preserved intact, and 3) virus-specific CD8� Tcells have in vivo killing capacity. Next, we demonstrate that at 8 mo post-virus inoculation, long-term CD8� T cells are capableof mediating a protective recall response against the establishment of �HV68 splenic latency. These observations provide evidencethat functional CD8� memory T cells can be generated and maintained during low-load �HV68 persistence. The Journal ofImmunology, 2007, 179: 141–153.

I mmunological memory is one of the hallmarks of the adap-tive immune system and it can be functionally defined as thestronger protective response of the host to secondary Ag

challenge (1, 2). It, thus, allows the immune system to respondmore vigorously to infectious pathogens that have been encoun-tered previously. Upon primary activation by Ag, CD8� T cellsfollow a program of proliferation and differentiation into effectorsthat control the infection (3). After this expansion phase, the ma-jority of Ag-specific CD8� T cells undergo programmed celldeath, leaving a population of memory CD8� T cells (4). A rapidand efficient recall response is partially due to the ubiquitous pres-ence of increased frequencies of memory T cells in peripheraltissues and secondary lymphoid organs (5). In addition, severalcharacteristics of memory T cell populations contribute to en-hanced secondary responses including the ability to self-renew inthe absence of Ag, higher activation status and reduced costimu-latory requirements, expression of lymphoid homing moleculesand homeostatic cytokine receptors, higher frequency than naiveprecursors, rapid proliferation upon secondary stimulation, and theability to maintain the integrity of the T cell repertoire (1, 6, 7).Nevertheless, some of these characteristics may be arbitrary (i.e.,markers, Ref. 8) or not universal (i.e., long lived, Ref. 9). Theheterogeneity of memory T cells in phenotype, function, andlocation has led to the idea that they can be divided in two major

subsets: effector (CD62LlowCCR7low) and central-memory(CD62LhighCCR7high) cells based on their expression of lym-phoid homing receptors (10).

Memory CD8� T cells during viral infections play a major rolein protection by rapid recognition and lysis of virus-infected cells.The contribution of memory CD8� T cells to recall responses iswell-defined during acute infections where Ag is cleared (11, 12).There is, however, little and contradictory information regardingmemory generation during persistent infections. This is of specialrelevance because persistent infectious diseases are a major healthconcern worldwide (13, 14). The current paradigm, mostly derivedfrom studies with lymphocytic choriomeningitis virus (LCMV)3

and HIV, is that persistent infection leads to some degree of CD8�

T cell effector dysfunction or deletion and lack of T cell memoryformation (15–19). CD8� T cell dysfunction during chronic infec-tion has been raised as one reason for pathogen persistence (20).This loss of function in the presence of persistent Ag follows aprogression in the sequence of cytotoxicity, IL-2, TNF-�, andIFN-� production, exhaustion, and deletion (20, 21). However,these findings appear to be at odds with situations where virusespersist and CD8� T cell functions are not apparently compromised(22–25), with reports of residual Ag presentation (26) or with apossible role for Ag in the maintenance of immunological memory(27). �-herpesviruses (�HV) are characterized by the establish-ment of lifelong asymptomatic infection, the persistence in �90%of the human population, and the association with a large list oflife-threatening conditions (28, 29). CD8� T cells are thought to bethe main effectors of long-term virus control (28, 30, 31). How doCD8� T cell populations maintain control of infection without

*Center for Vaccines and Immunity, Columbus Children’s Research Institute, Co-lumbus, OH 43205; and †College of Medicine, The Ohio State University, Columbus,OH 43210

Received for publication January 9, 2007. Accepted for publication April 27, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grant AI-59603and by Columbus Children’s Research Institute.2 Address correspondence and reprint requests to Dr. Emilio Flano, Center for Vac-cines and Immunity, Columbus Children’s Research Institute, Columbus, OH 43205.E-mail address: [email protected]

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus;�HV68, murine �-herpesvirus 68; LDA-PCR, limiting dilution nested PCR; RT, roomtemperature; d.p.i., days postinfection; m.p.i., months postinfection; MLN, mediasti-nal lymph node.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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pathogen recrudescence during persistent infections? Humans en-counter different pathogens through life of which a considerablenumber reach a persistent, parasitic, commensal, or a latency stateand the human system has to provide lifelong protection. Herpes-virus infections are a clear example: initial infection with CMV orEBV may occur early in childhood, which demonstrates that theimmune system is capable of controlling infection for 70 years ormore. Although persistent infections induce a continual evolutionof the function and numbers of T cells that are specific for theinfecting pathogen, there seems to be a substantial difference be-tween infections with impaired CD4� T cell help and/or a highantigenic load such as HIV or LCMV-clone 13 and infections withintact CD4� T cell help and low-load Ag persistence, such asherpesvirus infections (32, 33).

To understand more clearly how CD8� memory T cells aregenerated, maintained, and function during low-load persistent vi-ral infections, we studied their fate and function during the acuteand long-term response to a �HV infection. Using a murine modelof infection with �HV68, we show that Ag-specific CD8� T cellfunction is maintained during long-term, low-level viral and Agpersistence.

Materials and MethodsMice and viral infection

C57BL/6J and BALB/cJ mice were obtained from The Jackson Laboratory,Harlan Farms, or were bred at Columbus Children’s Research Institute(CCRI). �HV68, clone WUMS, was propagated and titered on monolayersof NIH3T3 fibroblasts. Mice were housed in BL2 containment under patho-gen-free conditions. The Institutional Animal Care and Use Committee atthe CCRI approved all the animal studies described here. Mice were anes-thetized with 2,2,2,-tribromoethanol and intranasally inoculated with 1000PFU �HV68 in 30 �l of HBSS.

Virus analysis

To determine the number of cells containing �HV68 genomes long-termpostinfection, a combination of limiting dilution analysis and nested PCR(LDA-PCR) was used (34). Splenocytes were isolated, RBC were lysed,and the number of cells per spleen was determined. Cells were seriallydiluted with NIH3T3 cells in 96-well plates, lysed, and viral DNA wasamplified by PCR using primers specific for �HV68 Orf50. A 2-�l aliquotof the product was reamplified using nested primers. The final product was

analyzed by ethidium bromide staining of DNA after electrophoresis in 3%agarose gel. This procedure was able to consistently detect a single copy oftarget sequence. Twelve replicates were assessed for each cell dilution andlinear regression analysis was performed to determine the reciprocal fre-quency (95% degree of confidence) of cells positive for �HV68 DNA. Atleast three independent experiments were used to determine the mean re-ciprocal frequency and SD of �HV68 DNA in splenocytes. Infectious cen-ter assays were performed as described (35).

Ag-presentation assays

Splenocytes were isolated and processed into single-cell suspensions usingcollagenase D (5 mg/ml) treatment for 45 min. Cells were next incubatedwith 2 mM PBS/EDTA for 10 min at room temperature to disrupt multi-cellular complexes. Cells were stained with an anti-CD11c Ab conjugatedto magnetic beads (Miltenyi Biotec) and separation columns were used topurify cells into CD11c� and CD11c� fractions. A total of 3 � 104 APCsfrom each fraction were plated with 1 � 105 �HV68-specific lacZ-induc-ible T cell hybridomas (49100.2 and 4943.4) (36) in 96-well plates intriplicate and incubated overnight. �-galactosidase activity was assessed inindividual wells as described (36). Background stimulation was determinedby using APCs from naive spleens as stimulators.

Flow cytometry analysis

Splenocytes were processed into single-cell suspensions as describedabove. Cells were stained with Fc-block (CD16/32) and then washed. Thecells were then stained with a combination of the following �HV68-spe-cific MHC tetramers: ORF6487–495/Db, ORF61524–531/Kb, ORF65131–140/Dd and M291–99/Kd, and Abs against CD62L (MEL-14), CD8 (53-6.7),CD43 (1B11), CD122 (TM-b1), CD127 (A7R34), CD44 (IM7), and CD4(GK1.5). All Abs were purchased from eBioscience with the exception ofCD43 (BD Pharmingen). MHC tetramers were generated as described (37)or obtained from the National Institutes of Health Tetramer Core Facility.The surface expression of CCR7 was analyzed using the recombinant li-gand CCL19 Ig followed by anti-human Fc-biotin and streptavidin-PE.This process can be slightly detrimental for the intensity of the tetramerstaining. Flow cytometry data were acquired on FACSCalibur or BD LSR(BD Biosciences) and analyzed using FlowJo software. Gates were setusing negative controls, isotype controls, and following the staining patternof each marker on the bulk lymphocyte and CD8 population. For any givenactivation marker and tetramer combination, all the analysis gates are iden-tical in size and position.

T cell avidity assays

Tetramer dilution. Single-cell suspensions were obtained as indicatedabove. Cells were plated in flasks coated with anti-mouse IgG plus IgM

FIGURE 1. Long-term infection is characterizedby low viral titers and low Ag persistence. A, Analysisof �HV68 titers during long-term latency. The numberof latently infected cells (left y-axis) in the spleen ofC57BL/6 (f) and BALB/c (�) mice was determined us-ing LDA-PCR assay at 5 m.p.i. The number of lyticallyinfected cells (ND, not detected, right y-axis) was de-termined using a modified plaque assay. Three individ-ual mice were independently analyzed at each timepoint; error bars represent SD. B, Analysis of �HV68antigenic load in mice during long-term latency. Spleencells from mice 4 m.p.i. were used as stimulators in anin vitro assay with two lacZ-inducible �HV68-specificT cell hybridomas to determine the number of APCscapable of presenting viral Ags on their cell surface.Left panel, Hybridoma 4943 specific for peptideORF61/Kb; right panel, hybridoma 49100 specific forpeptide ORF6/Db. Dendritic cells were purified by anti-CD11c Ab labeling and magnetic bead enrichment(75% purity).

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Abs (Jackson Immunochemicals) for 1 h to enrich for T cells. The nonad-herent cells were incubated with Fc-block (CD16/32) and washed. A totalof 2 � 106 cells were plated per well in 96-well plates and stained withORF6487–495/Db at 2-fold dilutions for 1 h at room temperature (RT). Cellswere washed and subsequently incubated with anti-CD8 Ab, washed, andthen fixed with 1% paraformaldehyde. The results were expressed as thepercentage of tetramer/CD8 double-positive cells over the log concentra-tion of tetramer. To compare groups within each experiment, the data werenormalized to the response at the highest concentration of tetramer. Theaffinity, KD, is derived from the slope by KD � 1/slope.Tetramer decay. Cells were processed, enriched by panning and Fc-blocked as described above. After staining with ORF6487–495/Db and anti-CD8 Ab for 1 h at RT, cells were washed twice and incubated in an excessof anti-Db 28-14-8s purified Ab and incubated at 37°C to allow tetramerdissociation. The anti-Db Ab was used to block rebinding of the tetramer tothe TCR. Decay was followed at intervals from 0 to 50 min at which timesthe cells were fixed with 1% paraformaldehyde. The results were expressedas the percentage of tetramer/CD8 double-positive cells over time. To com-pare groups within each experiment, the data were normalized to the per-centage of maximum tetramer binding over time. The half-life was derivedfrom the slope by t1/2 � ln2/slope.Intracellular cytokine staining. Single-cell suspensions were obtained asindicated above. A total of 2 � 106 cells/sample were incubated in com-

plete medium in the presence of IL-2 (10 U/ml), brefeldin A (10 �g/ml),and 10-fold dilutions of purified ORF6487–495 peptide for 5 h at 37°C. Aspositive control, cells were stimulated with PMA/ionomycin. After Fc-blocking, the cells were stained with Abs anti-CD44 and anti-CD8, fixed,permeabilized, and stained with anti-IFN-� or isotype control Abs. Thedata were expressed as the percentage of IFN-�/CD8 double-positive cellsover the log peptide concentration after subtracting the background obtainedfrom the negative controls. The data were normalized to the response at thehighest concentration of peptide. The EC50 was determined as the peptideconcentration provoking a response halfway between baseline and maximum.

In vivo cytotoxicity assay

Single-cell suspensions obtained from naive spleens were pulsed withORF6487–495 peptide or irrelevant influenza NP366–374 peptide (1 �g/106

cells) for 2 h at 37°C. The NP-pulsed control cells were stained with 50 nMCFSE and the relevant targets with 0.5 �M CFSE for 15 min at 37°C. Thecells were thoroughly washed, combined in a 1:1 ratio, and a total of0.5–1 � 107 cells were injected i.v. into mice. The presence of CFSE-positive cells was analyzed in spleen cell suspensions from the recipientmice 6 or 40 h later. Percent-specific killing was calculated according to theformula percent-specific killing � (1 � (ratio of naive recipients/ratio ofinfected recipients) � 100), where ratio � (number of CFSEhigh/number ofCFSElow).

FIGURE 2. Memory CD8� T cells predominate during long-term infection. Temporal kinetic analysis of the effector and memory CD8� T cellresponse to �HV68. A, Representative dot plots showing the CD62L/CD43 distribution of a population of virus-specific CD8 T cells (ORF61524–531/Kb)at different times after infection (14 days, 3 and 18 mo). Note the progressive increase in CD62L�CD43� �HV68-specific CD8� T cells. Cells havebeen previously gated as tetramer�CD8�. B, Evolution of the different populations of Ag-specific CD8� T cells during �HV68 infection. CD62L�

CD43�: f; CD62L�CD43�: Œ; CD62L�CD43�: F. Data are presented for ORF6487– 495/Db-specific CD8� T cells in spleen and lung. Similarkinetics were found for ORF61524 –531/Kb-specific CD8� T cells. C, All the Ag-specific CD8� T cell populations analyzed in spleen and MLN werehighly heterogeneous at 9 m.p.i. No significant differences were found between lytic-cycle epitopes (ORF61524 –531/Kb and ORF6487– 495/Db) andlatent-cycle epitopes (M291–99/Kd). All the analyses were performed in C57BL/6 and BALB/c mice. Three individual mice were analyzed at eachtime point; error bars represent SD.

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Adoptive cell transfers

Splenocytes from BALB/cJ mice at 8 mo post-�HV68 inoculation wereprocessed into single-cell suspensions as described above and stained withanti-CD4 and CD8 Abs. Cells were purified using a FACSVantage withDiva option and 4 � 105 purified cells (purity 99%) were transferred i.v.into naive BALB/cJ mice. The recipient and control mice were infectedwith �HV68 24 h later. Viral latency was determined in the spleen onday 16.

Statistical analysis

The data were fitted to linear and nonlinear regression lines. The form withthe highest R2 was used in further comparisons between groups. Early andlate groups were compared using two-way ANOVA with group and rep-licate as main effects while controlling for concentration or time. The extrasum-of-squares F test was used to compare the models and reject or not thesimpler null hypothesis model. Statistical analyses were run several timesif there were outliers in the data. One analysis included all observations. Asecond analysis was run excluding outliers that were outside a range of 3SDs. There was one observation dropped from the tetramer dilution stud-ies, none from the tetramer decay studies, and one from the IFN-� studies.The statistical analysis was performed using GraphPad Prism and SigmaStatsoftware.

Results�HV persistence: low antigenic load

�HVs are characterized by the establishment of lifelong asymp-tomatic infection by, among other strategies, persisting in a latentstate in memory B cells. There is, however, a low level of viralreactivation from latency that is thought to contribute to an activeprocess of infection of new cell reservoirs and to the release ofinfectious viral particles (28, 34, 38). Thus, �HVs represent a goodmodel to study immune responses during conditions of low-levelAg persistence. To study the low levels of viral latency during�HV68 persistent infection, we determined the viral load in thespleen during long-term �HV68 infection of mice. We used a sen-sitive LDA-PCR assay for �HV68 orf50 to detect the number ofcells carrying viral DNA. The data show that there were consis-tently �500 latently infected cells per spleen in C57BL/6 andBALB/c mice at 5 months postinfection (m.p.i.) (Fig. 1A). In ad-dition, we sought to determine the number of infectious viral par-ticles at the same times postinfection using a plaque assay. Thedata show that no infectious virus was detected (Fig. 1A), indicat-ing that there was lack of lytic virus or that the number of lyticviral particles at 5 m.p.i. is very low and thus, below the detectionlevel of this assay. Altogether, these data indicate that the viralload during long-term �HV68 carrier state is very low and suggestthat the levels of persistent Ag in the host must also be low. Todetermine the levels of persistent Ag during long-term infection,we used two lacZ�-inducible T cell hybridomas specific for twoclass-I-restricted lytic-phase epitopes from the ssDNA-bindingprotein (ORF6487–495/Db) and from the large RNase reductasesubunit (ORF61524–531/Kb) of �HV68. As shown in Fig. 1B, hy-bridoma stimulation with bulk spleen cells did not detect the pres-ence of viral Ags on 4 m.p.i. splenocytes. However, enrichment ofCD11c� cell fraction using magnetic beads induced the stimula-tion of �HV68-specific hybridomas. No stimulatory capacity couldbe detected in the CD11c� population. Together, these data dem-onstrate the during persistent �HV infection, a small population ofAPCs, likely dendritic cells, is capable of presenting lytic cycle-associated viral Ags on the cell surface in the context of MHCmolecules.

Generation of central-memory CD8� T cells during viruspersistence

Previous studies have shown that there is a shift in the compositionof the memory T cell pool from an effector-memory to a central-memory phenotype over time (11, 12, 39). However, during

chronic LCMV infection, the presence of persistent Ag is thoughtto prevent T cell memory generation (15). We sought to gain aclearer understanding of the generation and heterogeneity of dif-ferent subsets of memory CD8� T cells in lymphoid organs andthe respiratory tract during low-level Ag persistence using �HV68infection as a model. We performed a temporal kinetic analysis ofthe evolution of �HV68-specific CD8� T cells in two differentmouse strains: C57BL/6 and BALB/c. To track Ag-specific Tcells, we used tetramers against three different CD8� T cellepitopes (lytic cycle: ORF61524–531/Kb and ORF6487–495/Db, la-tent cycle: M291–99/Kd). We analyzed the level of cell surface ex-pression of the lymphoid homing molecule L-selectin (CD62L)and the activation-associated glycoform of CD43 (1B11) to dis-tinguish subpopulations of effector and memory CD8� T cells.Up-regulation of L-selectin expression on Ag-experienced cells isa common characteristic of memory T cells and their precursors (5,12). The activation-associated glycoform of CD43 (1B11) hasbeen extensively used to distinguish memory from effector T cellsin mice (40, 41). Our data show that Ag-specific CD8� T cellswith a central-memory phenotype (CD62LhighCD43low) could bereadily detected at 14 days postinfection (d.p.i.) in spleen and theirfrequency increased over time during the course of persistent in-fection (Fig. 2, A and B). Central-memory CD8� T cells consti-tuted between 15 and 50% of the �HV68-specific CD8� T cellsindependent of the tissue or epitope analyzed during long-terminfection (9 m.p.i.) (Fig. 2C). The kinetic analysis also shows thatthe effector CD8� T cell population (CD62LlowCD43high) con-tracts from day 14 postinfection until day 90 (Fig. 2B). This pro-cess is accompanied by a progressive increase in the frequency of

FIGURE 3. Flow cytometry analysis of Ag-specific CD8� T cells. A,Representative staining showing tetramer vs CD8 dot plots for each one ofthe tetramers used in this study. B, Distribution of CD122 and CD127staining on spleen cells; CD8� T cells and tetramer�/CD8� T cells. M291–99/Kd staining on day 28 is shown. Gates were set using negative controls,isotype controls, and following the staining pattern of each marker on thebulk lymphocyte and CD8� population. For any given activation marker,the analysis gates are identical in size and x-axis position. Numbers rep-resent the percentage of positive cells in each quadrant/gate.

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effector-memory T cells (CD62LlowCD43low). This shift in thecomposition of the T cells from effectors (CD43high) to memorycells (CD43low) stabilizes 3 mo postinfection and the frequency oftotal memory CD8� T cells plateaus and remains remarkably sta-ble until 18 mo postinfection, constituting 70–90% of the CD8� Tcells for any epitope analyzed. There is, however, a shift in thecomposition of the CD8� memory T cell pool over time that starts3 mo postinfection with central-memory T cells (CD62Lhigh

CD43low) gradually increasing their frequency while the effector-memory subset (CD62LlowCD43low) contracts (Fig. 2B). Similarkinetics were found for ORF6487–495/Db-specific CD8� T cells(data not shown). Altogether, the data show that virus-specificCD8� T cells primarily had an effector-memory phenotype andwere heterogeneous long-term post-virus inoculation in both lym-phoid organs and the lung. In addition, a population of virus-spe-cific CD8� T cells resembling a central-memory phenotype couldbe detected at early stages postinfection and progressively in-creased its frequency over time.

Our previous data suggest the possibility that memory CD8� Tcell precursors are being generated during the initial stages of theadaptive immune response to a �HV infection. To investigate thispossibility, we analyzed the expression of two of the hallmarks of

bona fide memory T cells on virus-specific CD8� T cells: homeo-static-cytokine receptors (IL-7 and IL-15) (15, 39) and lymphoidhoming molecules (L-selectin and CCR7) (5, 12). The strategy forthe gating of these populations is described in Fig. 3. The analysiswas performed 14 d.p.i., which marks the peak of �HV68 latencyand of viral Ag expression (36, 42). As shown in Fig. 4, A and B,subsets of �HV68-specific CD8� T cells expressing L-selectin,CCR7, and receptors for IL-7 and IL-2/IL-15� on their cell surfacecould be detected in bone marrow and spleen 2 wk post-virusinoculation. These data indicate that phenotypically “normal”memory CD8� T cell precursors are being generated during thepeak of the T cell response to a persistent �HV infection. It shouldbe noted that the analysis of C57BL/6 mice at 14 d.p.i. shows twopopulations of tetramer-positive cells, one which is tetramer lowand positive for L-selectin, IL-2/IL-15�, IL-7�, and CCR7, andone which is tetramer high and negative for the markers tested.Transient loss of tetramer binding and TCR/CD8 down-regulationhas been observed after T cell activation (43– 45) and could beexplained by the asymmetric T cell lymphocyte division afterAg stimulation (46). To determine whether memory T cell pre-cursors generated during primary infection had a central- oreffector-memory phenotype and their anatomical distribution,

FIGURE 4. CD8� memory T cells are generatedduring the early phase of infection. A and B, MemoryCD8� T cell precursors are detected in bone marrow(A) and spleen (B) of 14 d.p.i. �HV68-infectedC57BL/6 mice. The subset of Ag-specific cells precur-sors that express L-selectin, CD122, CD127, andCCR7 (x-axis) is shown inside the gate against thebinding of ORF61524–531/Kb and ORF6487–495/Db (y-axis). The numbers indicate the percentage of cells in-side the gate. The cells have been previously gated asCD8�tetramer�. C and D, Central and effector-mem-ory cells are present in lymphoid organs of infectedmice 14 days after virus inoculation. The number(C) of CD8�ORF6487– 495/Db� lymphocytes ex-pressing CD62LhighCD127high (central-memorycells), CD62LlowCD127high (effector-memory cells),and CD62LlowCD127low (effectors) and their fre-quency (D) among the CD8�ORF6487–495/Db� lym-phocyte population is shown in bone marrow, MLN,and spleen. Data correspond to six to nine individualmice per group. Error bars, SD.

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we analyzed simultaneously the presence of L-selectin andIL-7R on ORF6487– 495/Db-specific CD8� T cells in differentlymphoid tissues (Fig. 4C). Ag-specific CD8� T cells consistedof cells with an effector phenotype (CD62LlowCD127low), central-memory phenotype (CD62LhighCD127high), and effector-memoryphenotype (CD62LlowCD127high) in mediastinal lymph node (MLN),spleen, and bone marrow. The analysis shows that memory popula-tions constitute a significant fraction of the cells in the lymphoid tis-sues analyzed. Although most of the memory cells are found in thespleen, Fig. 4D shows that their frequencies are higher in bone mar-row and MLN than in spleen (CD62LhighCD127high: bone marrow8.7%, MLN 10.7%, and spleen 3.3%; CD62LlowCD127high: bonemarrow 9.8%, MLN 12.1%, and spleen 7.3%). Similar numbers andfrequencies were obtained using CD43 and CD62L to divide the sub-populations of Ag-specific cells (data not shown). Taken together,these results provide strong evidence that during primary �HV68 in-fection CD8� memory T cell precursors with a central-memory andeffector-memory phenotype are being generated within lymphoidtissues.

Memory CD8� T cells express homeostatic cytokine receptorsand lymphoid homing molecules during virus persistence

Continuous Ag stimulation prevents the expression of homeostaticcytokine receptors and lymphoid homing molecules on CD8�

memory T cells during LCMV persistence, which partially con-tributes to explain their dysfunction (15). Thus, we analyzed theexpression of L-selectin, CCR7, IL-2/IL-15� receptor, and IL-7Ron �HV68-specific CD8� T cells during long-term infection. Theanalysis of CD8� T cells specific for two lytic cycle epitopes(ORF61524–531/Kb, ORF6487–495/Db) during long-term infectionof C57BL/6 mice shows that virus-specific CD8� T cells havedown-regulated the expression of IL-2/IL-15� receptor, with onlya small fraction of the tetramer-positive cells expressing CD122 on

their cell surface (1–4%) (Fig. 5). However, the majority of thevirus-specific CD8� T cells show surface expression of the IL-7R(70–80%). In addition, a subpopulation of the Ag-specific CD8�

T cells express the lymphoid homing molecules L-selectin andCCR7 on their cell surface (20–30%). These data are in contrastwith results from other chronic infections (15) and indicate thatCD8� T cells expressing molecular determinants of central-mem-ory cells are maintained during persistent �HV68 infection. Todetermine whether the down-regulation of the IL-2/IL-15� recep-tor is specific for lytic cycle Ags or it also occurs on CD8� T cellsspecific for latent cycle epitopes, we did a temporal kinetic anal-ysis of �HV68-specific CD8 T cells in BALB/c mice comparingthe response of a lytic-epitope (ORF65131–140/Dd) and a latentepitope (M291–99/Kd). As shown in Fig. 6, CD8� T cells specificfor the latent epitope M291–99/Kd maintain the expression of the IL-2/IL-15� receptor over time. Lytic epitope-specific CD8� T cells,however, progressively down-regulate the surface expression of theIL-2/IL-15� receptor. Both populations of CD8� T cells maintain theexpression of IL-7R and L-selectin. Together, these data indicate thatduring long-term persistent �HV infection, memory CD8� T cellsretain the expression of homeostatic cytokine receptors (IL-7) andlymphoid homing molecules (L-selectin, CCR7) at their cell surface.However, CD8� T cells specific for lytic cycle Ags (ORF65131–140/Dd, ORF61524–531/Kb, and ORF6487–495/Db) specifically down-reg-ulate CD122 surface expression.

Ag-specific CD8� T cells maintain TCR affinity during viruspersistence

High-avidity CD8� T cells are thought to play a major role interminating viral infections (47, 48). The kinetics and affinity ofTCR-peptide/MHC interactions drive the selection of TCR clonesduring the course of an immune response resulting in an overallincreased affinity for Ag (49). This mechanistic model suggests

FIGURE 5. Lytic epitope-specificmemory CD8� T cells express L-se-lectin, CCR7, and IL-7� receptor butdown-regulate IL-2/IL-15� receptorexpression during long-term infec-tion. Analysis of bone marrow andspleen cells from mice at 9 m.p.i. with�HV68. Cells were previously gatedas CD8�tetramer�: ORF6487–495/Db

is shown on A and ORF61524–531/Kb

on B. The plots show tetramer stain-ing (y-axis) vs memory markers(CD62L, CD122, CD127, and CCR7on x-axis). Note that the expression ofIL-2/IL-15� receptor is down-regu-lated while the rest of memory mark-ers remain expressed at significantlevels. The numbers indicate the per-centage of cells inside the gate. Rep-resentative plots from three indepen-dent experiments with similar resultsare shown.

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that continuous Ag stimulation influences the CD8� T cell pool byinducing changes in TCR usage or affinity that translate in theselection of high-affinity immunodominant clones (50). Otherstudies show that high-avidity CD8� T cells are more susceptibleto apoptosis or clonal senescence under conditions of excessive Agload (51, 52) which may explain a decrease in immunodominance

and clonal dominance during memory to herpesvirus persistencecompared with primary responses (53). In addition, the down-reg-ulation of the IL-2/IL-15B receptor expression on lytic Ag-specificCD8� T cells during persistent �HV68 infection may be related toa decrease in clonal affinity. IL-15/IL-15R signaling mimics TCRcross-linking (54), activates CD8� T cells (55), and mediates avid-ity maturation of CD8� T cells at the single cellular level by in-creasing TCR functional affinity (56). Thus, it is possible that inthe presence of low levels of persistent Ag, high-avidity memoryCD8� T cells are being continuously stimulated by both Ag andIL-15 and driven to clonal exhaustion.

High-avidity CD8� T cells bind preferentially under conditionsof limited peptide/MHC complex availability (57, 58). To comparethe relative TCR affinity of ORF6487–495/Db-specific CD8� T cellsduring early (14–35 days) and long-term infection (at least3 m.p.i.), we performed a dose-response titration using tetramerdilution under conditions ranging from saturation to limiting avail-ability (Fig. 7A). The data show that the normalized titrationcurves for early and late time points postinfection were superim-posable (Fig. 7B). The analysis of the data indicated the fit was thesame for linear and nonlinear approaches. Thus, in this situation,the simpler linear model was chosen (R2-early: 0.9887, R2-late:0.9674). The slopes, elevations, and y intercepts of both curveswere not significantly different and a common slope (9.2409) andcommon y intercept for all the data (95.6591) could be determined.There was no effect of the time postinfection on the affinity oftetramer binding (KD-early: 0.1017, KD-late: 0.1157). These re-sults indicate that there was no significant change in the averageTCR peptide/MHC affinity over time between primary infectionand �HV68 persistence.

The half-life of the TCR-peptide/MHC complex is related to thestrength of signaling (59, 60) and the rate of dissociation of pep-tide-MHC tetrameric reagents from the T cell surface is related tothe off rate of the TCR-peptide/MHC interaction (49). Thus, we usedtetramer decay analysis to compare the binding of ORF6487–495/Db to�HV68-specific CD8� T cells during early and long-term in-fection by measuring the dissociation rate using flow cytometry(Fig. 7C). The normalized results show that there were no majordifferences in the rate of tetramer dissociation time betweenCD8� T cells analyzed at early and late time points duringinfection (Fig. 7D). The mathematical analysis indicated a bet-ter fit for the nonlinear one-phase exponential decay function(R2-early: 0.9481, R2-late: 0.9510), so that the functional formwas used in comparing groups. The comparison of fit indicatedthat the preferred model was one common curve fitting all datasets independently of group and a common half-life value wasdetermined as 17.19 min. (K: 0.0403). Altogether, the tetramerdilution and tetramer decay analyses demonstrate that Ag-spe-cific CD8� T cells exhibit similar TCR-peptide/MHC interac-tion affinities during primary and persistent �HV68 infection.These data suggest that persistent Ag expression during �HV68infection does not substantially modify the TCR affinity ofCD8� T cells.

Ag-specific CD8� T cells maintain functional avidity duringvirus persistence

The functional avidity of CD8� T cells is mediated by the inter-action between TCR/CD8� and peptide/MHC complexes but alsoby several other mechanisms such as the contribution of accessorymolecules in lipid raft recruitment of signaling proteins and reg-ulatory pathways (61–63). Persistent viral infections are thought tolead to different degrees of CD8� T cell dysfunction (15, 16, 33).

FIGURE 6. Latent epitope-specific CD8� T cells maintain the expres-sion of lymphoid homing molecules and homeostatic cytokine receptorsduring long-term infection. Contour plots show the temporal evolution ofmemory CD8� T cells in BALB/c mice for a latent epitope (M291–99/Kd,A) and for a lytic epitope (ORF65131–140/Dd, B). Spleen cells were previ-ously gated as CD8�tetramer�. Tetramer staining is plotted on the y-axisand the specific memory marker on the x-axis (CD122, CD127, CD62L).Note that memory CD8� T cells specific for the latent epitope maintainexpression of the memory markers while cells specific for the lytic epitopeprogressively lose surface expression of IL-2/IL-15� receptor by day 90after infection. Representative plots from three independent experimentswith similar results are shown. C, Bar diagrams show the frequency ofIL-2/IL-15�-expressing cells for the two epitopes described above. Errorbars, SD. �, p � 0.05.

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This progressive decrease in T cell function during viral persis-tence has been partially attributed to triggering of inhibitory path-ways (17, 64). To compare the functional capabilities of �HV68primary and long-term CD8� T cell responses, we sought to de-termine their effectiveness in IFN-� secretion or in cytotoxic kill-ing. These two functions were chosen because they characterizethe early (cytotoxicity) and final (IFN-�) stages of CD8� T cellfunctional impairment (20).

We measured the capacity of �HV68-specific CD8� T cells tosecrete IFN-� in response to saturating and limiting availability ofpeptide at early and late times postinfection. The data show intra-cellular IFN-� staining of splenic ORF6487–495/Db-specific CD8�

T cells (Fig. 8A). The data points show that the normalized titrationcurves for early and late time points postinfection overlapped (Fig.8B). A better fit of IFN-� production data over peptide concentra-tion was obtained with a nonlinear sigmoidal dose-response curveanalysis (R2-early: 0.8096, R2-late: 0.9082). The comparison of fitsindicated that the preferred model was one common curve fittingall data sets independently of group and a common peptide con-centration provoking a response halfway between baseline andmaximum was determined (EC50: 2.229 � 10�4). There were nosignificant differences in IFN-�-secreting capacity of each CD8� Tcell population defined as the negative log of the peptide concen-tration that resulted in 50% maximal IFN-� production. These dataindicate that Ag persistence during �HV infection has no detri-

mental effect on the functional capacity of virus-specific CD8� Tcells to produce IFN-� in response to antigenic stimulus.

We next compared the cytotoxic activity of ORF6487–495/Db-specific CD8� T cells directly in vivo in mice 3 wk, 3 mo, and 8mo postinfection. The elimination of ORF6487–495-loaded targetswas assessed 6 and 40 h after transfer. Specific target eliminationoccurred with faster and more efficient kinetics in mice at 3 wkpostinfection, which correlated with a higher frequency ofORF6487–495/Db-specific CD8� T cells (Fig. 9). Mice at 3 and 8mo postinfection had low specific killing rates 6 h after transfer(5–14% specific killing) but were capable of killing specific targetsafter a 40-h interval (65–88% specific killing). In addition, nomajor differences in specific killing were found between mice at 3and 8 mo postinfection. It should be noted that 1) during long-terminfection, the frequency of splenic ORF6487–495/Db-specific CD8�

T cells is 5- to 20-fold lower than during acute infection (Fig. 9)and 2) at 3 and 8 m.p.i., the �HV68-specific CD8� T cell poolpredominantly consists of effector-memory cells (as shown in Fig.2). Altogether, these data indicate that CD8� T cells during per-sistent �HV68 infection are capable of mounting a specific CTLresponse in vivo although with slower kinetics that during acuteinfection. The analysis of IFN-� production and cytotoxicity pro-vides evidence of the functional capacity of virus-specific CD8� Tcells during persistent infection.

FIGURE 7. The TCR affinity of virus-specific CD8� T cells does not change from primary infection to viral persistence. A and B, Measurement of TCRaffinity in CD8�ORF6487–495/Db� cells by peptide/MHC tetramer dilution analysis. Lymphocytes were obtained from spleens of mice infected 14 daysbefore (early group) or 3 mo before (late group), pooled, enriched by panning, and stained with serial 2-fold dilutions of ORF6487–495/Db tetramerconjugated to APC at concentrations ranging from 1/50 to 1/100,000. All the cells were subsequently stained with anti-CD8 Ab. Data are representativefrom three independent experiments with similar results. A, Representative FACS plots of the CD8-ORF6487–495/Db staining. B, Graphic representation ofthe normalized frequency of tetramer binding and linear regression analysis for the experiment shown in A. To normalize the groups, the data were expressedas the percentage of maximum tetramer binding. Inset, Raw data. C and D, Measurement of TCR affinity in CD8�ORF6487–495/Db� cells as determinedby peptide/MHC tetramer dissociation. Lymphocytes were sampled and processed as above, stained with ORF6487–495/Db tetramer, washed, saturated withanti-Db Ab, and incubated at RT to allow for tetramer dissociation. At different time points cells were sampled and stained with anti-CD8 Ab. Data arerepresentative from two independent experiments with similar results. C, Representative FACS plots of the CD8-ORF6487–495/Db staining at different timepoints during tetramer decay. D, Graphic representation of the normalized frequency of tetramer binding over time and nonlinear one-phase exponentialdecay regression analysis of the experiment shown in C. To normalize the groups, the data were expressed as the percentage of maximum tetramer binding.Inset, Raw data.

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CD8� T cells generated during long-term viral persistence canmediate protection to �HV68 latency during a recall response

Memory T cells mediate faster and more effective responses tosecondary pathogen challenge than naive T cells (1, 2). To deter-mine whether T lymphocytes from �HV68-immune mice were ca-pable of preventing the establishment of viral latency, we testedthe capacity of CD8� T cells from �HV68-infected mice to protectduring a recall response. CD8� T cells were FACS purified fromBALB/c mice that were intranasally inoculated with 1000 PFU of�HV68 8 mo before. During the sorting procedure, B cells wereexcluded by CD19 staining to avoid carrying latently infected Bcells in the purified populations. A total of 4 � 105 CD8� T cellsor a 1:1 mixture of CD4� and CD8� T cells were i.v. inoculatedin 300 �l of PBS into naive BALB/c recipients. The recipient micewere intranasally inoculated with �HV68 the following day andviral splenic latency was analyzed at the peak of latently infectedB cell expansion on day 16. The results show that adoptive transferof CD8� T cells from persistently infected mice reduces the num-ber of �HV68-infected cells during the establishment of splenic

latency (Fig. 10). In addition, the data revealed that the addition ofCD4� T cells did not improve the recall response of CD8� T cellsas measured by protection. These data show that memory CD8� Tcells from �HV68-infected mice can respond to a secondary viruschallenge by decreasing the number of latently infected cells in thespleen during the establishment of latency phase.

DiscussionPersistent infectious diseases are a major health concern (13, 14).The general consensus is that chronic viral infections lead to somedegree of CD8� T cell effector dysfunction or deletion and lack ofT cell memory formation (15–17). �HVs are very successfulpathogens that persistently infect �90% of the human populationand initial infection may occur early in childhood (28). In thisreport, we demonstrate that herpesvirus-specific central-memoryCD8� T cells develop during infection and that herpesvirus-spe-cific CD8� T cells maintain functional and protective capacitiesduring long-term persistent infection.

Our results show that during persistent infection with �HV68,there are consistently less than 500 latently infected cells perspleen. Despite this very low level of viral latency, splenic den-dritic cells but not B cells are capable of presenting viral lytic-phase epitopes to �HV68-specific CD8� T cell hybridomas. Thisfinding is unexpected as germinal center and memory B cells con-stitute the major viral reservoir during long-term latency (65, 66).It suggests that dendritic cells cross-present lytic-phase viral Agsduring �HV persistence and supports the existence of continuousviral reactivation during the latency phase of infection (38, 67, 68).Reduced levels of latency and continuous presentation of lytic-epitope Ags have relevant implications for the maintenance ofCD8� T cell memory. Low-level latency together with low viralgene expression (69) probably represents a very efficient immuneescape strategy and explains the low frequency of latent-phaseepitope-specific CD8� T cells during �HV68 persistence (70).Continuous presentation of lytic viral epitopes explains the de-layed generation of central-memory CD8� T cells and why effec-tors and effector-memory cells still constitute a large fraction of theCD8� T cell response during long-term latency (50–80%, Figs. 2,4, and 5). It is possible that these lytic epitope-specific CD8� Tcells partially represent secondary effectors or de novo generatedeffectors as result of virus reactivation. In this sense, it has beenrecently suggested that low-level TCR stimulation that occurs dur-ing HSV latency results in the maintenance of an activated butfunctional CD8-memory pool (71) and that newly recruited naiveCD8� T cells influence the heterogeneous CD8� T cell responseduring polyoma virus persistence (72).

Our data show that memory CD8� T cell precursors could bedetected at early stages during primary infection and that effector-memory cells constitute the dominant CD8� T cell subset onceprimary infection has been resolved. The kinetic analysis of the�HV68-specific CD8� T cell response shows that activated CD8�

T cells (CD62LlowCD43high) contract over a period of 3 mo post-viral inoculation. This slow process can be in part explained for theprolonged infectious mononucleosis-like syndrome in �HV68-in-fected mice (73) which results in exacerbated and prolonged CD8�

T cell activation and expansion (74, 75). In addition, �HV68 per-sistent replication and reactivation from latency (34, 76, 77) willcontribute to keep virus-specific CD8� T cells stimulated. Al-though the frequency of memory CD8� T cells remains stableduring long-term viral persistence, there is a shift in the ratio be-tween effector-memory and central-memory subsets, with central-memory cells progressively increasing their frequency over time.Selective expression of IL-7R identifies CD8� T cells that gener-ate memory cells (78, 79). In addition to this marker, we have also

FIGURE 8. The functional avidity of virus-specific CD8� T cells doesnot change from primary infection to viral persistence as measured by theirIFN-� production capacity. Lymphocytes were obtained from spleens ofthree to four mice infected 5 wk before (early group) or 3 mo before (lategroup), pooled, enriched by panning, and stimulated with different con-centrations of ORF6487–495 peptide. All the cells were subsequently pro-cessed for IFN-� intracellular cytokine staining and surface stained withanti-CD8 Ab. Data are representative from three independent experiments.A, Representative FACS plots of the intracellular IFN-� staining at twopeptide concentrations for the early and late groups. B, Graphic represen-tation of the normalized frequency of tetramer binding over time and non-linear sigmoidal dose response regression analysis of the experimentshown in A. To normalize the groups, the data were expressed as the per-centage of maximum IFN-� staining at 1 �g/ml. Inset, Raw data minusbackground (isotype-control intracellular staining).

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identified memory precursors using the expression of the IL-15Rand the lymphoid homing molecules L-selectin and CCR7 (15).Our data correlate with results from acute and persistent infectionsthat demonstrate that memory cell precursors exist at the peak ofthe CD8� T cell response (39, 79, 80). The generation and stabilityof CD8� T cell memory or CD8� T cell function during Ag per-sistence is poorly understood. It has been suggested that long-termT cell memory might not develop in conditions of chronic Agstimulation (6). Infections with high load of persisting Ag result inthe suppression of IL-7 and IL-15 receptor expression and down-regulation of lymphoid homing molecules on virus-specific CD8�

T cells, and this correlates with progressive T cell exhaustion (15,20, 81). Our results show that IL-7R, L-selectin, and CCR7 ex-pression are maintained on 20–70% of Ag-specific CD8� T cellsduring viral persistence 9 m.p.i. This represents a small fraction ofthe whole CD8� population compared with that of acute respira-tory infections, where most of the CD8� T cell population is con-stituted by memory cells (82). It is also possible that small differ-ences on the level of expression of phenotypic markers between

FIGURE 9. Long-term CD8� T cells maintain cyto-toxic capacity during persistent viral infection. Cytotox-icity was assessed by an in vivo CTL assays. Spleencells from naive mice were loaded with �HV68ORF6487–495 or with influenza NP peptides and stainedwith different amounts of CFSE before injection intonaive control mice or into mice at different time pointsafter �HV68 inoculation. Killing was calculated 6 h (leftcolumn) or 40 h (middle column) after transfer. Histo-grams show representative killing data from the analysisof individual naive mice and from mice at 3 wk, 3 mo,and 8 m.p.i. Bar diagrams show specific killing datafrom four to five individual mice per time point. Thefrequency of ORF6487–495/Db�CD8� T cells was deter-mined by FACS staining of spleen cells and is shown onthe right column as representative dot plots and as a bardiagram. Error bars, SD.

FIGURE 10. Long-term CD8� T cells can a mediate protective recallresponse to �HV latency. Spleen cells from BALB/c donor mice8 m.p.i. were sorted by FACS. A total of 4 � 105 immune CD8� or a1:1 mixture of CD4�/CD8� cells was adoptively transfer by i.v. injec-tion into naive mice. Control mice received no cells. Mice were chal-lenged with �HV68 24 h later. The number of latently infected spleencells was determined by an infectious center assay on day 16 afterchallenge. Error bars represent SD.

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memory cells generated during acute infections or �HV68 persis-tence may be biologically relevant. Nevertheless, virus-specificCD8� T cells with characteristics of memory cells are maintainedduring long-term persistent �HV68 infection, which suggests thatT cells may not need to fully rest from Ag exposure during con-ditions of low-level Ag persistence to differentiate into memory Tcells expressing lymphoid homing molecules and homeostatic cy-tokine receptors. It should be noted that differences in Ag load, Tcell precursor frequency, or TCR avidity can also influence theexpression of various phenotypic markers (such as CD62L andIL-7R) and the capacity to generate a diversity of cytokines (suchas IFN-�, TNF-�, and IL-2), suggesting that the phenotype ofmemory T cells is ultimately a consequence of molecular eventsoccurring during the Ag-driven phase of the response (83, 84).

IL-15 is implicated in CD8� T cell proliferation, survival, andavidity (56, 85, 86). Our data indicate that IL-2/IL-15� receptorexpression is down-regulated on lytic epitope-specific CD8� Tcells during long-term �HV68 persistence, which contrasts with itsexpression on latent epitope-specific CD8� T cells. Interestingly,EBV infectious mononucleosis induces a permanent deficit in IL-15R expression on CD8� T cells (87). It has been shown that�HV68-specific CD8� T cell maintenance can be independent ofIL-15 (88). The functional implications of impaired IL-15 signal-ing during �HV infections are unclear and we could not find func-tional deficiencies at the level of IFN-� production and cytotoxiccapacity on lytic epitope-specific CD8� T cells. However, func-tional and phenotypic differences between latent and lytic Ag-spe-cific CD8� T cells have been described elsewhere (89–91).

The interaction between herpesviruses and the host presents sev-eral specific characteristics that are relevant to the analysis of theCD8� T cell response: 1) continuous presence of persistent Agmay impact the evolution of the cognate response, 2) �HV persis-tence is characterized by low-level viral latency and antigenic load,and T cells should compete for this limited resource, and 3) anti-genic mutation is not likely to affect the T cell response. Our find-ings indicate that Ag persistence during �HV68 infection does notmodify TCR affinity and it has no detrimental effect on the func-tional capacity of virus-specific CD8� T cells to produce IFN-� orto kill in response to antigenic stimulus. These results are in con-cordance with data from murine CMV infection that suggest thatCD8� T cell avidity does not change over time (92) and withstudies that indicate that the TCR memory repertoire to persistentherpesvirus infections, although heterogeneous, can be remarkablystable (50, 93). Altogether, these findings derived from herpesvirusinfections argue against the idea that the continuous presence ofAg will eventually drive T cells into some degree of functionalexhaustion (16, 20, 21). These differences are likely to be ex-plained by the low-level Ag persistence during herpesvirus infec-tions. In this sense, during HSV latency in neurons, virus-specificCD8� T memory cells retain their capacity to produce IFN-� (94).Although the TCR usage between T cells during primary andmemory �HV responses is similar, there is a shift in clonotypesduring memory formation (95). �HV68 persistence has also beenassociated with enhanced effector functions when compared withnonpersistently infected mice (96). These conflicting results mayreflect differences in the experimental systems and differences be-tween human and murine studies. Regardless, these studies shedlight on the idea that CD8� T cells during herpesvirus persistencemaintain their functional ability to control infection and suggestthat low-level Ag persistence may not intrinsically be a detrimentalfactor for T cell function. It should also be noted that this processmight be mediated by the continuous recruitment of naive T cellsduring viral persistence (72). Our analysis of CD8� T cell protec-tion using adoptive transfers allowed us to determine the specific

contribution of long-term T cell populations to the control of virallatency. Although Ag persistence has been linked to poor CD8� Tcell cytotoxic and recall responses (15), this was not the case dur-ing �HV68 infection. CD8� T cells from persistently infectedmice were capable of mounting cytotoxic responses in vivo, albeitwith slower kinetics than during acute infection. These differencescan be explained by the reduced frequency of Ag-specific CD8� Tcells and the contraction of effector cells after 3 m.p.i. In addition,long-term CD8� T cells were capable of eliciting protectionagainst the establishment of viral latency after adoptive transfer,which supports the maintenance of CD8� T cell function during�HV68 persistence. Our findings indicate that protective memorycan be generated during herpesvirus infections and suggest that themodel of CD8� T cell linear differentiation that postulates central-memory T cells as the end of a maturation process (12) also applieswith an extended time frame to infections with low levels of per-sistent Ag. These findings have important implications for our un-derstanding of immune control and protection against pathogens orAgs that persist in the host and for the design of vaccinationstrategies.

AcknowledgmentsWe thank C. McAllister and the Morphology Core for assistance withFACS sorting, J. Haynes and the Biostatistics Core for help with the sta-tistical analysis, J. Cyster for CCL19 Ig, C. Walker for critical commentsto the manuscript, and M. Blackman for her continued support.

DisclosuresThe authors have no financial conflict of interest.

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