OX40- and CD27-mediated costimulation synergizes with anti-PD-L1 blockade by forcing exhausted CD8+ T cells to exit quiescence

of April 17, 2016.This information is current as

Quiescence T Cells To Exit+Forcing Exhausted CD8

PD-L1 Blockade by−Synergizes with Anti OX40- and CD27-Mediated Costimulation

Al-Shamkhani and Ronjon ChakravertyFreeman, Megan Sykes, Michael Croft, Aymen Ghorashian, Ben Carpenter, Clare L. Bennett, Gordon J.Noha Edwards, Lei Zhang, Shivajanani Sivakumaran, Sara Sarah L. Buchan, Teresa Manzo, Barry Flutter, Anne Rogel,

ol.1401644http://www.jimmunol.org/content/early/2014/11/15/jimmun

published online 17 November 2014J Immunol 

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The Journal of Immunology

OX40- and CD27-Mediated Costimulation Synergizeswith Anti–PD-L1 Blockade by Forcing Exhausted CD8+

T Cells To Exit Quiescence

Sarah L. Buchan,*,1 Teresa Manzo,†,‡,1 Barry Flutter,†,‡,1 Anne Rogel,* Noha Edwards,†,‡

Lei Zhang,†,‡ Shivajanani Sivakumaran,†,‡ Sara Ghorashian,†,‡ Ben Carpenter,†,‡

Clare L. Bennett,†,‡ Gordon J. Freeman,x Megan Sykes,{ Michael Croft,‖

Aymen Al-Shamkhani,*,2 and Ronjon Chakraverty†,‡,2

Exhaustion of chronically stimulated CD8+ T cells is a significant obstacle to immune control of chronic infections or tumors.

Although coinhibitory checkpoint blockade with anti–programmed death ligand 1 (PD-L1) Ab can restore functions to exhausted

T cell populations, recovery is often incomplete and dependent upon the pool size of a quiescent T-bethigh subset that expresses

lower levels of PD-1. In a model in which unhelped, HY-specific CD8+ T cells gradually lose function following transfer to male

bone marrow transplantation recipients, we have explored the effect of shifting the balance away from coinhibition and toward

costimulation by combining anti–PD-L1 with agonistic Abs to the TNFR superfamily members, OX40 and CD27. Several weeks

following T cell transfer, both agonistic Abs, but especially anti-CD27, demonstrated synergy with anti–PD-L1 by enhancing CD8+

T cell proliferation and effector cytokine generation. Anti-CD27 and anti–PD-L1 synergized by downregulating the expression of

multiple quiescence-related genes concomitant with a reduced frequency of T-bethigh cells within the exhausted population.

However, in the presence of persistent Ag, the CD8+ T cell response was not sustained and the overall size of the effector

cytokine-producing pool eventually contracted to levels below that of controls. Thus, CD27-mediated costimulation can synergize

with coinhibitory checkpoint blockade to switch off molecular programs for quiescence in exhausted T cell populations, but at the

expense of losing precursor cells required to maintain a response. The Journal of Immunology, 2015, 194: 000–000.

CD8+ T cell exhaustion resulting from excessive orchronic TCR stimulation poses a significant barrier to theimmune control of chronic infections or tumors (1). In

the exhausted state, tumor or viral Ag-specific CD8+ T cells be-come subject to multiple coinhibitory signals, for example via theprogrammed death (PD)-1 receptor, and lose functions in stepwisefashion (2). Ab-mediated blockade of single or multiple coinhi-bitory receptors can lead to restoration of CD8+ T cell functions.Indeed, early-phase clinical trials of Ab-mediated blockade of the

PD-1 pathway have already demonstrated significant efficacy intreating several tumor types (3), and there is now interest incombining this approach with other therapies to maximize thereversal of T cell exhaustion. When analyzed at a whole pop-ulation level, exhausted CD8+ T cells lack gene signatures asso-ciated with quiescence and possess disordered expression of genenetworks that regulate T cell functions (4). Responsiveness to PD-1 checkpoint blockade, however, depends upon a relatively qui-escent subpopulation of PD-1low CD8+ T cells maintained by theT-box transcription factor, T-bet, that retains the capacity to re-spond to Ag (5). In response to persistent Ag, proliferation of PD-1intT-bethigh precursors gives rise to PD-1high T-betlow terminallydifferentiated progeny that express high levels of another T-boxfamily member, Eomesodermin (Eomes) (5). Thus, the effect ofcoinhibitory blockade upon the overall composition of theexhausted repertoire, including the potential deleterious effects ofdriving terminal differentiation and replicative senescence in Ag-specific T cells, requires further study.In addition to initial TCR activation, productive T cell immunity

requires costimulation. Members of the TNFR superfamily, in-cluding 4-1BB, OX40, and CD27, are important costimulatoryreceptors [reviewed in (6)]. Individual or combinatorial costimu-latory signals via TNFR superfamily members have key roles inmaximizing clonal expansion, effector differentiation, and sur-vival of T cells (7, 8). For example, OX40 and CD27 costimula-tion trigger the assembly of intracellular signalosomes that inducesustained NF-kB activation and lead to upregulation of prosurvivalpathways in T cells (9, 10). Indeed, CD27- and OX40-mediatedsurvival of activated CD8+ T cells may be important in dictatingthe eventual size of the memory pool following contraction of theprimary response (11–15). Whereas poorly immunogenic tumors

*Cancer Sciences Unit, Faculty of Medicine, University of Southampton, SouthamptonSO17 1BJ, United Kingdom; †Transplantation Immunology Group, Cancer Institute,University College London, London NW3 2PF, United Kingdom; ‡Institute for Im-munity and Transplantation, University College London, London NW3 2PF, UnitedKingdom; xDepartment of Medical Oncology, Dana-Farber Cancer Institute, HarvardMedical School, Boston, MA 02115; {Columbia Center for Translational Immunol-ogy, Columbia University Medical Center, New York, NY 10032; and ‖Institute ofAllergy and Immunology, La Jolla, CA 92037

1S.L.B., T.M., and B.F. contributed equally to this work.

2A.A.-S. and R.C. are joint senior authors.

Received for publication July 2, 2014. Accepted for publication October 19, 2014.

This work was supported by Leukaemia and Lymphoma Research (to R.C., A.A.-S.,and S.L.B.), National Institutes of Health Grants P01 AI56299 and R01 A1051559(to G.J.F.), and National Institutes of Health/National Cancer Institute Grant P01CA111519 (to M.S.).

Address correspondence and reprint requests to Prof. Ronjon Chakraverty, Trans-plantation Immunology Group, Cancer Institute, University College London, LondonNW3 2PF, U.K. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: BMT, bone marrow transplantation; Eomes, Eome-sodermin; LCMV, lymphocytic choriomeningitis virus; Mh, MataHari; PD, pro-grammed death; PD-L1, PD ligand 1.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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or weakly replicating viruses fail to activate TNFR familyreceptors, enforcing costimulation experimentally through appli-cation of ligand fusion proteins or agonist Abs has shown thepotential to enhance both primary and recall immunity (6).The extent to which additional costimulation mediated via TNFR

family receptors is beneficial under conditions favoring exhaustivedifferentiation of T cells is less clear. In murine models of chroniclymphocytic choriomeningitis (LCMV) infection, physiologicalexpression of OX40 by virus-specific CD8+ T cells improves viralcontrol (16). In contrast, continuous signaling via CD27 is im-plicated in driving even more profound exhaustion of virus-specific effectors (17). Agonistic Ab-mediated costimulation via4-1BB can be detrimental or beneficial in promoting control ofchronic LCMV according to the precise treatment schedule (18).Thus, where expression of costimulatory ligands is already ele-vated or plentiful, driving further costimulation may have limitedvalue. However, exhaustive CD8+ T cell differentiation may alsooccur under conditions in which costimulatory ligand expressionis low, for example within tumors (19) or at late time points fol-lowing allogeneic stem cell transplantation (20). In the absence ofhelp, nonlicensed APCs may lack the repertoire of costimulatoryligands required for full generation of productive immunity; in thiscontext, coinhibitory signals could supervene earlier and accel-erate failure of chronically stimulated CD8+ T cells.In this study, we have tested the hypothesis that provision of

additional costimulation via TNFR family receptors under non-inflammatory conditions will aid restoration of functions to ex-hausted CD8+ T cells. We find that agonistic Abs to OX40 andespecially to CD27 synergize with anti–PD ligand 1 (PD-L1) byenhancing proliferation and effector cytokine generation. CD27-mediated costimulation synergized with coinhibitory checkpointblockade to switch off molecular programs for quiescence inexhausted T cell populations, but this occurred at the expense oflosing precursor cells required to maintain the response.

Materials and MethodsAnimals

Female C57BL/6 (B6) and B6.SJL (CD45.1) mice were purchased from theFrederick Cancer Research facility and bred in house. MataHari (Mh) TCR-transgenic mice on a B6.PL-Thya/Cy background were obtained fromJ. Dyson, Imperial College London (21). Ox402/2 mice were bred in house(22). All procedures were approved by local institutional research com-mittees and conducted in accordance with National Institutes of Healthor United Kingdom Home Office Animals (Scientific Procedures) Actof 1986.

PD-L1, OX40, OX40L, and CD27 Abs

Anti-mouse PD-L1 (10F.9G2), anti-OX40 (OX86), anti-CD27 (AT124-1), and their respective isotype controls have been described previously(23–26).

Bone marrow transplantation and adoptive T cell transfer

Male B6 mice received 9 Gy irradiation, followed by receipt of 5 3 106

female B6 bone marrow cells. Seven days later, 1 3 106 Mh CD8+

transgenic T cells with or without additional 2.5 3 106 female B6 poly-clonal CD4+ T cells were transferred to bone marrow transplantation(BMT) recipients (20). Individual CD4+ and CD8+ donor populations wereselected using CD4/CD8 T cell isolation kits (Miltenyi Biotec). Uptake ofBrdU was used to examine the turnover of cells in vivo at later time pointsby administration of 0.8 mg/ml BrdU (Sigma-Aldrich) in the water ofrecipient mice over 7 d prior to analysis.

Abs and flow cytometry

The following cell surface Abs (purchased from eBioscience, San Diego,CA) and their respective isotype controls were used: anti-CD8a (53-6.7),anti-CD27 (LG.7F9), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD107a(1D4B), anti-Thy1.1 (HIS51) anti–PD-1 (J43), and anti-OX40 (OX86).

Anti-Vb8.3 TCR (1B3.3) was purchased from BD Biosciences (Oxford,U.K.). Direct intracellular staining was carried out using anti-perforin(OMAK-D), anti–T-bet (4B10), anti-Eomes (Dan11mag), and isotypecontrols, purchased from eBioscience (San Diego, CA), or anti-granzymeB (GRB05) from Life Technologies. Following brief peptide restimulation,intracellular staining was performed using anti–IFN-g (XMG1.2) or anti–TNF-a (MP6-XT22) together with the appropriate isotype controls (BDBiosciences, Oxford, U.K.). Intranuclear staining for BrdU was carried outusing anti–BrdU-allophycocyanin flow kit (BD Biosciences), according tothe manufacturer’s instructions. To detect CD107a, cells were restimulatedfor 4 h in the presence or absence of 1 mM UTY peptide with Golgi-Stop(BD Pharmingen) in the presence of anti-CD107a or isotype control.Cells were then resurface stained with anti-CD107a or isotype. Flowcytometric analysis was performed on a LSRFortessa or FACs Canto II(BD Biosciences), and cell counting was performed on a Coulter Counter(Beckman Coulter).

Intracellular cytokine staining

Cells from spleen or blood were analyzed for Ag-specific IFN-g or TNF-arelease by ex vivo intracellular staining. Briefly, cells were cultured in theabsence or in the presence of 1 mM of the peptide UTY (WMHHNMDLI)or irrelevant peptide (OVA SIINFEKL) for 4 h (ProImmune or PeptideProtein Research), and brefeldin A (GolgiPlug; BD Pharmingen) wasadded either at the start or for the last 2 h. Cells were then surface stainedfor CD8 and Vb8.3, or relevant congenic markers, fixed, permeabilized,and then analyzed for intracellular cytokine staining by addition of theappropriate Ab.

Gene expression analysis

mRNA was isolated using the RNeasy Micro Kit, followed by cDNAgeneration using the RT2 PreAMP cDNA Synthesis Kit (both Qiagen).Gene expression analysis was performed using the RT2 Profiler T CellAnergy and Immune Tolerance PCR Array by quantitative PCR usingActb, Gusb, and Hsp90ab1 housekeeping genes for normalization(Qiagen). Raw threshold data generated by PCR was uploaded toa dedicated web portal for further analysis (http://www.sabiosciences.com/pcrarraydataanalysis.php).

Statistical analyses

Statistical analyses were performed using the unpaired t test (two tailed). Ap value ,0.05 was considered to be significant (*p , 0.05, **p , 0.01,***p , 0.001).

ResultsAgonistic costimulation via OX40 synergizes with anti–PD-L1to recover functions of helpless, exhausted CD8+ T cells

We have shown previously that TCR-transgenic Mh CD8+ T cells(specific for the male Ag, UTY) become exhausted followingdelayed transfer to male, MHC-matched BMT recipients (20).Administration of anti–PD-L1 can partially rescue the functions ofexhausted Mh CD8+ T cells under conditions in which they arecotransferred with polyclonal female CD4+ T cells (20). BecauseCD4+ T cells can protect against exhaustion in models of chronicviral infection (27), we first evaluated whether exhaustion ofhelpless Mh CD8+ T cells could similarly be reversed followinganti–PD-L1. As shown in Fig. 1A, both CD4 helped and unhelpedMh CD8+ T cells (Thy1.1+ Vb8.3+) demonstrated similar reduc-tions in their capacity to produce IFN-g over time following theirtransfer to male B6 BMT recipients. However, Mh CD8+ T cellexpression of PD-1 was higher in the unhelped versus the helpedexperimental group (Fig. 1B), and, although anti–PD-L1 treatmentcould enhance proliferation from baseline in helpless Mh CD8+

T cells, it had little effect upon generation of IFN-g (Fig. 1C). Thiswas in contrast to helped conditions, in which, as we have pre-viously reported (20), anti–PD-L1 treatment increased both pro-liferation and IFN-g generation. Because the amount of male Agremains constant throughout the experiment, these data show thatCD4+ T cells can protect CD8+ T cells from exhaustion througheffects that are independent of any change in overall Ag load.

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In initial experiments to examine the effect of additional co-stimulation upon rescue of exhausted Mh CD8+ T cells, we firstevaluated the expression of TNFR family members that are up-regulated in response to TCR activation (OX40, 4-1BB). Com-pared with naive input cells, we observed increased expression ofOX40, which was equivalent upon both helped and unhelpedCD8+ T cells at day 42 following transfer (Fig. 2A). To test theeffect of enforcing OX40 costimulation in helped compared withunhelped Mh CD8+ T cells, we administered an agonist anti-OX40mAb (OX86) on day 35 following T cell transfer, alone or incombination with anti–PD-L1 given on days 36 and 39, andevaluated the response at day 42. Under both helped and unhelpedconditions, anti-OX40 given alone had no effect upon proliferation(as estimated by BrdU incorporation) or IFN-g production by MhCD8+ T cells (Fig. 2B, 2C, Supplemental Fig. 1A). However,when anti-OX40 was combined with anti–PD-L1 blockade, weobserved synergistic increases in Mh CD8+ T cell proliferationthat were evident in helpless, but not helped cells (Fig. 2B, 2C). Inthe helpless setting, the combination of anti–PD-L1 and anti-OX40 also synergized to increase the absolute numbers of IFN-g+

Mh CD8+ T cells in the spleen, although these effects were rela-tively modest when evaluated on a per-cell basis (Fig. 2B, 2C,Supplemental Fig. 1A). The synergy for effector cytokine ex-pression in the combined treatment group was limited to IFN-g;anti-OX40 alone had no effect upon TNF-a generation, whereasanti–PD-L1 induced a minor increase from baseline with no fur-ther increase upon the addition of anti-OX40 (Supplemental Fig.1B). No increases were observed in the frequency of Mh CD8+

T cells dual staining for IFN-g and TNF-a in any treatment group(Supplemental Fig. 1B). Because OX40 is expressed constitutivelyon murine T regulatory cells and is inducible on other cells, in-cluding NK and NKT cells (7), we also tested whether the effectof combined anti–PD-L1 and anti-OX40 would still occur underconditions in which Mh CD8+ T cells expressed OX40, but otherimmune cell populations did not. Thus, we transferred unhelpedMh CD8+ T cells to male BMT recipients, as set out in Fig. 2B,but reconstituted irradiated male recipients with female OX402/2

bone marrow. Under these experimental conditions, anti–PD-L1treatment alone had no effect, whereas anti-OX40 and anti–PD-L1induced significant increases in absolute numbers of IFN-g+ Mh

CD8+ T cells, indicating an intrinsic effect of costimulation uponthe transferred CD8+ T cell population (Supplemental Fig. 1C).

Agonistic costimulation via CD27 is more effective than OX40in the initial reversal of CD8+ T cell exhaustion

CD27 is another TNFR family member that differs from OX40 inthat its expression is constitutive upon naive CD8+ T cells. As

shown in Fig. 3A, CD27 was expressed to a similar extent upon

both input naive and unhelped exhausted Mh CD8+ T cells at day

42 (Fig. 3A). In a similar approach to the experiments outlined in

Fig. 2, we applied agonistic anti-CD27 alone or in combination

with anti–PD-L1 to male BMT recipients adoptively transferred

with unhelped Mh CD8+ T cells 5 wk earlier. In contrast to anti-

OX40, we found that anti-CD27 given alone significantly im-

proved proliferation of unhelped Mh CD8+ T cells, although it had

no effect upon IFN-g generation (Fig. 3B, 3C, Supplemental Fig. 1).

When anti-CD27 was combined with anti–PD-L1, additive effects

upon proliferation and synergistic increases in IFN-g expression

were noted (Fig. 3B, 3C, Supplemental Fig. 1A). The synergy for

effector cytokine expression in the combined treatment group was

limited to IFN-g; both anti-CD27 and anti–PD-L1 alone induced

minor increases in TNF-a generation, with no further increases

in the combined treatment group (Supplemental Fig. 1B). No

increases were observed in the frequency of Mh CD8+ T cell

dual staining for IFN-g and TNF-a in any treatment group

(Supplemental Fig. 1B). Similar to our findings with anti-OX40

and anti–PD-L1, synergy between anti-CD27 and anti–PD-L1 was

only observed under helpless conditions (Supplemental Fig. 1D).

To compare the effects of combining anti–PD-L1 with each TNFR

Ab, we performed direct comparisons of anti-OX40 or anti-CD27

in combination with anti–PD-L1. As shown in Fig. 4, anti-CD27

and anti-OX40 in combination with anti–PD-L1 were similarly

effective in promoting a proliferative response in Mh CD8+ T cells

and in enhancing effector function as evaluated by CD107a ex-

pression. However, anti–PD-L1 plus anti-CD27 were more ef-

fective than anti–PD-L1 and anti-OX40 in enhancing the effector

function of Mh CD8+ T cells as evaluated by IFN-g upon peptide

stimulation (Fig. 4).

FIGURE 1. The effect of CD4+ T cell help upon the

development of CD8+ T cell exhaustion. A total of 1 3 106

CD45.1+ Mh transgenic CD8+ T cells was transferred with

or without 2.5 3 106 female B6 CD4+ T cells, 1 wk after

lethal irradiation of B6 male mice and reconstitution with

female B6 bone marrow. (A) Graph shows mean percent-

age 6 SEM of Mh CD8+ T cells that produced IFN-g in

response to UTY peptide on days 8 and 42 after T cell

transfer. Data are pooled from two independent experi-

ments (helped, n = 5–6/group; unhelped, n = 3–12/group).

(B) Graph showing mean percentage 6 SEM of PD-1high

Mh CD8+ T cells (n = 5/group). (C) Irradiated male

recipients were given 200 mg anti–PD-L1 blocking Ab on

days 36 and 39 following T cell transfer (n = 5, +CD4;

n = 6, no CD4) or isotype control (n = 6, +CD4; n = 7, no

CD4) before analysis on day 42. From left to right, graphs

show mean percentage 6 SEM of Mh CD8+ T cells that

had incorporated BrdU, mean percentage 6 SEM of

Mh CD8+ T cells that produced IFN-g, and mean 6SEM absolute numbers of Mh CD8+IFN-g+ cells/

spleen. Data are pooled from two independent experi-

ments. Statistical comparisons performed using two-

tailed, unpaired Student t test: *p , 0.05, **p , 0.01,

***p , 0.001.

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Molecular profiling of T cell responses to TNFR costimulationin the presence or absence of coinhibitory checkpoint blockade

To identify potential mechanisms underlying the synergy betweenOX40 and CD27 agonistic Abs and anti–PD-L1, we extractedmRNA from flow-sorted Mh CD8+ T cells derived from mice, 7 dfollowing the commencement of individual or combined Abcombinations (day 42, n = 4 mice/group) and then performedquantitative RT-PCR for 84 individual genes linked to T celltolerance and effector functions. As shown in the heat map inFig. 5A, the gene expression profiles of Mh CD8+ T cells derivedfrom mice receiving anti-CD27 and anti–PD-L1 segregated fromthe other groups, although there was some overlap, primarily withthe anti-CD27 group and the combined anti-OX40/anti–PD-L1group. Within the subset of genes showing reduced transcriptionin the anti-CD27 and anti–PD-L1 group, we found that a signifi-cant number was involved in quiescence and/or anergy, includinggenes encoding transcriptional repressors (Foxp1, Foxp3, Egr2,Egr3, Ing4), E3 ubiquitin ligases (Itch, Rnf128), and other genesencoding proteins preventing TCR-proximal signaling (Dgka,Dgkz) or providing coinhibitory signals (Ctla4, Pdcd1, Btla).Using a .2.0-fold reduction cutoff, we evaluated how these geneswere affected in each of the experimental groups (Fig. 5B). All of

the treatment groups were characterized by a core signature ofreduced Egr3 expression and, with the exception of the anti–PD-

L1 group, reduced Foxp3 expression. Egr2, an Egr3-related

transcription factor gene required for the induction of several other

anergic factors (28–30), was reduced in the anti-CD27 group and

both the combined treatment groups. In addition, the combined

anti-CD27 and anti–PD-L1 group showed reductions in the ex-

pression of a more extensive set of anergy-related genes (Fig. 5B).

The number of downregulated genes was not as wide in the

combined anti-OX40 and anti–PD-L1 group despite the demon-

stration of significant synergy in terms of proliferation; however,

this group was additionally characterized by reduced expression of

the coinhibitory receptor Btla (an effect that was also seen, but to

a lesser extent in the anti-OX40 group; Fig. 5B and data not

shown). To evaluate this gene expression pattern in more detail,

we examined the expression of a panel of 10 genes that have been

shown individually to be necessary for anergy or quiescence (28–

30). When anti-CD27 was combined with anti–PD-L1, the entire

panel of anergy and quiescence genes showed reduced expression

with a clear synergistic pattern. In the combined anti-OX40 and

anti–PD-L1 group, marked synergy was observed for Egr2 down-

regulation with further additive effects upon Rnf128 repression.

FIGURE 2. Effect of agonistic anti-OX40

and/or blocking anti–PD-L1 Ab upon helped

or unhelped donor Mh CD8+ T cell effector

functions. (A) Representative histograms showing

OX40 expression upon gated Mh CD8+ T cells

(open histograms) in recipient spleens on day 42

following transfer to irradiated B6 male mice

(mean % OX40+ was 10.5 6 2.2 in helped versus

11.4 6 2.4 in unhelped; naive, 1.5 6 0.5). Filled

histograms show OX40 staining of endogenous

cells in same host. (B) Male BMT recipients were

given anti-OX40 i.p. day 35 following Mh CD8+

T cell transfer (with or without CD4+ T cells, n =

4/group) or 200 mg anti–PD-L1 blocking Ab i.p.

days 36 and 39 (n = 5, +CD4; n = 6, no CD4) or

both Abs (n = 6, +CD4; n = 7, no CD4). Control

mice received the same number of i.p. injections

with the relevant isotype control (n = 6–7). Top

two rows, Representative contour plots show IFN-g

production by helped Mh CD8+ T cells following

exposure to UTY peptide, with gates set according

to irrelevant peptide and representative histograms

showing the percentage of Mh CD8+ T cells

incorporating BrdU. Bottom two rows, Repre-

sentative contour plots for IFN-g production and

BrdU incorporation following transfer of un-

helped Mh CD8+ T cells. (C) From left to right,

graphs show mean percentage 6 SEM of Mh

CD8+ T cells that had incorporated BrdU, mean

percentage 6 SEM of Mh CD8+ T cells that

produced IFN-g, and mean 6 SEM absolute

numbers of Mh CD8+IFN-g+ cells/spleen. Data are

pooled from two independent experiments. Sta-

tistical comparisons performed using two-tailed,

unpaired Student t test: *p , 0.05, **p , 0.01,

***p , 0.001.

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In contrast, with the exception of Egr3, none of the anergy geneswas downregulated in the PD-L1 group. We also extended thisanalysis to evaluate the entire gene set using a .2.0-fold changecutoff (Supplemental Fig. 2). Using these criteria, it was note-worthy that the changes in the anergy- and quiescence-relatedgenes were not paralleled by reciprocal increases in the expres-sion of genes encoding effector molecules or the transcriptionalregulators that induce them when considered at a whole pop-ulation level. Indeed, the expression of several regulator genesrequired for effector differentiation (Jak3, Gata3, Stat6, Tbx21,Icos, Irf4, Fos, Jun) was in fact reduced in Mh CD8+ T cells de-rived from mice receiving anti-CD27 and anti–PD-L1.

Loss of T-bethighEomeslow cells by combined treatment withanti-CD27 and anti–PD-L1 leads to eventual contraction of theeffector pool

In the model of CD8+ T cell exhaustion following chronic LCMVinfection, a quiescent T-bethighEomeslowPD-1low precursor pop-ulation is required for maintaining residual immunity to Ag andfor restoration of functions in response to PD-1 checkpointblockade (5, 31). Because anti-CD27 alone, and in combinationwith anti–PD-L1, switched off a number of anergy- or quiescence-related genes, we wanted to determine how this would impactupon the precursor population and maintenance of immunity long-term. We therefore conducted experiments in which we tracked

Mh CD8+ T cell function in relation to T-bet and Eomes expres-sion at both early and late time points following Ab treatment.Using the same treatment schedule as Fig. 3, but starting Abtreatment at day 59 following unhelped T cell transfer, we againobserved a synergistic increase in the frequency and absolutenumbers of peripheral blood Mh CD8+ T cells in the combinedanti-CD27 and anti–PD-L1 group (Fig. 6A, 6B). As in previousexperiments, the combined Ab treatment led to a synergistic in-crease in the frequency of IFN-g+ cells upon peptide restimulationon day 65 with a similar trend for absolute numbers (Fig. 6B). Wethen evaluated early changes in T-bet and Eomes expression in theresponding Mh CD8+ T cell populations from each group. In thecontrol group, peripheral blood Mh CD8+ T cells were hetero-geneous with respect to T-box transcription factor expression,retaining a significant pool of T-bethighEomeslow precursor-likecells that constituted ∼40% of the entire population anda smaller, terminally differentiated T-betlowEomeshigh subset of∼10% (Fig. 6C). Although there were no differences in the surfaceexpression of CD27 in T-bethigh versus T-betlow cells (data notshown), we reasoned that anti-CD27 would primarily target theformer population because of its greater replicative potential (5).Indeed, anti–PD-L1 and anti-CD27 treatment induced a rapid re-duction in the frequency of T-bethighEomeslow Mh CD8+ T cells,and, as predicted by their precursor-product relationship (5),this was linked to a reciprocal increase in the frequency of

FIGURE 3. Effect of agonistic anti-CD27 and/or blocking anti–PD-L1 Ab upon unhelped Mh CD8+ T cell effector functions. (A) From experiments of

similar design to those in Fig. 2, representative histograms show CD27 expression upon naive input Mh CD8+ T cells or on day 42 (open histograms)

following transfer to irradiated male mice and isolation from recipient spleens. Filled histograms show isotype control staining. Numbers indicate mean

fluorescence intensity. (B) BMT recipients were given anti-CD27 on day 35 following unhelped Mh CD8+ T cell transfer (n = 8) or 200 mg anti–PD-L1

blocking Ab on days 36 and 39 (n = 9) or both Abs (n = 9). Control mice received the same number of i.p. injections with the relevant isotype control (n =

7). Top, Representative contour plots show IFN-g production by Mh CD8+ T cells following exposure overnight to UTY peptide, with gates set according to

irrelevant peptide. Bottom, Representative histograms showing the percentage of Mh CD8+ T cells incorporating BrdU. (C) Graphs show mean percentage 6SEM of Mh CD8+ T cells that had incorporated BrdU, mean percentage 6 SEM of Mh CD8+ T cells that produced IFN-g, and mean 6 SEM absolute

numbers of Mh CD8+IFN-g+ cells/spleen. Data are pooled from two independent experiments. Statistical comparisons performed using two-tailed, unpaired

Student t test: *p , 0.05, **p , 0.01, ***p , 0.001.

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T-betlowEomeshigh cells; intermediate changes as compared withthe controls were observed in the single anti-CD27 or anti–PD-L1groups (Fig. 6C).In spleens from control mice, we observed a decline over time

in the relative frequency of Mh CD8+ T cells that were T-bethigh

Eomeslow versus T-betlowEomeshigh, consistent with ongoing Agactivation and terminal differentiation (Supplemental Fig. 3). Todetermine the long-term effects of anti-CD27 and/or anti–PD-L1upon Mh CD8+ T cell function and the balance between precursorversus terminally differentiated subsets, we re-evaluated IFN-gsynthesis and T-bet/Eomes expression in recipient spleens at day120 after T cell transfer, 61 d following anti-CD27 and/or anti–PD-L1 administration (Fig. 6D–F). Despite the initial rescue of theresponse following combined treatment, the frequency of MhCD8+ T cells capable of producing IFN-g at day 120 in responseto relevant peptide was further reduced compared with the control(2.5-fold) or singly-treated anti–PD-L1 group (4-fold) (Fig. 6D).The absolute number of total and IFN-g+ Mh CD8+ T cells/spleenwas also significantly lower in the combined anti-CD27/anti–PD-L1 group compared with the control (2-fold) and anti–PD-L1group (17-fold) (Fig. 6D and data not shown); indeed, we notedsimilar reductions in the pool size of functional Mh CD8+ T cellswithin the single anti-CD27 Ab group. In contrast, the absolute

numbers of IFN-g+ Mh CD8+ T cells/spleen in the single anti–PD-L1 Ab-treated mice were greater than controls, indicating a long-term protective effect of this Ab against exhaustion. To determine

how spleen Mh CD8+ T cell function at day 120 correlated withexpression of T-bet and Eomes, we performed intracellularstaining for each transcription factor. Consistent with the hy-

pothesis that anti-CD27 was driving further loss of precursorsrequired for maintaining Ag responsiveness, the surviving MhCD8+ T cells in both the combined anti-CD27 and anti–PD-L1group and the single anti-CD27 group showed significant skewing

to a terminally differentiated T-betlowEomeshigh phenotype witha relative loss of cells that were T-bethighEomeslow (Fig. 6E).Furthermore, in a pooled analysis of all the groups, we observed

a significant negative correlation between the percentage of cellsthat were Eomeshigh and the percentage of cells capable of gen-erating IFN-g (Fig. 6F). Thus, additional CD27 costimulation

enables temporary rescue of exhausted CD8+ T cells, but at theexpense of driving their terminal differentiation and eventual lossof function.

DiscussionIn this study, we have shown that anti-OX40– and anti-CD27–mediated costimulation synergized with coinhibitory checkpoint

blockade to restore functions of exhausted, helpless CD8+ T cells.When combined with anti–PD-L1, stimulation of T cells throughOX40 and especially CD27 primarily acted to block molecularprograms for anergy or quiescence, driving CD8+ T cells to un-

dergo rapid proliferation and terminal differentiation. However,transient improvements in effector functions occurred at the ex-pense of loss of precursor cell populations capable of sustaining

the response.As in models of chronic viral infection, unhelped HY-specific

CD8+ T cells become more exhausted than helped cells and less

amenable to rescue through anti–PD-L1 blockade alone. Thesefindings are also consistent with the concept that CD4+ T cells areless prone to exhaustive differentiation than CD8+ T cells (32) andthus capable of providing helper signals at the priming and/or

maintenance phase of the response (27). Because the amount ofmale Ag is fixed during the development of HY-specific CD8+

T cell exhaustion following BMT, the capacity of CD4+ T cells to

mitigate against CD8+ T cell exhaustion is independent of anyeffect upon the overall Ag load. Lack of help was linked to higherexpression of PD-1 upon the exhausted Mh CD8+ T cell reper-

toire, and this is likely to explain the relative failure of helplesscells to respond to anti–PD-L1. The effect of CD4+ T cells uponPD-1 expression could be direct, for example through the syn-thesis of IL-2, which can limit upregulation of PD-1 upon CD8+

memory T cells (33), or indirect, through licensing of APCs (34).Lack of CD4+ T cell help is also a major obstacle to successfulimmunotherapy of cancer (35). In a tumor model characterized by

ineffective Ag presentation via MHC class II (36), we have alsoobserved synergy between anti-OX40 and anti-CD27 with anti–PD-L1. Thus, following vaccination with irradiated B16 mela-

noma cells expressing Flt3-ligand, addition of either anti-OX40 oranti-CD27 to anti–PD-L1 synergized to enhance tumor infiltrationby endogenous T cells and significantly delay tumor growth(S. Buchan and A. Al-Shamkhani, unpublished data). The finding

that tumor-reactive CD8+ T cells within human melanomas fre-quently coexpress PD-1 and TNFR family costimulatory molecules(37) suggests that this approach may have clinical application.OX40- or CD27-mediated costimulation activates several

pathways downstream of the TCR that are inhibited via PD-1signaling, including PI3K-Akt, NF-kB, and NFAT (38). In addi-

FIGURE 4. Comparison of agonistic anti-CD27 and anti-OX40 in

combination with anti–PD-L1 Ab upon unhelped Mh CD8+ T cell effector

functions. Experimental design as set out in Figs. 2 and 3. Graphs show

mean percentage 6 SEM of Mh CD8+ T cells that had incorporated BrdU,

mean percentage 6 SEM of Mh CD8+ T cells that expressed surface

CD107a or intracellular IFN-g upon restimulation, and mean 6 SEM

absolute numbers of Mh CD8+IFN-g+ cells/spleen. Data are pooled from

two independent experiments (n = 7–9/group). Statistical comparisons

performed using two-tailed, unpaired Student t test: *p , 0.05, **p ,0.01, ***p , 0.001.

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tion, PD-1 ligation also interrupts proximal TCR signalingthrough mechanisms that require recruitment of Src homologyregion 2 domain–containing phosphatase 2 to an immunoreceptortyrosine-based switch motif contained within its cytoplasmic do-main (39). For the most part, we observed that the interactionbetween OX40- and especially CD27-induced costimulation wassynergistic with anti–PD-L1, indicating that TNFR family–mediated signaling was acting on molecular pathways distinctfrom those inhibited by PD-1. The lack of synergy in the helpedsetting would also imply that signaling via OX40 or CD27 issomehow suboptimal in unhelped CD8+ T cells, although whetherthis is a T cell–intrinsic effect and/or the result of reduced li-gand expression requires further study. Following cotreatmentwith anti-CD27 and anti–PD-L1 especially, several genes en-coding proteins promoting anergy or quiescence were sharply

downregulated. This finding was unexpected because the tran-scriptional programs that underlie exhaustion and anergy are oftenviewed as being distinct (40), with lack of quiescence being a keyproperty assigned to exhausted T cells when the population is ex-amined as a whole (4). Indeed, the behavior of the Mh CD8+ T cellsupon adoptive transfer to male recipients did not parallel in vivomodels of anergy in which T cell functions are impaired almostimmediately upon Ag encounter (41). Instead, more akin to modelsof exhaustion, the functions of Mh CD8+ T cells were progressivelylost over time (Supplemental Fig. 4). Nevertheless, as the proin-flammatory effects of irradiation diminish, it is possible that reducedlevels of costimulation (signal 2) also induce overlapping anergicmolecular programs that contribute to the observed T cell dysfunction.When evaluated at a whole population level, we did not observe

reciprocal changes within the anergy and effector gene sets in

FIGURE 5. Effect of anti-OX40 and anti-CD27 alone or in combination with anti–PD-L1 upon expression of genes linked to tolerance or effector function.

Experimental design as set out in Figs. 2–4. On day 42, unhelped Mh CD8+ T cells were flow sorted to high purity from recipient spleens and mRNA extracted

(n = 3 mice in controls; n = 4 mice/group in Ab treatment groups). (A) Heat map showing quantitative RT-PCR for 84 genes tested (seeMaterials and Methods).

Color code for each group shown within figure. (B) Venn diagram showing pattern for .2.0-fold reduction in anergy/quiescence genes according to treatment

group. (C) Graphs showing fold change expression compared with controls for a panel of 10 anergy quiescence-related genes. The p values were calculated

based on an unpaired t test of the replicate 2(2d Ct) values for each gene in the control group and treatment groups: *p , 0.05, **p , 0.01, ***p , 0.001.

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T cells following combined coinhibitory blockade and enforcedCD27-mediated costimulation. Reduced expression of anergy-related factors would be predicted to enhance TCR-proximalsignaling leading to downstream activation of Ras, ERK, andJNK pathways that in turn induce effector differentiation (29).However, several downstream master regulators of effector geneexpression were in fact moderately downregulated in the com-bined treatment group when evaluated directly ex vivo despite thetransiently increased capacity of T cells to undergo degranulationor generation of IFN-g upon restimulation. Without performingsingle-cell analyses of gene expression, we cannot exclude thepossibility that the lack of reciprocity reflects the fact that thepopulation evaluated is mixed, including cells that remainexhausted as well as those with restored functions. Alternatively,the quantitative PCR may miss the effects of posttranscriptionalregulation of the relevant genes. However, recent studies have alsorevealed different patterns of connectivity in exhausted comparedwith effector or memory cells, such that specific transcriptionfactors (e.g., T-bet) can possess highly variable transcriptionalinteractions according to their cellular context (4). Thus, removal

of anergy factors and enhanced TCR-coupled signal transductionmay be insufficient to fully restore effector programs that havebecome disconnected in the setting of continual TCR stimulation.Although combined anti-CD27 and anti–PD-L1 led to robust

increases in proliferation and measurable gains in effector func-tion upon restimulation, these effects were not sustained. It isknown that tolerant cells induced to undergo proliferation in re-sponse to lymphopenia also transiently improve their effectorfunctions but then switch back to their tolerant state as epigeneticchanges required for tolerance are re-established (42); it is pos-sible that a similar mechanism applies in the setting of exhaustion.However, an alternative and nonexclusive mechanism suggested inthis study is that the loss of quiescence factors induced by anti-CD27 with or without anti–PD-L1 eventually leads to a loss ofprecursor cells and consequent reductions in the total pool size ofAg-specific T cells as effectors reach their Hayflick limit andundergo replicative senescence (43). The fact that the effector poolwas also eventually diminished in the group treated with anti-CD27 Ab alone suggests that reductions in only a subset ofanergy factors are sufficient for the loss of T-bethigh precursors to

FIGURE 6. Effect of agonistic anti-CD27 with or without anti–PD-L1 Ab upon unhelped Mh CD8+ T cell T-box factor expression and long-term effector

functions. Experimental design as set out in Fig. 3, except that anti-CD27 was given on day 59 and anti–PD-L1 on days 59 and 62, with isotype control Abs

given on the same days (n = 4/group). (A) Frequency of blood Mh CD8+ T cells (as percentage of live gate) following indicated Ab treatment. Statistical

comparisons are for combined treatment group versus isotype control. (B and C) Analyses performed on day 65 in peripheral blood. (B) Left, Mean 6 SEM

absolute number of blood Mh CD8+ T cells. Middle, Mean percentage 6 SEM of blood Mh CD8+ T cells that produced IFN-g. Right, Mean 6 SEM

absolute number of Mh CD8+IFN-g+ cells/ml blood. (C) Graphs showing mean percentage of blood Mh CD8+ T cells that were T-bethighEomeslow (left) or

T-betlowEomeshigh (right) on day 65 following transfer. (D–F) Analyses performed on day 120 in spleen. (D) Mean percentage 6 SEM of Mh CD8+ T cells

that produced IFN-g (left) and mean 6 SEM absolute numbers of Mh CD8+IFN-g+ cells/spleen (right) at day 120 following transfer. (E) Graphs showing

mean percentage of spleen Mh CD8+ T cells that were T-bethighEomeslow (left) or T-betlowEomeshigh (right) on day 120 following transfer. (F) Scatter plot

showing correlation between frequency of IFN-g+ Mh CD8+ T cells (x-axis) versus frequency of cells that were Eomes+ (y-axis). Data are representative

of two independent experiments with similar design. Statistical comparisons performed using two-tailed, unpaired Student t test: *p , 0.05, **p , 0.01,

***p , 0.001.

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occur. Future studies addressing the potential of combined coin-hibitory checkpoint blockade and costimulation therapies willtherefore need to examine in detail how restoration of exhaustedT cell functions affects the composition of the emerging repertoireaccording to markers of replicative potential such as T-box tran-scription factor expression and telomere length. The risk that suchtherapies deplete the effector pool still further may be greatestwhen levels of Ag and/or costimulation are already high.

AcknowledgmentsWe thank Hans Stauss for helpful comments on the manuscript.

DisclosuresG.J.F. has patents and receives patent royalties on the PD-1 pathway. The

remaining authors have no financial conflicts of interest.

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