Selective expansion of a subset of exhaustedCD8 T cells by �PD-L1 blockadeShawn D. Blackburn*, Haina Shin*, Gordon J. Freeman†, and E. John Wherry*‡

*Immunology Program, The Wistar Institute, Philadelphia, PA 19104; and †Department of Medical Oncology, Dana-Farber Cancer Center, Boston, MA 02115

Edited by James P. Allison, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved August 14, 2008 (received for review February 14, 2008)

Programmed death-1 (PD-1) regulates T cell exhaustion duringchronic infections. Blocking the PD-1:PD-ligand (PD-L) pathwayreinvigorates exhausted CD8 T cells. Exactly how blockingPD-1:PD-L interactions improves T cell immunity, however, remainsunclear. PD-1:PD-L blockade could reprogram all exhausted T cellsto become antiviral effectors. Alternatively, this blockade mightselectively expand a subset of exhausted T cells. We have identifiedtwo subpopulations of exhausted CD8 T cells during chronic viralinfection in mice. One subset of exhausted CD8 T cells is rescued by�PD-L1 blockade, whereas the other subset appears more termi-nally differentiated and responds poorly to PD-1:PD-L blockade.Blocking PD-1:PD-L interactions reduces spontaneous apoptosisand enhances expansion and protective immunity of the rescuablesubset, but not the more terminally differentiated subset of ex-hausted CD8 T cells. These results have implications for predictingclinical responses to PD-1-based therapeutic interventions and forunderstanding T cell dynamics during persisting infections.

chronic infection � lymphocytic choriomeningitis virus � T cell exhaustion �PD-1 � T cell memory

Recent studies have revealed an important role for thenegative regulatory molecule programmed death-1 (PD-1)

in T cell exhaustion during chronic viral infections (1). PD-1, amember of the CD28:CTLA-4 family of costimulatory andcoinhibitory receptors, contains both immunotyrosine inhibitorymotif (ITIM) and immunotyrosine switch motif (ITSM) signal-ing motifs, recruits the phosphatase Shp-2, and can deliverinhibitory signals (1). PD-1 interacts with two ligands, PD-ligand1 (PD-L1), expressed by a wide variety of cells, and PD-L2,expressed mainly by macrophages and DC (1). A role for PD-1in regulating T cell responses to chronic viral infections wasidentified by using lymphocytic choriomeningitis virus (LCMV)infection of mice (2). PD-1 was highly overexpressed on ex-hausted CD8 T cells from chronically infected animals comparedwith functional memory CD8 T cells from mice that had resolvedacute infection. In vivo blockade of the PD-1:PD-L pathwayduring chronic LCMV infection led to a dramatic increase in thenumber and functionality of virus-specific CD8 T cells andenhanced control of infection (2). Other animal models of viralinfection also supported a major role for the PD-1 pathway inregulating antiviral T cell responses (3, 4). These observationswere quickly extended to primates and humans. Simian immu-nodeficiency virus (SIV)-, HIV-, hepatitis C virus (HCV)-, andhepatitis B virus (HBV)-specific CD8 T cells express elevatedlevels of PD-1 compared with CD8 T cells specific for nonper-sisting pathogens such as influenza virus or vaccinia virus (5–12).In vitro blockade of PD-1:PD-L interactions reverses exhaustionof HIV-, HBV-, and HCV-specific T cells, and the proliferativecapacity of these virus-specific T cell populations is dramaticallyimproved. In light of these recent findings, PD-1 has emerged asnot only a major regulator of T cell exhaustion during chronicinfection, but also as an important potential therapeutic target.

At least two models have been proposed for the mechanism ofreversal of exhaustion by PD-1:PD-L blockade (13). In onemodel, PD-1:PD-L blockade reprograms all exhausted T cells,converting them to functional antiviral effector T cells. In a

second model, PD-1:PD-L blockade selectively expands and/orenhances function of a subset of exhausted CD8 T cells. In thepresent work, we have addressed this question directly. We haveidentified two subsets of exhausted CD8 T cells during chronicLCMV infection in mice that differ in expression of PD-1 andCD44. One subset that expressed intermediate levels of PD-1and high levels of CD44 (PD-1IntCD44Hi) could be reinvigoratedby blocking PD-1:PD-L interactions. In contrast, the secondsubset was PD-1Hi but CD44Int and responded poorly to PD-1pathway blockade. Thus, our data suggest that the cellularmechanism of reinvigorated T cell responses during chronic viralinfection is by selective expansion of one subset of exhaustedCD8 T cells, whereas another subset is more terminally differ-entiated and unable to be rescued by PD-1 pathway blockadealone. These observations indicate that defining the populationsof exhausted CD8 T cells that can and cannot respond toPD-1-based therapeutics could be relevant for future clinicalinterventions.

ResultsIdentifying Subsets of Exhausted LCMV-Specific CD8 T Cells. Infectionof adult mice with the Armstrong strain (Arm) of LCMV causesan acute infection that is cleared by day 8–10 after infection,resulting in a highly functional population of effector andsubsequently, memory CD8 T cells (14). LCMV clone 13, incontrast, causes a chronic infection with viremia for 60–80 daysand exhaustion of CD8 T cells (14). All known T cell epitopes areshared between these two strains of LCMV and exhausted CD8T cells responding to LCMV clone 13 infection can be compareddirectly with functional CD8 T cells of the same specificitygenerated after acute LCMV Arm infection (14). We evaluatedPD-1 expression on LCMV-specific CD8 T cells after Arm vs.clone 13 infection by using two different clones of �PD-1antibodies. The RPM1–30 clone of �PD-1 reveals a highermaximal expression and thus a broader range of PD-1 expressioncompared with the J43 clone used in many previous studies (Fig.1A). Although DbGP33-specific CD8 T cells from Arm immunemice were predominantly PD-1Lo, two levels of higher PD-1expression were observed on DbGP33-specific CD8 T cells fromchronically infected mice (Fig. 1B). The vast majority of thevirus-specific CD8 T cells found during chronic LCMV infectionwere PD-1Int or PD-1Hi. Thus, the RPM1–30 antibody allowsvirus-specific CD8 T cells to be defined as PD-1Lo, PD-1Int, orPD-1Hi rather than PD-1� or PD-1� (Fig. 1 A and B). Interest-ingly, the two subsets of exhausted CD8 T cells found duringchronic LCMV infection also differed in their expression ofCD44 (Fig. 1B). Gating on the PD-1Int subset revealed high levels

Author contributions: S.D.B. and E.J.W. designed research; S.D.B. and H.S. performedresearch; G.J.F. contributed new reagents/analytic tools; S.D.B. and E.J.W. analyzed data;and S.D.B. and E.J.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

‡To whom correspondence should be addressed. E-mail: jwherry@wistar.org.

This article contains supporting information online at www.pnas.org/cgi/content/full/0801497105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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of CD44 (PD-1IntCD44Hi; blue overlay in Fig. 1B Right), whereasthe PD-1Hi subset was CD44Int (red overlay in Fig. 1B Right).Decreased CD44 expression by exhausted CD8 T cells has beennoted (14, 15), although the significance of this decrease has notbeen clear. The PD-1Hi subset also expresses slightly morecytotoxic T lymphocyte antigen-4 (CTLA-4) compared with thePD-1Int subset [supporting information (SI) Fig. S1], althoughprevious studies suggest a minimal role for CTLA-4 on ex-hausted CD8 T cells (2, 16). It is also worth pointing out that theRPM1–30 clone reveals low levels of PD-1 expressed byDbGP33-specific memory CD8 T cells from LCMV Arm im-mune mice compared with CD44Lo CD8 T cells, consistent with�2–4-fold higher PD-1 mRNA in LCMV-specific memory CD8T cells compared with naïve CD8 T cells (data not shown). BothPD-1Int and PD-1Hi subsets of GP33-specific CD8 T cells fromchronically infected mice are functionally exhausted, and at day30 after infection only 14.8 � 1.5% and 6.4 � 1.3% of theGP33-specific PD-1Int and PD-1Hi CD8 T cells, respectively,from the spleen (SP) coproduced IFN-� and TNF-� in 5 hwhereas �85% of GP33-specific memory CD8 T cells fromLCMV Arm immune mice were able to coproduce these cyto-kines (data not shown). A small population of PD-1Lo virus-specific CD8 T cells is often present in chronically infected mice,but these PD-1Lo CD8 T cells still express higher levels of PD-1than LCMV-specific memory CD8 T cells from Arm immunemice and are functionally compromised (Fig. 1 and data notshown). At later times during chronic infection (�60 days after

infection) the SP often consisted of largely PD-1IntCD44Hi

exhausted T cells, whereas in the same mice other tissues suchas the bone marrow (BM) contained exhausted CD8 T cells thatwere PD-1HiCD44Int (Fig. 1C). Overlaying the SP- (Fig. 1C Right,blue overlay) and BM-derived (Fig. 1C Right, red overlay)DbGP33� CD8 T cells with CD44 expression reveals largelydistinct subsets of exhausted CD8� T cells are present in thesetissues �60 days after infection. Thus, exhausted CD8 T cellsfound during chronic LCMV infection could be divided into aPD-1IntCD44Hi and PD-1HiCD44Int subsets.

The PD-1IntCD44Hi Exhausted CD8 T Cell Subset Had Greater Prolifer-ative Potential and Was More Responsive to PD-1 Blockade in Vivothan the PD-1HiCD44Int Subset. We next wanted to compare directlythe properties of PD-1IntCD44Hi and PD-1HiCD44Int subsets ofexhausted LCMV-specific CD8 T cells in vivo. We took advan-tage of the fact that the RPM1–30 antibody does not block PD-1interactions with its ligand (17) and thus can be used to separatesubsets of exhausted CD8 T cells based on the level of PD-1expression. Exhausted LCMV-specific CD8 T cells from the SPof chronically infected mice were separated into PD-1Int andPD-1Hi subsets by using the RPM1–30 antibody (Fig. 2A). Equalnumbers of GP33-specific PD-1IntCD44Hi or PD-1HiCD44Int

CD8 T cells were adoptively transferred to naïve congenicrecipient mice (Fig. 2 A). Recipient mice were then challengedwith LCMV clone 13. The PD-1Int GP33-specific CD8 T cellsexpanded significantly more vigorously upon challenge than thePD-1Hi GP33-specific CD8 T cells (Fig. 2 B and C). In theseexperiments the total donor (Ly5.2�) CD8 T cell population alsoexpanded dramatically more in the case of the PD-1Int donorpopulation, reflecting additional LCMV-specific CD8 T cellpopulations in the donor pool (Fig. 2D). It is important to pointout that the responsiveness of both PD-1Hi and PD-1Int GP33-specific CD8 T cells was far less than the response of PD-1Lo

GP33-specific CD8 T cells from LCMV Arm immune mice (Fig.2C Inset), consistent with the more vigorous responsiveness ofmemory T cells compared with exhausted CD8 T cells (2, 18).

To compare how the two exhausted CD8 T cell subsetsresponded to PD-L1 blockade, some recipient mice were treatedwith �PD-L1 for the first 7 days after viral challenge. Blockingthe PD-1 pathway significantly improved the responsiveness ofthe PD-1Int subset, and the number of DbGP33-specific CD8 Tcells increased significantly compared with untreated mice (Fig.2C). In contrast, there was little improvement in the response ofthe PD-1Hi subset in the presence of PD-L1 blockade in anyexperiment. Similar results were observed for the total donorLy5.2� CD8 T cell population (Fig. 2D). (Note that many of theDbGP33 tetramer� Ly5.2� CD8� T cells are specific for otherLCMV epitopes and will also respond in this experimentaldesign.) This differential responsiveness of PD-1Int and PD-1Hi

subsets of exhausted CD8 T cells was unlikely caused by incom-plete blockade because treatment with 5-fold higher doses ofblocking antibody (1 mg per injection) did not improve theresponsiveness of the PD-1Hi subset, but had a similar impact onthe PD-1Int subset, consistent with saturation of this pathway bythe blockade (data not shown).

Differential Proliferative Potential of Tissue-Defined Exhausted CD8 TCell Subsets. To examine the responsiveness of subsets of ex-hausted CD8 T cells in vivo by using an approach that did notdepend on separation by using an antibody against PD-1, we tookadvantage of the anatomical differences of PD-1IntCD44Hi andPD-1HiCD44Int subsets described in Fig. 1. We next testedwhether or not LCMV-specific CD8 T cells from the SP (PD-1Int) vs. BM (PD-1Hi) differed in their response to challengeinfection in vivo and whether PD-L1 blockade had a differentialimpact on these tissue-defined subsets of exhausted CD8 T cells.Equal numbers of LCMV DbGP33-specific CD8 T cells (Ly5.2�)

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Fig. 1. Identification of subsets of exhausted CD8 T cells. (A) CD8� CD44� Tcells from Arm vs. clone 13-infected mice (day 30 after infection) were stainedwith either the J43 clone or the RPM1–30 clone of �PD-1. (B) Splenocytes fromeither LCMV Arm or clone 13-infected mice (day 30 after infection) werestained with �PD-1 clone RPM1–30 and gated on CD8� T cells. PD-1Hi (red) andPD-1Int (blue) DbGP33� CD8� T cells were overlaid on total CD8 T cells (black;Right) from the same mice to assess overlap of these populations and toillustrate differences in CD44 expression. (C) Lymphocytes from SP or BM ofclone 13-infected mice (day 90 after infection) were stained with �PD-1 cloneRPM1–30. Plots are gated on CD8� T cells. DbGP33� CD8� T cells from the SPor BM. DbGP33� CD8� T cells from SP (blue) or BM (red) were overlaid totalCD8� splenocytes (black; Right).

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from each location were adoptively transferred to naïve congenic(Ly5.1�) mice (Fig. 3A). In these experiments, recipient micewere challenged with Listeria monocytogenes expressing theLCMV GP33 epitope (LMGP33) to assess the responsiveness ofthe exhausted CD8 T cell subsets to a second pathogen (Fig. 3A).Thus, recipients of PD-1IntCD44Hi CD8 T cells from the SP orPD-1HiCD44Int CD8 T cells from the BM were infected withLMGP33 in the presence or absence of �PD-L1 antibody. Theresponse of the SP-derived (PD-1Int) GP33-specific CD8 T cellswas �2-fold greater at 2 weeks after challenge compared withthe BM-derived PD-1Hi CD8 T cells (Fig. 3B). PD-L1 blockadehad virtually no impact on the proliferative expansion of thePD-1HiCD44Int exhausted CD8 T cell subset from the BM. Incontrast, blocking the PD-1 pathway significantly improved theresponsiveness of PD-1Int virus-specific CD8 T cells from the SP(Fig. 3B). Tissue homing or residence was unlikely to account forthese differences because the absolute number of BM-derivedvirus-specific CD8 T cells was reduced in the SP and BM ofrecipient mice (Fig. 3C).

Protective Immunity of Subsets of Exhausted CD8 T Cells. We nextexamined protective immunity after LCMV or LMGP33 rechal-lenge by using the experimental designs described above. Threedays after challenge with LCMV clone 13 viral titers weredetermined in the SP of recipient mice that did or did not receivePD-1:PD-L blockade. Recipients of PD-1Int exhausted CD8 Tcells controlled LCMV clone 13 challenge significantly betterthan recipients of PD-1Hi exhausted CD8 T cells (Fig. 4A). Inaddition, PD-1:PD-L blockade led to a significant enhancementof viral control by the PD-1Int exhausted CD8 T cell subset, butthis blockade had no impact on viral control by the PD-1Hi subset

(Fig. 4A). To examine protective immunity by a single definedspecificity of exhausted CD8 T cells, mice receiving equalnumbers of GP33-specific exhausted CD8 T cells of each subsetwere challenged with LMGP33. In the absence of PD-1:PD-Lblockade, the PD-1IntCD44Hi subset was �6-fold more efficientat bacterial control compared with the PD-1HiCD44Int subset(Fig. 4B). Again, PD-L1 blockade had essentially no impact onpathogen control by the PD-1HiCD44Int subset, but dramaticallyimproved protective immunity by the PD-1IntCD44Hi subset ofexhausted GP33-specific CD8 T cells (Fig. 4B). Thus, whetherdefined phenotypically or anatomically, subsets of exhaustedCD8 T cells responded differently to blockade of the PD-1:PD-Lpathway upon antigen encounter in vivo. Blockade of the PD-1:PD-L pathway in vivo during challenge infection had littleimpact on the expansion of the PD-1Hi subset of exhausted CD8T cells, but rather this blockade selectively augmented theexpansion and protective immunity of the PD-1Int subset ofexhausted CD8 T cells.

�PD-L1 Blockade Preferentially Rescues PD-1Lo/Int Exhausted CD8 TCells from Apoptosis. PD-L1 blockade in vivo alone or in combi-nation with therapeutic vaccination reduces the level of apopto-sis of exhausted CD8 T cells (19). In addition, higher levels ofPD-1 expression correlate with an increased rate of apoptosis inHIV-specific CD8 T cells, and engaging PD-1 on SIV-specificCD8 T cells increases cell death (6, 20). Based on these obser-vations, we examined the PD-1Lo, PD-1Int, and PD-1Hi subsets ofvirus-specific CD8 T cells from chronically infected mice fordirect ex vivo annexin V and 7AAD staining (Fig. 5A). Thepercentages of annexin V� and annexin V� 7AAD� CD8 T cellsincreased in a stepwise fashion on PD-1Lo, PD-1Int, and PD-1Hi

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Fig. 2. In vivo PD-L1 blockade enhanced expansion of splenic PD-1Int, but not the PD-1Hi subset of exhausted CD8 T cells. (A) PD-1Hi and PD-1Int splenocytes fromclone 13-infected (day 30 after infection) Ly5.2� mice were column-purified. Each fraction yielded �90% purity for PD-1Hi and PD-1Int. Note that the PD-1Int

fraction contained a small percentage of PD-1Lo cells, but these cells expressed higher PD-1 levels compared with memory CD8 T cells. DbGP33� CD8� T cells (6 �104) were adoptively transferred to Ly5.1 naïve recipient mice followed by rechallenge with LCMV clone 13 with or without �PD-L1 antibody treatment (200 �gi.p. on days 0, 3, and 6 after infection). As a control, LCMV-specific memory CD8 T cells from Ly5.2� LCMV Arm immune mice were transferred to Ly5.1� naïverecipients and rechallenged. (B–D) On day 7.5 after rechallenge, recipient mice were killed, and total donor Ly5.2� or DbGP33� Ly5.2� CD8� T cells were identifiedby flow cytometry (n � 3–5 mice per group, data are representative of three similar experiments).

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CD8 T cells from chronically infected mice, suggesting that thelevels of cell death increased in vivo as PD-1 levels increased.These results suggest that PD-1:PD-L1 blockade could rescuethose CD8 T cells that express the highest levels of PD-1 fromapoptosis. Thus, we developed an assay to evaluate spontaneouscell death of exhausted CD8 T cell subsets. By examining T cellsthat incorporated a dye indicating cell death (vivid red; VR) andstaining for active caspase 3 (ACasp3) as an indicator of earlystages of apoptosis, cells in late (VR� ACasp3 �/�) vs. early(VR� ACasp3�) stages of cell death could be identified (Fig.5B). In the following studies, the analysis is restricted to VR�ACasp3� cells that are likely in the process of dying. By usingthis assay, higher levels of PD-1 expression on LCMV-specificCD8 T cells correlated directly with increased ACasp3 afterculture for 5 h at 37°C (Fig. 5C). The level of spontaneouscaspase 3 activation was highest in the PD-1Hi subset comparedwith the PD-1Int subset of LCMV-specific CD8 T cells fromchronically infected mice, consistent with previous reports forHIV- and SIV-specific CD8 T cells (6, 20). The small subset ofPD-1Lo GP33-specific CD8 T cells present in the chronically

infected mice displayed a lower level of ACasp3 staining, but thislevel was still greater than that found in GP33-specific CD8 Tcells from LCMV Arm immune mice (Fig. 5C). Thus, sponta-neous ex vivo caspase 3 activation was increased in exhaustedCD8 T cells compared with memory CD8 T cells, and the levelof ACasp3 staining increased as PD-1 levels increased.

To determine whether blocking the PD-1:PD-L pathwayinfluenced death of exhausted CD8 T cell subsets, CD44Hi/Int

CD8 T cells were sorted into PD-1Hi, PD-1Int, and PD-1Lo subsetsand incubated 5 h at 37°C in medium. Increased PD-1 expressionagain correlated with higher levels of ACasp3 in these highlypurified CD8 T cell populations. However, �PD-L1 blockadepreferentially rescued the PD-1Int and PD-1Lo subsets fromcaspase 3 activation but had minimal effect on the PD-1Hi subset(Fig. 5 D and E). Memory CD8 T cells from LCMV Arm immunemice showed little spontaneous apoptosis, but also little changein cell death in the presence of PD-L1 blockade consistent withthe low expression of PD-1 by this population. Together, theseresults suggest that not only are PD-1Int/Lo exhausted CD8 T cellsless susceptible to induction of cell death than PD-1Hi exhaustedsubset, but they are also more likely to benefit from �PD-L1blockade by greater survival.

DiscussionWe identified subsets of exhausted CD8 T cells based on PD-1and CD44 expression and tissue distribution. Our results supporta model in which one subset of exhausted CD8 T cells foundduring chronic viral infection is rescuable by blocking PD-1signals whereas the other subset is more terminally differenti-ated and is not rescued by blocking PD-1:PD-L interactions. Thedifferential responses of the two subsets of exhausted CD8 Tcells had a substantial impact on not only the expansion of thesevirus-specific CD8 T cells, but also on protective immunity invivo. Thus, our data support a model in which blockade ofPD-1:PD-L interactions selectively rescues one subset of ex-hausted antiviral CD8 T cells and suggest that restoration ofantiviral immunity by PD-1:PD-L blockade does not need toinduce reprogramming of all exhausted CD8 T cells to achievea beneficial effect. In this regard it is worth noting that even inprevious studies a subpopulation of exhausted CD8 T cells wasobserved that failed to respond and proliferate after PD-1:PD-Lblockade (2). The present results also suggest that it should bepossible to predict which T cell responses and which chronicallyinfected subjects will respond most effectively to therapeuticintervention with PD-1:PD-L blockade based on the level ofPD-1 expression and/or distribution of exhausted T cell subsets.

What prevents reversal of exhaustion for the PD-1HiCD44Int

subset of exhausted CD8 T cells? Although incomplete blockadein vivo is one possibility, we believe that this is unlikely becausesimilar results were observed even when 5-fold higher amountsof blocking antibody were used (data not shown). Anotherpossibility is that CD8 T cells expressing the highest levels ofPD-1 are already committed to cell death, such that PD-1:PD-Lblockade at this point is insufficient to rescue their survival. Athird possibility is that the PD-1HiCD44Int subset of exhaustedCD8 T cells is restrained by additional regulatory pathways orcellular deficiencies. Our recent gene expression studies ofexhausted CD8 T cells point to a number of additional pathwaysthat may contribute to T cell exhaustion, including other inhib-itory molecules, potential signaling changes, and metabolicdefects that are likely to compromise the ability of exhaustedCD8 T cells to respond to stimulation (15). During HIV infec-tion, a second inhibitory receptor CTLA-4 can be overexpressedby exhausted CD4 T cells, and a complex pattern of PD-1 andCTLA-4 coexpression existed for these HIV-specific CD4 T cells(16). Although CTLA-4 is slightly elevated on the PD-1Hi

compared with the PD-1Int subset of exhausted CD8 T cells, it isunclear how CTLA-4 or other pathways associated with T cell

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(SP-derived), but not the PD-1Hi (BM-derived) subset of exhausted CD8 T cells.(A) PD-1Hi CD8� T cells were derived from the BM and PD-1Int CD8� T cells fromthe SP of clone 13-infected (day 90 after infection) Ly5.2� mice. CD8 T cellsfrom BM and SP were column-purified, CD8� T cell populations were normal-ized to contain 6 � 104 DbGP33� CD8�T cells, and cells were adoptivelytransferred to Ly5.1 naïve recipient mice followed by rechallenge withLMGP33 (3 � 104 cfu) with or without �PD-L1 antibody treatment (200 �g i.p.on days 0, 3, and 6 after infection). (B) Ly5.2� DbGP33� CD8� T cells weremonitored in the blood of recipient mice after LMGP33 rechallenge (n � 3–5mice per group, data are representative of five experiments). (C) Total donorDbGP33� Ly5.2� CD8� T cell number was assessed in recipient SP and BM onday 18 after challenge.

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dysfunction mentioned above regulate the two exhausted T cellsubsets defined by in the present work. CTLA-4 appears to havea minimal role on exhausted CD8 T cells (2, 16), but we wouldpredict that additional regulatory pathways or cellular defectswould be more prominent in the PD-1HiCD44Int vs. PD-1IntCD44Hi subset of exhausted CD8 T cells. Even for the lessterminally differentiated exhausted CD8 T cells, additionalnegative regulatory pathways and cellular defects may bepresent. Future studies will be necessary to examine these issues.However, both increased apoptosis and additional inhibitorypathways or metabolic deficiencies should be amenable tocomplementary types of therapeutic intervention to enhance theeffectiveness of PD-1:PD-L blockade.

Restoration of T cell responses by PD-1:PD-L blockade is apromising approach to enhance immunity to persisting infectionsand tumors. Our data indicate that there is heterogeneity in Tcells responding to a chronic viral infection. PD-1:PD-L block-ade has a dramatic effect restoring immunity and rescuingresponsiveness of one subpopulation of exhausted LCMV-specific T cells, but another subset of exhausted CD8 T cellsexists that is less efficiently rescued by blocking this inhibitorypathway alone. During SIV infection it appears that three levelsof PD-1 expression could also be present. Velu et al. (21)demonstrated that by 12 weeks after infection there were PD-1�

and PD-1� CD8 T cells, but the SIV tetramer� CD8 T cellsexpressed the highest level of PD-1 even among the PD-1� CD8T cells (21) similar to the PD-1Lo, PD-1Int, and PD-1Hi exhaustedCD8� T cells described here for mice. Radziewicz et al. (10)found a similar expression pattern in human HCV infectionwhere the HCV-specific CD8 T cells in some subjects clearlyexpressed a higher level of PD-1 compared with the rest of thePD-1� CD8 T cells. Thus, it is likely that populations of PD-1Lo,PD-1Int, and PD-1Hi CD8 T cells also exist in humans. However,the precise phenotypic characteristics of exhausted T cellssubsets could vary in different infections or even anatomicalsites. Indeed, recent studies suggest that although the circulatingpool of HCV-specific CD8 T cells can express PD-1, the level ofPD-1 is substantially lower in the PBMC compared with the levelobserved in the liver (22), and similar observations have beenreported for different nonlymphoid tissues compared withthe blood during SIV infection (21). Moreover, circulatingPD-1Lo/Int HCV-specific CD8 T cells in humans respond moreefficiently to PD-1:PD-L blockade in vitro compared with theintrahepatic PD-1Hi subset. Together, these observations fromhuman and primate studies suggest that our findings regardingsubsets of exhausted CD8� T cells in mice could have directrelevance to human chronic viral infections. Thus, the subsets of

exhausted CD8 T cells present in different settings may havesubstantial relevance to predicting the outcome of future clinicalinterventions during chronic infections.

Overall, our findings suggest that reversal of exhaustion doesnot need to operate through functional reprogramming of allexhausted T cells. Rather, selective expansion of a subset ofexhausted T cells, although not excluding changes in per cellfunction, appears to be a major mechanism for improved T cell

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Fig. 4. Protective immunity by subsets of exhausted CD8 T cells in the absence or presence PD-L1 blockade. (A) PD-1Hi and PD-1Int splenocytes were purifiedas in Fig. 2, and 1 � 106 LCMV-specific CD8� T cells [normalized based on responsiveness via ICS to a pool of 20 known LCMV CD8 T cell epitopes described byKotturi et al. (24)] were transferred to Ly5.1 naïve recipient mice followed by rechallenge with LCMV clone 13 (2 � 106pfu i.v.) with or without �PD-L1 antibodytreatment as in Fig. 2. As a control, Ly5.1� mice that received no donors cells were also infected. Mice were killed on day 3 after infection. and LCMV quantifiedin the SP. (B) Adoptive transfers were performed as described in Fig. 3. Recipients were then rechallenged with LMGP33 (1.5 � 104 cfu i.v.) with or without �PD-L1antibody (200 �g i.p. on days 0 and 3 after infection). Mice were killed on day 4 after infection, and LMGP33 was quantified in the SP.

Arm PD1 PD1 PD10

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Fig. 5. Differential inhibition of apoptosis of exhausted CD8 T cell subsets byPD-1:PD-L blockade. (A) Splenocytes from clone 13-infected mice (day 30 afterinfection) were isolated and stained with 7-AAD and annexin V directly exvivo. (B) ACasp3 was used as an indicator of apoptosis in live cells. Dead cellswere excluded by using an amine-reactive dye (vivid red), and ACasp3 wasdetected by intracellular staining. (C) Splenocytes from clone 13-infected micewere incubated at 37°C for 5 h in medium. ACasp3 was detected in liveDbGP33� CD8 T cells. (D and E) PD-1Hi, PD-1Int, and PD-1Lo exhausted CD8 T cellssubsets were sorted by flow cytometry and incubated at 37°C for 5 h with orwithout �PD-L1 (10 �g/ml), and ACasp3 was detected in the live cell subset byflow cytometry (data are representative of three independent experiments).Note that PD-1 expression did not change in 5 h in vitro (data not shown).Similar results were observed in the presence of added peptide (data notshown).

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responses after PD-1:PD-L blockade during chronic viral infec-tion. These results also suggest that additional strategies mightbe necessary to restore responsiveness of the PD-1Hi subset ofexhausted T cells. Defining the subpopulations of exhaustedCD8 T cells present before PD-1:PD-L blockade interventionand devising strategies to rescue the subset of exhausted CD8 Tcells refractory to this blockade are important future goals.

MethodsMice, Virus, Infections, and PD-L1 Blockade. Four- to six-week-old C57BL/6 micewere purchased from The Jackson Laboratory. LCMV Arm and clone 13,recombinant L. monocytogenes (LMGP33), strains were used as described (14,23). Mice were infected with LCMV Arm (2 � 105 pfu) i.p. or with LCMV clone13 (2 � 106 pfu) i.v. PD-1:PD-L blockade was performed as described in ref. 2.All mice were used in accordance with IACUC guidelines.

Flow Cytometry, Intracellular Cytokine Staining (ICS), and Apoptosis Assays.MHC class I peptide tetramers were made and used as described in ref. 14.Antibodies were purchased from ebioscience or BD Biosciences. Lymphocyteswere stained and analyzed as described (14, 23). ICS was performed as de-

scribed (14, 23). In apoptosis assays, cells were stained first with the aminereactive dye vivid red (Molecular Probes) and CD8, CD44, and PD-1. Cells werefixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and stainedintracellularly with an anti-active caspase 3 antibody. In direct ex vivo assays,apoptosis was detected by using annexin V–FITC and 7-AAD (BD Bioscience).

Cell Purification and Adoptive Transfers. CD8 T cells were purified to �90%purity by using magnetic beads (Miltenyi Biotec) or by FACS sorting. PD1Hi andPD1Int populations were separated by using �-PD-1 PE (RPM1–30) and anti-PEmagnetic beads, resulting in PD1Hi and PD1Int populations of �90% purity(MACS beads; Miltenyi Biotec). In each individual experiment, identical num-bers of DbGP33 tetramer� CD8 T cells were adoptively transferred to eachrecipient mouse. For apoptosis assays, cells were separated as described aboveor by FACS sorting.

ACKNOWLEDGMENTS. We thank Dan Barber, Dave Masopust, Kyong-MiChang, and the members of the Wherry laboratory for helpful discussions. Thiswork was supported by National Institutes of Health Grants AI071309 (toE.J.W.), AI56299 (to G.J.F.), and HHSN 26620050030C (to E.J.W. and G.J.F.) andby the Commonwealth Universal Research Enhancement Program, Pennsyl-vania Department of Health (to E.J.W.).

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2. Barber DL, et al. (2006) Restoring function in exhausted CD8 T cells during chronic viralinfection. Nature 439:682–687.

3. Iwai Y, Terawaki S, Ikegawa M, Okazaki T, Honjo T (2003) PD-1 inhibits antiviralimmunity at the effector phase in the liver. J Exp Med 198:39–50.

4. Isogawa M, Furuichi Y, Chisari FV (2005) Oscillating CD8(�) T cell effector functionsafter antigen recognition in the liver. Immunity 23:53–63.

5. Day CL, et al. (2006) PD-1 expression on HIV-specific T cells is associated with T cellexhaustion and disease progression. Nature 443:350–354.

6. Petrovas C, et al. (2006) PD-1 is a regulator of virus-specific CD8� T cell survival in HIVinfection. J Exp Med 203:2281–2292.

7. Trautmann L, et al. (2006) Up-regulation of PD-1 expression on HIV-specific CD8� T cellsleads to reversible immune dysfunction. Nat Med 12:1198–1202.

8. Zhang JY, et al. (2007) PD-1 up-regulation is correlated with HIV-specific memory CD8�

T cell exhaustion in typical progressors, but not in long-term nonprogressors. Blood109:4671–4678.

9. Urbani S, et al. (2006) PD-1 expression in acute hepatitis C virus (HCV) infection isassociated with HCV-specific CD8 exhaustion. J Virol 80:11398–11403.

10. Radziewicz H, et al. (2006) Liver-infiltrating lymphocytes in chronic human hepatitis Cvirus infection display an exhausted phenotype with high PD-1 and low CD127 expres-sion. J Virol 81:2545–2553.

11. Boni C, et al. (2007) Characterization of hepatitis B virus (HBV)-specific T cell dysfunc-tion in chronic HBV infection. J Virol 81:4215–4225.

12. Boettler T, et al. (2006) Expression of the interleukin-7 receptor � chain (CD127) onvirus-specific CD8� T cells identifies functionally and phenotypically defined memoryT cells during acute resolving hepatitis B virus infection. J Virol 80:3532–3540.

13. Blattman JN, Greenberg PD (2006) PD-1 blockade: Rescue from a near-death experi-ence. Nat Immunol 7:227–228.

14. Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R (2003) Viralpersistence alters CD8 T cell immunodominance and tissue distribution and results indistinct stages of functional impairment. J Virol 77:4911–4927.

15. Wherry EJ, et al. (2007) Molecular signature of CD8� T cell exhaustion during chronicviral infection. Immunity 27:670–684.

16. Kaufmann DE, et al. (2007) Up-regulation of CTLA-4 by HIV-specific CD4(�) T cellscorrelates with disease progression and defines a reversible immune dysfunction. NatImmunol 8:1246–1254.

17. Matsumoto K, et al. (2004) B7-DC regulates asthmatic response by an IFN-�-dependentmechanism. J Immunol 172:2530–2541.

18. Wherry EJ, Blattman JN, Ahmed R (2005) Low CD8 T cell proliferative potential andhigh viral load limit the effectiveness of therapeutic vaccination. J Virol 79:8960 –8968.

19. Ha SJ, et al. (2008) Enhancing therapeutic vaccination by blocking PD-1-mediatedinhibitory signals during chronic infection. J Exp Med 205:543–555.

20. Petrovas C, et al. (2007) SIV-specific CD8� T cells express high levels of PD1 and cytokinesbut have impaired proliferative capacity in acute and chronic SIVmac251 infection.Blood 110:928–936.

21. Velu V, et al. (2007) Elevated expression levels of inhibitory receptor programmeddeath 1 on simian immunodeficiency virus-specific CD8 T cells during chronic infectionbut not after vaccination. J Virol 81:5819–5828.

22. Nakamoto N, et al. (2008) Functional restoration of HCV-specific CD8 T cells by PD-1blockade is defined by PD-1 expression and compartmentalization. Gastroenterology134:1927–1937.

23. Wherry EJ, et al. (2003) Lineage relationship and protective immunity of memory CD8T cell subsets. Nat Immunol 4:225–234.

24. Kotturi MF, et al. (2007) The CD8� T cell response to lymphocytic choriomeningitisvirus involves the L antigen: Uncovering new tricks for an old virus. J Virol81:4928 – 4940.

Blackburn et al. PNAS � September 30, 2008 � vol. 105 � no. 39 � 15021

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