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T Cell Exhaustion+Sufficient to Drive CD8Chronic Antigen Stimulation Alone Is

Yvonne M. Mueller and Peter D. KatsikisChristine M. Bucks, Jillian A. Norton, Alina C. Boesteanu,

http://www.jimmunol.org/content/182/11/6697doi: 10.4049/jimmunol.0800997

2009; 182:6697-6708; ;J Immunol

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Chronic Antigen Stimulation Alone Is Sufficient to Drive CD8�

T Cell Exhaustion1

Christine M. Bucks, Jillian A. Norton, Alina C. Boesteanu, Yvonne M. Mueller,and Peter D. Katsikis2

The failure of CD8� T cells to respond to chronic infection has been termed “exhaustion” and describes the condition in whichCD8� T cells exhibit reduced differentiation, proliferation, and effector function. CD8� T cell exhaustion has been extensivelystudied in the murine model of chronic infection, lymphocytic choriomeningitis virus (LCMV). Although LCMV-based studieshave yielded many interesting findings, they have not allowed for discrimination between the roles of cytokine- and Ag-drivenexhaustion. We have created a system of chronic Ag stimulation using murine influenza A virus that leads to exhaustion andfunctional disability of virus-specific CD8� T cells, in the absence of high viral titers, sustained proinflammatory cytokine pro-duction and lymphocyte infection. Our findings show that Ag alone is sufficient to drive CD8� T cell impairment, that Ag-drivenloss of virus-specific CD8� T cells is TRAIL mediated, and that removal of Ag reverses exhaustion. Although programmed death1 was up-regulated on chronic Ag-stimulated CD8� T cells, it played no role in the exhaustion. These findings provide a novelinsight into the mechanisms that control functional exhaustion of CD8� T cells in chronic infection. The Journal of Immunology,2009, 182: 6697–6708.

I t is firmly established that during the course of many acuteviral infections, the adaptive immune response, in particularCD8� T cells, is critical for effective viral clearance (1–3).

The failure of CD8� T cells to fully function and eliminate virusmay be responsible, in part, for the establishment of chronic viralinfection. Indeed, defective CD8� T cell function has been docu-mented in HIV (4–7), hepatitis B (8, 9) and hepatitis C (10, 11)chronic viral infections. Recognized T cell defects span a broadrange of functions, from impaired differentiation, proliferation, andeffector function (5, 12–14) to increased susceptibility to apoptosis(15). Given the global significance of chronic viral infections, it isincreasingly important to understand the defects that exist in ex-hausted CD8� T cells, as well as the mechanisms that lead to thisexhaustion.

There are several mouse models of chronic viral infection thatlead to the development of functionally inactive, exhausted effec-tor and memory CD8� T cells. CD8� T cell exhaustion has beendocumented following infection with lymphocytic choriomeningi-tis virus (LCMV)3 (16–20), murine hepatitis (21) and polyomavi-rus (22). Among these, LCMV infection is one of the most prom-inently studied chronic infections. During LCMV infection,

chronic infection is achieved by infection of wild-type mice withhigh doses of rapidly replicating virus (18–20), or by infection ofimmunosuppressed mice that lack functional CD4� T cells (16,23), or perforin (24). Under these conditions, there is a profoundloss of virus-specific CD8� T cell expansion, and an accompany-ing loss of IL-2, IFN-�, and TNF-� cytokine production by CTLsthat do survive (19, 20). Exhaustion impacts multiple facets ofvirus-specific CD8� T cell function. Accordingly, exhaustedCD8� T cells also exhibit a loss of cytotoxic function (19), anddecreased mitogen induced proliferation (23).

Although many significant findings have been made usingLCMV infection, previous studies have been unable to examinethe contribution of individual factors to CD8� T cell exhaustion.LCMV is characterized by the presence of sustained high viralloads, proinflammatory cytokine burst, and lymphocytic viral tro-pism (25, 26). Their combined presence prevents the delineation ofthe relative contribution of individual factors to clonal exhaustion,and therefore the capability of any of these individual events todrive exhaustion remains unknown. To address this issue, we cre-ated a novel system of chronic Ag stimulation, using the influenzaA virus. In this model system, CD8� T cells are repeatedly stim-ulated, in vivo, with live influenza virus. Live influenza virus wasused, rather than inactivated or repeated peptide stimulation, toconserve all aspects of physiologically relevant priming of CD8�

T cells. The continuous stimulation of CD8� T cells, in the ab-sence of high viral load and cytokine burst, allowed direct exam-ination of the contribution of chronic Ag stimulation to the devel-opment of CD8� T cell exhaustion. Our data indicate that chronicAg stimulation alone is sufficient to induce CD8� T cell exhaus-tion, as shown by the failure of virus-specific CD8� T cells tore-expand or produce IFN-� upon viral rechallenge. Interestingly,the virus-specific CD8� T cell response was restored eitherthrough a period of Ag rest or through inhibition of the TRAILapoptosis pathway. Taken together, these data indicate that Agalone is sufficient to drive CD8� T cell exhaustion and may sug-gest that during more complex chronic infection, Ag stimulationshould be a primary target for control.

Department of Microbiology and Immunology, Drexel University College of Medi-cine, Philadelphia, PA 19129

Received for publication March 27, 2008. Accepted for publication March 18, 2009.

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 by Grants R01 AI62437, AI46719, and AI66215 awardedby the National Institutes of Health (to P.D.K.).2 Address correspondence and reprint requests to Dr. Peter D. Katsikis, Department ofMicrobiology and Immunology, and Institute for Molecular Medicine and InfectiousDisease, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA19129. E-mail address: [email protected] Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; DC,dendritic cell; i.n., intranasal; NP, nuclear protein; MLN, mediastinal lymph node;MFI, mean fluorescence intensity; TCID50, 50% tissue culture infective dose; Tg,transgenic; PD-1, programmed death 1; PD-L1, programmed death ligand 1.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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Materials and MethodsMice and reagents

Female, 8-wk-old, C57BL/6 mice were purchased from The Jackson Lab-oratory and used in all Ag stimulation and intranasal (i.n.) infections de-scribed. Female 8-wk-old OT-I TCR transgenic (Tg) mice, a gift from H.Shen (University of Pennsylvania, Philadelphia, PA), were bred at DrexelUniversity College of Medicine (Philadelphia, PA) and used as donors forH1N1 WSN-OVA experiments. For TRAIL and MRL/lpr Fas-deficientstudies, 8-wk-old TRAIL knockout mice (Amgen), MRL/lpr (The JacksonLaboratory), or C57BL/6 control mice were Ag-stimulated and rechal-lenged as described below. All mice were maintained in an American As-sociation of Laboratory Animal Care-accredited facility and acclimatizedfor 1 wk before use. All experiments were completed following Institu-tional Animal Care and Use Committee approval. Chronic Ag stimulationand i.n. influenza rechallenge were achieved using H1N1 A/Puerto Rico/8/34 (PR8) or H3N2 HKx31 (x31), a gift from W. Gerhardt (University ofPennsylvania, Philadelphia, PA) or recombinant OVA-expressing H1N1A/WSN/33 (WSN-OVA), a gift from D. Topham (University of Rochester,Rochester, NY).

Chronic Ag stimulation and influenza infection

Female C57BL/6 mice were i.p. primed with 3 � 106 50% tissue cultureinfective dose (TCID50) influenza A virus H1N1 strain PR8 or 1 � 106

TCID50 WSN-OVA. Control mice received a single i.p. injection on day 0;chronic mice received two injections per week for a minimum of 4 wk. Fori.n. rechallenge, mice were anesthetized with Avertin (2,2,2 tribromoeth-anol) at 240 mg/kg (Acros Organics) and then i.n. infected with 0.2 TCID50

x31 or 40 TCID50 WSN-OVA. For chronic Ag rest studies, mice werechronically i.p. primed with WSN-OVA for 30 days and then Ag rested for45 days before influenza i.n. rechallenge.

OT-I naive T cell and day 30 virus-specific cell adoptivetransfer

A total of 1 � 105 OT-I OVA257–264 TCR Tg T cells were suspended insterile injectable saline, and i.v. transferred to naive C57BL/6 mice. Trans-ferred mice were then either single or chronic Ag-stimulated as previouslydescribed. For rechallenge, splenocytes were isolated from the spleens ofWSN-OVA single or chronic Ag-stimulated mice. Splenocytes were sur-face stained to identify OVA TCR Tg V�2 and V�5.1 double positive(eBioscience) CD8� T cells, as previously described (27). A total of 5 �104 day-30 virus-specific CD8� T cells were then i.v. transferred, in 100 �lof sterile injectable saline solution, to naive C57BL/6 mice. At 24 h aftertransfer, host mice were i.n. challenged with influenza virus as described.For tracking studies, day-30 virus-specific CD8� T cells (Thy1.2�) wereCFSE-labeled and transferred to naive Thy1.1� mice. Recipients were i.n.challenged with virus on day 1 after transfer, or remained uninfected.

Determination of influenza viral titers and tissue cytokines

Influenza viral titers and proinflammatory cytokine production were deter-mined in the lung, spleen, kidney, and liver by real-time PCR. Tissues werefrozen at �80°C in 1 ml of TRIzol/50 mg tissue (TRI-Reagent; MolecularResearch Center). At time of RNA isolation, tissues were thawed on iceand homogenized, and RNA was extracted according to Molecular Re-search Center specifications. Fragmented RNA was removed using QiagenRNeasy kit. For viral titers, purified RNA was reverse transcribed to cDNAusing random hexamers and matrix protein-specific primer (5�-TCT AACCGA GGT CGA AAC GTA-3�). The resulting cDNA samples were used,in triplicate, for real-time PCR. For the reverse-transcribed assay, matrixprotein-specific primers and FAM probe were used in combination with 2XTaqman Universal PCR mix (Applied Biosystems). Primers used were asfollows: 900 nM influenza matrix protein (sense) 5�-AAG ACC AAT CCTGTC ACC TCT GA-3�, (antisense) 5�-CAA AGC GTC TAC GCT GCAGTC C-3� and 200 nM influenza probe FAM-5�-TTT GTG TTC ACGCTC ACC GT-3�-TAMRA. For cytokine measurements, cDNA was re-verse transcribed and assayed in triplicate by real-time PCR. For real-timeassay, IL-1-, IL-6-, IL-12-, and TNF-�-specific primers were used in com-bination with 2X SYBR Universal PCR mix. Primers for cytokine were asfollows: IL-1 (sense) 5�-CAA CCA ACA AG GAT ATT CTC CAT G-3�and (antisense) 5�-GAT CCA CAC TCT CCA GCT GCA-3�; TNF-�(sense) 5�-CCA GAC TCT TCC CTG AGG TG-3� and (antisense) 5�-CAAGGT AGA GAG GCC AGG TG-3�. For IL-6 and IL-12, RT2 qPCR Primerassay was used (SABiosciences). The specificity of all primer binding wasconfirmed by SYBR labeling and dissociation analysis. ABI Prism 7900HTsequence detection system with SDS 2.2.1 software (Applied Biosystems)was used for detection and analysis. Viral loads were calculated based on

influenza viral stock standard curve and are reported as TCID50 U/tissue.Cytokine production was determined as fold change over healthy tissueusing the equation (2CT)�1 influenza dosed/(2CT)�1 healthy control.

Lymphocyte preparation

Lung, mediastinal lymph node (MLN), and spleen single cell suspensionswere prepared as previously described, by Halstead et al. (28). In brief,lung tissue was digested with 3 mg/ml collagenase A and 150 �g/mlDNase I in RPMI 1640 containing 5% FBS, for 2 h at 37°C. Digested tissuewas filtered over a 40-�m nylon mesh, the mononuclear cell populationwas purified over Lympholyte-M density gradient (CedarLane Laborato-ries). MLN were disrupted by passing through a 40-�m nylon mesh andfiltered over 20-�m nylon mesh. The spleen was disrupted under 20-�mnylon mesh. Cells were washed one time in RPMI 1640 containing 5%FBS. RBC were lysed through addition of 900 �l of sterile dH2O to thesplenocyte pellet. Lysis was stopped with 100 �l of sterile 10X PBS and9 ml of RPMI 1640 containing 5% FBS; cells were washed and filteredbefore counting. Femur bones were harvested and flushed with RPMI 1640containing 3% to remove lymphocytes. Cells were washed, and RBC werelysed as described for spleen. One lobe of liver was harvested and me-chanically disrupted under mesh, then digested in 3 mg/ml collagenase I for1 h at 37°C. The cells were then purified by Percoll gradient. Live and deadcounts of mononuclear cells from all tissues was performed by acridineorange (3 �g/ml) ethidium bromide (5 �g/ml) stain and UV lightmicroscopy.

Flow cytometry

Virus-specific CD8� T cells were identified by multicolor flow cytometry,by either MHC class I H-2Db nuclear protein (NP)366–374 tetramer staining,provided by and produced with the assistance of J. Altman (Emory Uni-versity School of Medicine, Atlanta, GA) or TCR-specific anti-V�2-FITCor anti-V�2-allophycocyanin and anti-V�5.1-PE (eBioscience) staining.Additional phenotyping was performed using the following surface andintracellular Abs: anti-CD8-PerCP (BD Biosciences), anti-CD8-AlexaFluor 405 (Caltag Laboratories), anti-CD69-PE-Cy7 (BD Biosciences), anti-CD25-allophycocyanin (eBioscience), anti-CD279-FITC (eBioscience),and anti IFN-�-allophycocyanin (eBioscience). Cells were stained as pre-viously described (28) and fixed in 1% paraformaldehyde. Stained cellswere acquired using a FACSCalibur or FACSAria (BD Biosciences) andanalyzed using Flow Jo (Tree Star).

Peptide stimulation

A total of 1 � 106 lung mononuclear cells were cultured for 6 h at 37°Cin the presence of 10 �g/ml brefeldin A and the presence or absence of 1�g/ml OVA257–264 peptide (SIINFEKL) or 1 �g/ml NP366–374 (ASNENMETM). For subdominant epitopes, cells were cultured with M1128–135

(MGLIYNRM) or nonstructural protein NS2114–121 (RTFSFQLI). All pep-tides were purchased from ANASPEC. Cells were intracellular stained forIFN-� production and analyzed as described in flow cytometry methods.

Programmed death (PD)-1 studies

Single or chronic stimulated C57BL/6 mice were treated with functionalgrade, anti-mouse B7H-1, clone MIH5, or with control rat IgG2a isotype(eBioscience). Mice were treated with 100 �g of anti-B7H-1 or isotypecontrol every third day from days 0–30 of single or chronic Ag stimulation.At the completion of Ag stimulation, the frequency and absolute number ofday-30 virus-specific memory cells was assessed by flow cytometry. Equalnumbers of day-30 virus-specific cells from isotype or anti-B7H-1-treatedsingle or chronic mice were transferred to naive mice. Recipient mice wererechallenged with influenza virus as previously described and harvested atthe peak of the secondary response on day 7.

Statistical analysis

JMP statistical analysis program (SAS) was used to perform Student’s ttest, nonparametric Wilcoxon signed-rank test for paired samples and Sha-piro-Wilk W test for normality. A value of p � 0.05 was consideredsignificant.

ResultsChronic influenza A stimulation does not induce systemicinfection or inflammation

To examine whether chronic Ag stimulation, in the absence ofsustained inflammation, induced physical deletion and/or exhaus-tion of virus-specific CD8� T cells we developed a system of

6698 CHRONIC Ag STIMULATION DRIVES CD8� T CELL EXHAUSTION


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chronic Ag stimulation that used murine adapted influenza virus.Control mice received a single injection of influenza A virus,whereas chronic mice received two injections per week for a min-imum of four consecutive weeks. For both single and chronic Agstimulation, virus was delivered by i.p. injection of either 3 � 106

TCID50 murine adapted influenza A strain H1N1 A/PR/8 (PR8) or1 � 106 TCID50 genetically modified A/WSN/33 expressing Kb-restricted OVA257–264 (WSN-OVA). The i.p. injection was chosenas the route of delivery because it does not result in pulmonaryinfection and disease.

Because live infectious virus was used to achieve chronic Agstimulation of CD8� T cells, we first sought to determine whethersystemic infection was induced following repeated i.p. delivery ofinfluenza virus. To assess this, mice were either i.p. injected (i.p.primed) or i.n. infected with PR8 virus (3 � 106 TCID50 and 3TCID50, respectively) and harvested on days 1, 3, and 5 after virusdelivery. Weight loss was used as an indicator of productive in-fluenza infection and was recorded daily for both i.n. and i.p. in-jected mice during the first 5 days following Ag delivery. The i.p.primed mice maintained constant weight, whereas i.n. infectedmice rapidly lost up to 22% of their body weight over 5 days (Fig.1A). The absence of weight loss in i.p. primed mice, in comparisonto i.n. infected mice, suggested that i.p. priming did not induceproductive infection or inflammation. To directly examine whetheror not productive infection was established in i.p. primed mice, the

viral load was quantified using real-time PCR. The mRNA wasisolated from lungs, spleen, liver, intestine, and kidney at days 1,3, and 5 after PR8 delivery, and the conserved matrix 1 protein ofinfluenza virus was quantified by real-time PCR to determine thequantity of virus in each tissue. Influenza virus primarily infectspulmonary epithelial cells and alveolar macrophages; therefore asexpected, the lungs of i.n. infected mice exhibited a 4 log increasein virus over the 5-day time period, indicating that productive in-fection and virus replication had occurred (Fig. 1B). In contrast,there was a limited quantity of viral RNA in the first 3 days in thelungs and spleen of mice that received influenza virus through i.p.injection. Due to both the very high 3 � 106 TCID50 i.p. dose used,and the virus decay kinetics, the presence of virus in the lungs onday 3 most likely reflects virus circulating in the lung vasculatureand not lung parenchyma infection. By day 5, virus was below 10TCID50 in all tissues examined, indicating that i.p. injection ofinfluenza virus did not result in a productive systemic infection(Fig. 1B).

Because proinflammatory cytokines may induce bystander Tcell activation (29), increase dendritic cell (DC) maturation (30),and increase rate of CD8� T cell contraction (31), we were inter-ested in determining the cytokine profile after priming with PR8virus. Specifically, we determined transcriptional changes inseveral proinflammatory cytokines produced during influenza in-fection, including IL-1, IL-6, IL-12, and TNF-�. The presence of

FIGURE 1. The i.p. priming withPR8 did not lead to morbidity, sys-temic infection, cytokine burst, orchronic inflammation. C57BL/6 micewere i.n. infected or i.p. primed with3 TCID50 or 3 � 106 TCID50 influ-enza virus strain PR8. A, Weights ofi.n. infected and i.p. primed micewere recorded daily (n � 5 mice/group). B, Viral load was measuredby real-time PCR in the lung, spleen,kidney, and intestine on days 1, 3, and5 after virus delivery (n � 3 mice/group for each time point). C, Thelevel of mRNA was quantified, by re-al-time PCR, for IL-1, IL-6, IL-12,and TNF-� in the lung, spleen, intes-tine, and liver of i.p. primed or i.n.infected mice. D, Cytokine produc-tion (mRNA) quantified at day 30 af-ter single or chronic Ag stimulation.Data are fold increase over healthyuninfected control lung mRNA (n �3 mice/group per time point).

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these cytokines was determined by real-time PCR in the spleen andlungs of uninfected healthy control, i.p. primed, and i.n. infectedmice. In the lungs of i.n. infected animals, both IL-1 and TNF-�mRNA expression were substantially increased relative to expres-sion in healthy uninfected lungs (32.44 � 0.34 fold and 19.06 �0.64 fold over healthy controls by day 3, for IL-1 and TNF-�,respectively) (Fig. 1C, top right). IFN-� also increased a maxi-mum of 2.74 � 0.74 fold over uninfected healthy lungs by day 1(data not shown). The limited increase in IFN-� is not surprisinggiven that influenza virus NS1 protein has been shown to inhibitIFN-� production (32). In striking contrast, there was no signifi-cant increase in cytokine production in the lungs of i.p. primedmice (Fig. 1C, top left). This finding is particularly significant be-cause virus was detected in the lungs of i.p. primed mice at days1 and 3 postinjection. The absence of inflammatory cytokines, de-spite the presence of virus, most likely indicates an absence ofinfection in the lungs of i.p. primed mice.

Because virus was i.p. delivered for chronic Ag stimulation, thecytokine profile in peripheral tissues was also determined. Formost cytokines and tissues examined, the greatest increase in cy-tokine mRNA was observed 1 day after virus delivery (Fig. 1C,bottom). The kinetics of IL-6 and IL-12 expression in the spleenvaried from other cytokines examined, with IL-6 and IL-12 ex-pression peaking at day 3 (1.31 � 0.45 and 2.8 � 0.36, fold in-crease in IL-6 and IL-12, respectively). Importantly, these early,transient peaks of inflammation were not sustained, and transcriptwas comparable to naive controls by day 5.

Although i.p. injections of virus induced only transient andsmall increases in proinflammatory cytokines, it was important toexclude that repeated delivery of virus in chronic stimulated micedid not result in differences in cytokine production. Therefore, wedetermined the cytokine profile in the peripheral tissues of singleand chronic Ag-stimulated mice at day 30. The profile of IL-1,IL-6, IL-12, and TNF-� expression was comparable between sin-gle and chronic Ag-stimulated mice, in all tissues examined (Fig.1D), suggesting that chronic stimulation did not result in a chronicinflammation. With the exception of IL-6 in the lungs of single andchronic Ag-stimulated mice, all cytokine amounts were less than2-fold increased over uninfected healthy controls. Perhaps mostimportantly, there was no statistically significant difference inmRNA transcripts between single and chronic Ag-stimulated micefor any of the cytokines examined.

The repeated delivery of virus could impact the number or abil-ity of DC and thus indirectly affect the CD8� T cell response.Therefore, we determined the frequency and phenotype of DC inthe spleens of single and chronic Ag-stimulated mice by flow cy-tometry. At day 30, DC from both single and chronic Ag-stimu-lated mice were comparable in terms of frequency and phenotype.The number of CD11c�CD11b� and CD11c�CD11b� DC inthe spleens of chronic Ag-stimulated mice were not statisticallydifferent from single prime controls (CD11c�CD11b�: 1.97 �0.33 � 105 vs 2.23 � 0.4 � 105; CD11c�CD11b�: 6.63 �1.9 � 105 vs 5.67 � 1.4 � 105 for chronic vs single, respec-tively, n � 3 mice/group). Mean fluorescence intensity (MFI) ofCD86 expression was also similar between these mice(CD11c�CD11b�: 88.5 � 12 vs 98.5 � 17.3;CD11c�CD11b�: 96 � 15 vs 123 � 16; chronic and single,respectively, in n � 3 mice/group). The MFI of MHC class IIexpression on these DC was also comparable between singleand chronic Ag-stimulated mice (CD11c�CD11b�: 1260 �22.4 vs 1221.3 � 126.6; CD11c�CD11b�: 2204 � 152 vs2123 � 174; chronic and single, respectively, n � 3 mice/group). These data indicate that the chronic Ag stimulation reg-iment did not negatively impact DC numbers or phenotype.

Taken together, these results indicate that repeated i.p. de-livery of virus did not result in sustained systemic infection orproinflammatory cytokine production and does not alter DCnumbers or phenotype. Therefore, this system allowed us tostudy the effects of chronic Ag stimulation on virus-specificCD8� T cell development and secondary expansion in the ab-sence of chronic inflammation.

Reduced quantity of virus-specific CD8� T cells after chronicAg stimulation

Having demonstrated that i.p. injection of live influenza A virusdid not lead to sustained cytokine production or systemic infection,we sought to determine whether chronic Ag stimulation alonecould induce defects in the development of virus-specific CD8� Tcells. To test this, 1 � 105 naive CD8� T cells from TCR Tg OT-Imice were transferred i.v. to naive C57BL/6 mice. Recipients thenreceived either a single i.p. injection of WSN-OVA virus (106

TCID50) or were chronic Ag-stimulated, by i.p. injection two timesa week for four consecutive weeks. After 30 days, virus-specificCD8� T cells in the spleen were examined by multicolor flowcytometry. Interestingly, chronic Ag-stimulated mice exhibited anearly 2-fold reduction in frequency and number of virus-specificCD8� T cells, at day 30, when compared with single prime controlmice (frequency: 4.94 � 0.46% and 2.61 � 0.12% in single andchronic Ag-stimulated, respectively; absolute number: 3.32 �0.32 � 105 and 1.54 � 0.15 � 105 in single and chronic Ag-stimulated, respectively; n � 20 mice/group and n � 23 mice/group, p � 0.001) (Fig. 2, A and B). Kinetic analysis of virus-specific CD8� T cells in single and chronic Ag-stimulated miceindicated that significant differences in frequency and absolutenumber occurred as early as 14 days after initial priming (Fig. 2C).These kinetic data suggest that chronic Ag-stimulated cells mayundergo an increased rate of contraction relative to single primecontrols. Closer examination of the day 30 virus-specific chronicAg-stimulated CD8� T cells showed that they exhibited skewedmemory differentiation compared with single prime controls. Spe-cifically, there was a significantly greater frequency of memory,CD44�CD62L� virus-specific CD8� T cells in single prime con-trols compared with chronic Ag-stimulated mice (55.4 � 4.0% vs32.9 � 3.8% single and chronic, respectively; p � 0.001, n � 9mice/group) (data not shown). Interestingly, chronic Ag-stimu-lated CD8� T cells were evenly distributed between CD44� andCD44� memory cells (32.9 � 3.8% and 36.1 � 6.7% CD44� andCD44� memory, respectively; n � 9 mice/group) (data notshown).

Previous studies using LCMV, indicate that virus-specificCD8� T cells in chronically infect mice have altered migration inboth lymphoid and nonlymphoid tissues, when compared withacutely infected animals (20). Therefore, we examined the distri-bution of virus-specific CD8� T cells in the MLN, bone marrow,lung, and liver. In contrast to LCMV (20), the decreased numberof virus-specific cells in the spleen did not reflect impaired migra-tion of chronically stimulated cells, as similar decreases were ob-served in the MLN, bone marrow, lung, and liver (Fig. 2D). Im-portantly, this loss of chronically stimulated cells was observed inboth frequency and absolute number when compared with singleprime controls.

To determine whether the reduction in day-30 virus-specificCD8� T cells was restricted to the OVA TCR Tg system, endog-enous CD8� T cells underwent chronic Ag stimulation with theinfluenza A virus strain PR8. After 30 days, the virus-specificpopulation in the spleen was identified by tetramer staining forinfluenza immunodominant epitope-specific NP366-positive T



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cells. Interestingly, there was no significant decrease in the fre-quency (Fig. 3) or absolute number (Fig. 3B) of NP366-positiveCD8� T cells (frequency: 2.08 � 0.32% and 1.89 � 0.3% singleand chronic, respectively; absolute number: 2.49 � 0.44 � 105 and2.44 � 0.51 � 105 single and chronic, respectively; n � 12 mice/group). There are several possible reasons for this disparity. Pre-vious studies indicate that the initial precursor frequency may dic-tate the rate of development and phenotype of memory CD8� Tcells (33). Therefore, transfer of OT-I TCR Tg T cells at a higherprecursor frequency, may have changed the rate of expansion andcontraction of the virus-specific CD8� T cell population, com-pared with priming of endogenous CD8� T cells. A second pos-sibility may be the inability of the host to generate new OVA-specific CD8� T cells during the chronic stimulation. Duringchronic Ag stimulation with PR8 virus, new thymic CD8� T cellemigrants can be generated, and replenish dying chronic Ag-stim-ulated cells. In contrast, there is a finite population of transferredOT-I TCR Tg cells that can be stimulated by WSN-OVA. If stim-ulation induces death, the population cannot be replenished byendogenous CD8� T cells, and will be reduced. Indeed, a recent

report by Vezys et al. (34) elegantly showed that chronic infectionresults in a heterogeneous population of virus-specific CD8� Tcells that is maintained by the continuous release of naive cellsfrom the thymus.

Deficient quality of virus-specific CD8� T cells followingchronic Ag stimulation

Having observed only limited changes to the quantity of day-30virus-specific CD8� T cells, in both frequency and number, wewere interested in determining the effect that chronic Ag stimula-tion had on the quality of these cells. Specifically, we sought todetermine whether chronic Ag stimulation altered the ability ofvirus-specific CD8� T cells to respond to secondary viral chal-lenge. Because the size of the memory population directly influ-ences the magnitude of the secondary response (35), and chronicAg stimulation with WSN-OVA resulted in a 2-fold reduction inthe number of day 30 virus-specific CD8� T cells, it was necessaryto correct for the difference in population size before influenzarechallenge. Normalization was achieved by transferring an equalnumber (5 � 105) of day-30 chronic Ag-stimulated or single primevirus-specific CD8� T cells to naive C57BL/6 mice. Recipientmice were then i.n. challenged with 40 TCID50 WSN-OVA. Micewere harvested at the peak of the secondary response on day 7, andthe virus-specific CD8� T cells in the lung, draining MLN, andspleen were examined by multicolor flow cytometry.

Despite transfer of an equal number of day-30 single or chronicAg-stimulated virus-specific CD8� T cells, there was a significantreduction of virus-specific CD8� T cells in the lungs of chronicAg-stimulated mice, at the peak of the secondary response. In sin-gle prime controls, 30.2 � 2.89% of the CD8� T cells in the lungwere virus-specific, whereas chronic Ag-stimulated mice had only5.2 � 1.3% virus-specific CD8� T cells (n � 9 mice/group, p �

FIGURE 2. Chronic Ag stimulation reduced the quantity of day-30 vi-rus-specific CD8� T cells. OT-I transferred C57BL/6 mice were single orchronic stimulated with WSN-OVA. On day 30, the frequency of virus-specific CD8� T cells was determined by flow cytometry. A, Representa-tive flow cytometry virus-specific CD8� T cells (gated on CD8� T cells).B, Reduction in virus-specific CD8� T cells was observed in pooled fre-quency and absolute number (n � 15 mice/group, p � 0.001). C, Kineticanalysis of virus-specific OT-I cells during 30 days of priming (n � 3mice/group). D, Reduction of chronic CD8� T cells in lymphoid and non-lymphoid tissues indicates migration is not impaired.

FIGURE 3. Chronic Ag stimulation of endogenous CD8� T cells withPR8 did not significantly reduce the day-30 population. C57BL/6 micereceived either a single i.p. injection or chronic Ag stimulation with influ-enza A virus strain PR8. After 30 days of stimulation, spleens were har-vested and the frequency and absolute number of NP366-positive CD8� Tcells was assessed by multicolor flow cytometry. A, Representative FACSplots (left) show that there was no significant reduction in the frequency ofday-30 virus-specific CD8� T cells in chronic Ag-stimulated mice com-pared with single prime controls. Pooled frequency (right) indicates a trendtoward a decrease in chronic stimulated CD8� T cells, though no signif-icance is reached. B, Pooled absolute numbers indicate that independent ofchronic Ag stimulation the number of day 30 virus-specific CD8� T cellsremains comparable.



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0.002) (Fig. 4, A and B). Correction for absolute numbers illus-trated that the defect was not only in frequency, but also in num-ber, as the lungs of single prime control mice contained 5.35 �0.88 � 105 virus-specific CD8� T cells, whereas the lungs ofchronic mice had 10-fold less, with only 0.5 � 0.89 � 105 virus-specific CD8� T cells (n � 9 mice/group, p � 0.001) (Fig. 4B).The population was similarly reduced in the draining MLN and thespleen (data not shown). The reduction in the secondary lymphoid

tissues and lung confirmed that the loss of chronic Ag-stimulatedCD8� T cells, relative to single prime controls, was not due toaltered migration.

Importantly, the significant reduction in the frequency and ab-solute number of the day-7 effector population was observedwhether WSN-OVA or PR8 was used for chronic Ag stimulation.Therefore, this event was not TCR Tg-restricted. An equal numberof PR8 single or chronic Ag-stimulated CD8� T cells were trans-ferred to naive C57BL/6 mice. As before, mice were then i.n.rechallenged with influenza A virus and harvested at the peak ofthe secondary response on day 7. In the lungs of single primerecipients, virus-specific CD8� T cells represented 17.56 � 2.2%of the total CD8� T cells, whereas in chronic mice, only 7.45 �1.22% of the CD8� T cells were virus-specific (n � 12 mice/group, p � 0.0001) (Fig. 4, C and D). Additionally, the absolutenumber of chronic-stimulated virus-specific CD8� T cells was re-duced greater than 3-fold when compared with single prime con-trols (6.77 � 1.31 � 105 and 2.01 � 0.43 � 105 single and chronicstimulated, respectively, n � 12 mice/group, p � 0.001) (Fig. 4D).Given that an equal number of day-30 virus-specific CD8� T cellswere transferred to naive mice, these data strongly indicated thatchronic Ag-stimulated virus-specific CD8� T cells were defectivein quality.

During influenza infection, CTLs exert several important functionsincluding production of IFN-�. Following ex vivo OVA257–264 pep-tide stimulation, the frequency of IFN-�-producing day-7 pulmo-nary effectors from chronic Ag-stimulated mice was significantlyreduced compared with single prime controls (data not shown),confirming the tetramer- and TCR-specific surface staining results.More importantly, however, chronic Ag-stimulated CD8� T cellsexhibited significantly decreased IFN-� production on a per cellbasis when compared with single prime controls (MFI � 1447single prime Ag-stimulated and MFI � 995 chronic Ag-stimu-lated CD8� T cells, n � 6 mice/group, p � 0.001) (Fig. 5). Thisfinding indicated that qualitative defects exist beyond reducedexpansion or survival because the surviving virus-specificCD8� T cells also exhibited a significant impairment in IFN-�cytokine production.

Beyond characterizing the response to the dominant epitope, wealso determined the effect of chronic Ag stimulation on subdomi-nant epitopes. In some chronic infections, skewing of dominancein CD8� T cell responses has been observed, whereby immuno-dominant responses are lost and subdominant responses becomemore prevalent (19, 21, 36). When chronic Ag stimulation alonewas used to induce exhaustion of CD8� T cells, both immuno-dominant and subdominant responses were suppressed, although

FIGURE 4. Chronic Ag stimulation reduced the quality of virus-spe-cific CD8� T cells. An equal number of single or chronic Ag-stimulatedday-30 virus-specific CD8� T cells were transferred to naive C57BL/6mice. Recipient mice were i.n. challenged with 40 TCID50 WSN-OVA andharvested at the peak of the secondary response. Representative flow cy-tometry plots (A) and pooled frequencies and absolute numbers (B), show-ing a significant decrease in virus-specific CD8� T cells in the lung ofchronic mice compared with single prime mice. Each symbol represents asingle animal. Representative flow cytometry plots (C) and pooled frequen-cies and absolute numbers (D) show that chronic Ag stimulation with PR8 alsoresults in a significant reduction in responding virus-specific CD8� T cellsrelative to single prime controls, following i.n. viral rechallenge.

FIGURE 5. Chronic Ag-stimulated day 7 secondary effector CD8� T cellsproduced less IFN-�. IFN-� production by chronic Ag-stimulated virus-spe-cific CD8� T cells was defective compared with controls, after ex vivo stim-ulation with OVA257–264 peptide. A, Representative histogram (gated on CD8�

T cells) shows the reduction in IFN-� production by chronic Ag-stimulatedCD8� T cells relative to single prime controls. B, Each symbol represents asingle animal, pooled MFI of IFN-�-producing cells (gated on CD8� T cells)indicates decreased function of chronic Ag-stimulated CD8� T cells comparedwith single prime controls (n � 6 mice, p � 0.001).



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the most profound effect was observed in the immunodominantNP366–374 epitope. Indeed, ex vivo peptide stimulation of day-7pulmonary effectors with peptides for conserved M1128–135 andnonstructural protein NS2114–121 resulted in robust IFN-� produc-tion by single prime CD8� T cells, whereas a markedly reducedresponse was observed in chronic Ag-stimulated CD8� T cells(Fig. 6).

To confirm that chronic CD8� T cells did not simply undergoapoptosis immediately after transfer, day-30 CD8� T cells fromsingle and chronic Ag-stimulated mice were CFSE-labeled andtransferred to naive hosts. New recipients were i.n. rechallengedwith influenza virus or left uninfected. In the absence of viral in-fection, the frequency of CFSE-positive OT-I� T cells in thespleen of single and chronic recipient mice was comparable, up to5 days after transfer (Fig. 7A). The low frequency of CFSE-posi-

tive cells, 4–6% of OT-I population, found at day 5 is reasonablegiven that the endogenous frequency of V��V�� cells of the na-ive host is �1%. The identification of comparable CFSE-positivepopulations between the two groups suggests that, at least earlyafter transfer, the survival is similar. After rechallenge, all donorOT-I� cells found in the lungs of single prime mice were CFSE-negative, indicating proliferation, whereas in the lungs of chronicmice donor OT-I� cells, although capable of surviving, fail torobustly expand in response to restimulation (Fig. 7B).

Recovery of function by Ag rest of chronic Ag-stimulatedday-30 cells

If the loss of CD8� T cell quantity and function is Ag-driven, wereasoned that removal of Ag might reverse the virus-specificCD8� T cell exhaustion. Previous studies in LCMV (37) and HIV

FIGURE 6. Chronic stimulation resulted in a loss of dominant and subdominant epitope responses. C57BL/6 mice received either a single i.p. prime orchronic stimulation for 30 days with 3 � 106 TCID50 PR8. An equal number of single or chronic stimulated virus-specific CD8� T cells were transferredto naive mice. Recipient mice were i.n. rechallenged with x31 and harvested at the peak of the secondary response on day 7 (n � 3 mice/group). Pulmonarylymphocytes were stimulated ex vivo with peptides to dominant (NP366–374) or subdominant epitopes (M1128–135 or NS2114–121) for 6 h and intracellularstained for IFN-� production. Representative flow cytometry plots show that both dominant and subdominant epitope specific responses were reduced inchronic mice (bottom) compared with single prime controls (top).

FIGURE 7. Comparable survival of single and chronic stimulated CD8� T cells after transfer to naive recipients. Day 30 single or chronicAg-stimulated CD8� T cells were CFSE-labeled and transferred to naive recipients. Recipients remained uninfected or were rechallenged i.n. withWSN-OVA virus. Mice were harvested on days 5 (healthy controls) and day 7 (virus-infected). A, Comparable frequency of CFSE-positive OT-I�

T cells were found in the spleens of naive recipients receiving either single or chronic stimulated CD8� T cells. B, In the lungs of day 7 rechallengedmice, single prime CD8� T cells exhibited robust expansion, resulting in an absence of CFSE-positive cells. Chronic Ag-stimulated CD8� T cellsfailed to expand in number, and did not divide. Histograms depict CFSE stain on cells are gated on donor OT-I� T cells based on Thy1.2 and V�/V�expression.



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(38–40) show that in the absence of Ag, CD4� and/or CD8� Tcell responses can be partially restored, and suggest that the im-pairment may not be permanent. To address the issue of revers-ibility in the chronic Ag influenza system, 1 � 105 OT-I Tg T cellswere transferred to naive C57BL/6 mice. Recipient mice were theneither single or chronic Ag-stimulated with WSN-OVA, as previ-ously described. After 30 days of Ag stimulation, the chronic de-livery of virus was discontinued and mice were “Ag-rested” for 45days. Mice were harvested after 45 days of rest, and an equalnumber of virus-specific CD8� T cells were transferred to naiveC57BL/6 mice. Recipient mice were rechallenged i.n. with 40TCID50 WSN-OVA, and harvested at the peak of the secondaryresponse on day 7. Following Ag rest, virus-specific CD8� T cellsin the lungs of single prime and chronic Ag-rested mice were com-parable both in frequency (37.1 � 2.39% and 38.1 � 4.88%; n �3 mice/group) (Fig. 8A) and number (1.35 � 0.2 � 106 and 1.06 �0.2 � 106; n � 3 mice/group) (Fig. 8B). Equivalent responses, insingle and chronic Ag-rested CD8� T cells, were also observed inthe draining MLN and spleen of infected mice (data not shown).Additionally, the quality of the virus-specific CD8� T cells wasrecovered in chronic rested mice. When pulmonary lymphocyteswere ex vivo stimulated with OVA257–264 peptide, and intracellu-lar stained for IFN-�, there was no significant difference betweensingle prime and chronic rested CD8� T cells in the frequency orMFI of IFN-�-producing cells (Fig. 8C). These data suggested thatthe exhausted state of CD8� T cells in this model is not irrevers-ibly programmed and can be recovered following Ag rest.

Inhibitory signaling through PD-1 does not mediate Ag-inducedCD8� T cell exhaustion

In response to activation, CD8� T cells up-regulate inhibitory sig-naling molecules to block continued activation and proliferationresponses. Chronic infection can result in similar up-regulation ofinhibitory molecules, and prominent among them is PD-1. Recentevidence suggests that enhanced PD-1 expression on virus-specificCD8� T cells may be partially responsible for the CD8� T cellexhaustion observed in LCMV (41) and HIV (42–45) infections.Therefore, we sought to determine whether under conditions ofchronic Ag stimulation alone, the impairment in virus-specificCD8� T cell function and expansion was due to enhanced expres-sion of PD-1. Examination at day 30 showed that PD-1 expressionwas significantly increased, in comparison to single prime con-trols, on both total and virus-specific chronic Ag-stimulated CD8�

T cells (Fig. 9A). Because CD8� T cells from chronic Ag stimu-

lation mice exhibited increased PD-1 expression, PD ligand-1 (PD-L1) binding to PD-1 was blocked by treating both single andchronic Ag-stimulated mice with anti-B7H-1 (anti-PD-L1) duringthe first 30 days of Ag stimulation. The efficiency of anti-PD-L1treatment was confirmed by examining the bulk lymphocyte cellabsolute number. After PD-L1 blockade, lymphocytes in singleand chronic Ag-stimulated recipients were significantly increasedcompared with isotype-treated controls (data not shown). Despiteblockade of PD-1 signaling, there was no significant increase in thesize of the virus-specific chronic Ag-stimulated CD8� T cell pop-ulation at day 30 (Fig. 9B). Additionally, when rechallenged, therewas no recovery of expansion or function of the anti-B7H-1-treatedchronic Ag-stimulated virus-specific CD8� T cells in comparison toanti-B7H-1 single primed CD8� T cells or isotype-treated chronicstimulated controls (n � 5 mice/group, p � 0.005) (Fig. 9C). Al-though PD-1 expression increased following chronic Ag stimulation,PD-1 inhibitory signaling did not appear to mediate the defects ob-served in secondary rechallenge. These findings may indicate an im-portant distinction between the role PD-1 plays when chronic stimu-lation occurs under an inflamed vs a noninflamed state.

TRAIL signaling mediates deletion of virus-specific CD8�

T cells

Because the elevated expression of PD-1 had no functionalrole in the depression of chronic Ag-stimulated CD8� T cells,we wanted to determine whether these cells were more suscep-tible to death than their single prime counterparts. Specifically,we examined the death-inducing pathways, TRAIL and Fas. Toaddress the role of these pathways in the defects observed dur-ing rechallenge, C57BL/6, Fas-deficient MRL/lpr, or TRAILknockout mice were single or chronic Ag-stimulated withinfluenza virus strain PR8. After 30 days, single or chronicstimulated virus-specific CD8� T cells were transferred to na-ive C57BL/6 mice. Transferred mice were rechallenged i.n.with 0.2 TCID50 x31 influenza A virus and harvested, on day 7at the peak of the secondary response. As previously shown, inthe lungs of C57BL/6 mice, there was a greater than 2-foldchange in frequency between single and chronic stimulated vi-rus-specific CD8� T cells (Fig. 10A). A coordinate 3-foldchange in the absolute number was observed when single andchronic stimulated virus-specific populations were compared(10.09 � 1.36 � 105 and 3.24 � 0.4 � 105 single and chronic,respectively, n � 6 mice/group, p � 0.001) (Fig. 10, A and B).On day 7 of secondary rechallenge, Fas signaling-deficient mice

FIGURE 8. Chronic Ag-stimulated virus-specific CD8� T cells recovered function after Ag rest. A total of 1 � 105 OT-I TCR Tg T cells were transferred tonaive C57BL/6 mice. Mice were single or chronic stimulated with 1 � 106 TCID50 WSN-OVA, by i.p. injection, for 30 days. On day 30, chronic animals wereAg rested for 45 days, and an equal number of single or chronic Ag-rested virus-specific CD8� T cells were transferred to naive C57BL/6 mice, which were theni.n. challenged with WSN-OVA and harvested on day 7. A, Representative flow cytometry (gated on CD8� T cells) from lungs of single or chronic rested infectedmice is shown. As indicated by V�2�V�5.1� cells, chronic Ag-rested CD8� T cells expanded equivalently to single prime animals. B, Pooled absolute numbersindicate that Ag rest recovers not only frequency, but also the number of chronic rested virus-specific CD8� T cells (n � 3 mice/group). C, Representative flowcytometry histogram (gated on CD8� T cells) shows equivalent MFI of IFN-� production after ex vivo stimulation with OVA254–267-specific peptide.



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also exhibited a 2-fold change virus-specific CD8� T cells fre-quency when single and chronic stimulated animals were com-pared (Fig. 10A). Correction for absolute number showed thatFas-deficient chronic Ag-stimulated virus-specific CD8� Tcells were still significantly reduced relative to Fas-deficientsingle prime controls (7.24 � 1.29 � 105 and 3.66 � 0.75 �105 single and chronic, respectively, n � 7 mice/group, p �0.04) (Fig. 10, A and B). This reduction was confirmed by exvivo stimulation with influenza-specific peptide NP366 –374,which resulted in significantly less IFN-� production by thechronic Ag-stimulated CD8� T cells from C57BL/6 or Fas-deficient mice, compared with their single prime counterparts(data not shown). On the basis of our data, it did not appear thatFas-mediated death had a substantial role in the loss of chronic-stimulated CD8� T cells.

Chronic Ag stimulation in TRAIL knockout mice yielded sub-stantially different results. Following transfer of day 30 virus-spe-cific CD8� T cells, and i.n. rechallenge, chronic Ag-stimulatedCD8� T cells from C57BL/6 wild-type controls were significantlydecreased in frequency and absolute number compared with singleprime controls. Comparison of the frequency and absolute numberof single prime and chronic Ag-stimulated virus-specific CD8� Tcells show that single prime responses were 2.33 � 0.35 and4.82 � 0.81 fold greater than responses from chronic stimulatedanimals, respectively (Fig. 10C). Direct comparison of the absolutenumber of T cells shows that chronic stimulated virus-specificCD8� T cells were significantly lower than single prime controls(n � 9 mice/group for chronic stimulated and n � 6 mice/groupfor single CD8� T cells, p � 0.03) (Fig. 10D). Interestingly, in theabsence of TRAIL, chronic stimulated CD8� T cells were nolonger significantly reduced in frequency or number when com-

pared with TRAIL-deficient single prime controls. Indeed, the ra-tio of single prime to chronic stimulated virus-specific cells, infrequency and absolute number, was near 1, indicating that theresponses were comparable (0.98 � 0.21 and 1.03 � 0.28,frequency and absolute number, respectively) (Fig. 10C). Directcomparison of the absolute number confirms this, as the virus-specific population from chronic stimulated TRAIL knockout micewas 3.04 � 1.06 � 105 and from single primed mice was 2.13 �0.62 � 105 (n � 6 mice/group) (Fig. 10D). TRAIL knockoutchronic stimulated virus-specific CD8� T cells not only respondedto rechallenge at levels comparable to single prime controls, butwas significantly greater than the chronic Ag-stimulated virus-spe-cific CD8� T cell from C57BL/6 mice (Fig. 10D). Ex vivo stim-ulation of TRAIL knockout single prime and chronic Ag-stim-ulated CD8� T cells resulted in comparable IFN-� production,confirming the previously presented tetramer data (data notshown). The findings in rechallenged TRAIL knockout and Fassignaling-deficient virus-specific CD8� T cells strongly indi-cate that the inability of chronically stimulated virus-specificCD8� T cells to respond to rechallenge is mediated by TRAIL-induced apoptosis.

DiscussionDuring the course of several different chronic infections, virus-spe-cific CD8� T cells become functionally exhausted and incapable ofclearing virus from the infected host (4–11, 13–15, 46). Althoughmuch effort has been exerted in determining the phenotype and func-tional deficits of exhausted CD8� T cells, there is a paucity of studiesexamining the individual factors that contribute to CD8� T cell ex-haustion. Indeed, several classic mouse models of chronic infectionused to characterize exhausted CD8� T cells are marked by high viral

FIGURE 9. PD-1 did not mediatechronic Ag-induced CD8� T cell ex-haustion. To assess the functional sig-nificance of enhanced PD-1 expres-sion on chronically stimulated CD8�

T cells, 1 � 105 OT-I TCR Tg T cellswere transferred to naive C57BL/6mice. After transfer, mice received ei-ther single or chronic Ag stimulationwith WSN-OVA virus along with 100�g of rat IgG2a isotype or anti-B7H-1Ab every third day during the 30-daystimulation period. A, Representativeflow cytometry plots show signifi-cantly increased PD-1 expression onboth total and virus-specific chronicAg-stimulated CD8� T cells. B, After30 days, the absolute number of vi-rus-specific cells was determined byflow cytometry. There is no signifi-cant recovery in the spleen number ofanti-B7H-1-treated chronic Ag-stim-ulated CD8� T cells in comparison tosingle prime controls. C, Followingtransfer and i.n. rechallenge, we ob-served no significant recovery ofchronic Ag-stimulated CD8� T cellsthat were treated with anti-PD-L1.Lung numbers shown.



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loads, sustained proinflammatory cytokine production, and/or lym-phocyte infection (16–22), all of which may contribute to exhaustion.Studies using these infections have led to many important findingsthat define our understanding of exhausted CD8� T cells (16–24).However, these models are unable to provide information about thesignificance of Ag stimulation alone in the development of exhaustedCD8� T cells. In this study, we used murine-adapted influenza Avirus to provide chronic Ag stimulation to CD8� T cells, in the ab-sence of lymphocyte infection, sustained high viral loads, and overtinflammation. Importantly, the chronic Ag stimulation we achieved isdistinct from repeatedly stimulating with inactivated virus or peptidealone. Although it has been shown, in vitro, that heat- or UV-inacti-vated influenza virus can be used for CTL priming (47, 48), the mostefficient mechanism of CTL priming is through the use of live virus(47, 48). The use of repeated peptide stimulations was avoided be-cause it prohibits exploration of the CTL response to subdomi-nant epitopes. Additionally, with both inactivated and peptideinduced stimulation, cross-presentation appears to be a primarymechanism of priming (47, 48). Through use of live infectiousvirus, we could preserve classical Ag processing, costimulationevents, and examination of responses to multiple viral epitopes.

The repeat virus injections did not result in chronic inflam-mation, as determined by proinflammatory cytokine production,and did not affect the phenotype and number of DC. There wasno difference in IL-1, IL-6, IL-12, and TNF-� expression in any

of the tissues examined after a single i.p. injection or after 30days of repeated stimulation. Because DC expression of CD86and MHC class II did not differ in chronically stimulated ani-mals, it is likely that DC are not functionally impaired. Al-though our studies demonstrate that transferred chronicallystimulated Ag-specific CD8� T cells in naive hosts exhibit ex-pansion defects, suggesting that these defects are intrinsic to theCD8� T cells, we cannot exclude that the some function of DCis affected in chronically stimulated animals and contributes tothe CD8� T cell defect. Such a function of DC could be re-quired to keep CD8� T cells “healthy,” but it does not appearto relate to maturation or CD86 expression of DC.

Using this chronic stimulation system, we determined that TCRstimulation by chronic Ag alone was sufficient to induce exhaus-tion of CD8� T cells, as exhibited by their inability to respond toviral rechallenge. Chronic Ag-stimulated CD8� T cells displayeddefects in expansion and cytokine production following in vivorechallenge with influenza virus and suggest a reduced quality ofthe virus-specific CD8� T cells. When equal numbers of singleprime or chronic Ag-stimulated day-30 virus-specific CD8� Tcells were transferred into naive mice and i.n. rechallenged,chronic virus-specific CD8� T cells exhibited a profound reduc-tion in frequency and absolute number at the peak of the secondaryresponse. The impairment in virus-specific CD8� T cell popula-tion extended beyond a decrease in number, and also resulted inthe functional loss of IFN-� production. This loss was identified inCTLs responding to both dominant and subdominant epitopes. It isimportant to note, that the loss of subdominant epitope responsesmay actually be an underestimate. Because subdominant responseswere only identified by cytoplasmic staining for IFN-�, a loss inMFI would prevent their identification as subdominant responders.The defects observed after chronic Ag stimulation alone are sim-ilar to those documented during other chronic infections (16, 17, 19,20, 23), and may indicate that much of what is driving the clonalexhaustion of CD8� T cells during chronic infection is Ag and con-tinuous TCR stimulation. Our findings in mice are supported by ourstudies in SIV-infected macaques, which suggest that Ag is drivingSIV-specific CD8� T cell exhaustion (49).

Although many defects observed were consistent with previ-ous observations (16, 17, 19, 20, 23), we also observed uniqueproperties of chronic stimulated CD8� T cells distinct frompreviously published reports. Chief among the distinctions wasthe ability of impaired chronic stimulated CD8� T cells to com-pletely recover after Ag rest and respond to rechallenge at lev-els comparable to single prime controls. This suggests that thecapability to generate fully functional memory CD8� T cells isnot irreversibly lost by the exhausted CD8� T cells, and thatonce TCR stimulation is removed, memory can be generated.Our data suggest that during chronic Ag stimulation the effectorphase is perpetuated. Although long-term maintenance of theseeffectors most likely requires the presence of constant Ag (50,51), we have shown that once Ag is removed, productive mem-ory can be generated. In a recent report, neither cytokine pro-duction nor inflammation was sufficient to sustain homeostaticproliferation of chronic CD8� T cells in the absence of Ag (50),and it was concluded that in the absence of Ag, chronic CD8�

T cells cannot be maintained. However, despite the absence ofhomeostatic proliferation of chronic CD8� T cells, a small sur-viving population of virus-specific CD8� T cells remained (50).The capacity of these surviving Ag-independent chronic mem-ory CD8� T cells to respond to viral rechallenge has never beenexamined (50, 51). Our findings of functional recovery after Agrest suggest that despite a reduction in number, in the absenceof Ag chronic memory, CD8� T cells developed during LCMV

FIGURE 10. TRAIL-induced apoptosis mediated the loss of chronicstimulated virus-specific CD8� T cells in rechallenge. C57BL/6, TRAILknockout or MRL/lpr Fas-deficient mice were single or chronic stimulatedfor 30 days with PR8. After 30 days, mice were harvested and an equalnumber of NP366–374 tetramer-specific CD8� T cells were transferred tonaive C57BL/6 mice, which were rechallenged and harvested on day 7. A,The NP366-positive CD8� T cell response in single primed C57BL/6 andMRL/lpr Fas-deficient mice was increased 2-fold and greater than 3-fold infrequency and absolute number when compared with chronic Ag-stimu-lated animals. Data are shown as the ratio of single prime stimulated tochronic Ag-stimulated CD8� T cells, where 1 is an equivalent responsebetween single and chronic stimulation. B, Pooled absolute numbers indi-cate that the absence of Fas does not restore chronic CD8� T cells (n � 7mice/group, p � 0.04). C, Comparison of the ratio of single prime tochronic stimulated frequency and absolute number of virus-specific CD8�

T cells indicates that in the absence of TRAIL, chronic CD8� T cellsrecover; 1 is an equivalent response between single and chronic stimula-tion. D, Pooled absolute number indicates that in the absence of TRAIL,chronic Ag-stimulated virus-specific CD8� T cells are comparable to sin-gle prime controls (n � 6 mice/group).



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infection may still recover function. Thus the inability ofchronic CD8� T cells to undergo homeostatic proliferation inthe absence of Ag may not necessarily correlate with an inabil-ity of surviving cells to adequately respond to viral rechallenge.

A second important distinction between the exhaustion observedin chronic Ag-stimulated CD8� T cells vs other models of chronicinfection was the role of inhibitory molecule PD-1. Recently, anumber of reports have indicated that PD-1 expression is increasedin chronically stimulated CD8� T cells and that signaling throughPD-1:PD-L1 is responsible, at least in part, for functional deficitsof exhausted CD8� T cells (41–45, 52, 53). Following chronic Agstimulation alone, we observed a significant increase in expressionof PD-1 on both total and virus-specific CD8� T cells. The in-creased PD-1 expression on total CD8� T cells was particularlystriking, and could suggest nonspecific activation of CD8� T cells.Alternatively, the increased expression on total CD8� T cells mayhave been induced by IFN-� produced by Ag-specific effectors(54). Indeed, the link between IFN-� and PD-1 expression hasrecently been established in patients with Mycobacterium tuber-culosis (54). Despite the increased PD-1 expression, blockade ofsignaling during the priming phase yielded no significant effect onthe ability of chronic stimulated CD8� T cells to expand or func-tion in response to i.n. rechallenge. Although the frequency ofday-30 PD-1-expressing cells was increased in chronic stimulatedCD8� T cells compared with single prime controls, the MFI ofPD-1 between the two groups was not statistically different. Perhapsin this case, it is not only the frequency of PD-1-expressing cells thatis important, but also the intensity of the receptor expression on thosecells. The disparity in findings between our chronic Ag stimulationsystem and other models of chronic infection may also point to im-portant differences in the development of exhausted CD8� T cells inthe presence or absence of overt inflammation.

Finally, because inhibitory PD-1 expression did not seem to beregulating the loss of chronic Ag-stimulated CD8� T cell function,we examined the role of TRAIL and Fas death pathways. Follow-ing transfer of Fas-deficient single or chronic stimulated CD8� Tcells to naive mice, there was no significant change in the abilityof chronic Ag-stimulated CD8� T cells to respond to viral rechal-lenge. However, when chronic Ag stimulation was initiated inTRAIL knockout mice, both single and chronic stimulated virus-specific CD8� T cell responses were comparable. Perhaps evenmore importantly, TRAIL-deficient chronic CD8� T cells weresignificantly greater in absolute number than their wild-typechronic CD8� T cell counterparts. A role for TRAIL-mediatedapoptosis has been suggested in the development of helplessCD8� T cells (55) and in CD8� T cell exhaustion (24). HelplessCD8� T cells, those CD8� T cells primed in the absence of CD4�

T cells, are frequently compared with exhausted CD8� T cells asthey share a variety of functional defects (56–60) and may havesimilar mechanisms of exhaustion and deletion. Previous studiesindicate that increased susceptibility of helpless CD8� T cells toapoptosis is TRAIL-mediated (55, 61), at least at early stages ofCD8� T cell memory, and can be overcome in the presence ofTRAIL decoy receptors (55). Our findings of increased suscepti-bility to TRAIL-mediated death following chronic influenza Astimulation, in combination with previous findings, provoke thequestion of whether chronic Ag stimulation results in a loss ofCD4� T cell help during priming. Although this is one possibility,a recent study refutes the relationship between TRAIL and thedevelopment of helpless CD8� T cell memory (62). Listeriamonocytogenes infection of double-deficient (MHC class II�/�

TRAIL�/�) mice showed that the absence of TRAIL failed torecover the helpless CD8� T cell memory response (62). Addi-tionally, because helpless CD8� T cells exhibit a permanent de-

fect, and chronic Ag-stimulated CD8� T cells were restored afterAg rest, one could argue that the defect seen in this study is not dueto helplessness. Further study will be necessary to determinewhether the effects of longer duration of chronic Ag stimulationcan be overcome in the absence of TRAIL.

We have shown that chronic Ag stimulation alone is sufficient forthe induction of CD8� T cell exhaustion and subsequent deletion ofthis population. Contrary to other models of chronic infection inwhich deletion and exhaustion is induced in the context of inflamma-tion and lymphocyte infection, the exhaustion induced by chronic Agstimulation alone was reversible by Ag withdraw and not mediated byinhibitory PD-1 signaling. Rather, the loss of virus-specific CD8� Tcells appears to be mediated by TRAIL apoptosis. These data point toa critical importance of understanding the conditions under whichexhausted CD8� T cells are derived. If inflammation and viral loadare successfully controlled we may be able to better use current ther-apies, or develop other therapies, which would allow for recovery ofexhausted CD8� T cell function.

AcknowledgmentsWe thank Annie Borowski, Constantinos Petrovas, and Duc Do for helpfulsuggestions and technical support.

DisclosuresThe authors have no financial conflict of interest.

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