IL10 and PD-1 Cooperate to Limit the Activity of Tumor ... · Microenvironment and Immunology IL10...

Microenvironment and Immunology

IL10 and PD-1 Cooperate to Limit the Activity ofTumor-Specific CD8þ T CellsZhaojun Sun1, Julien Fourcade1, Ornella Pagliano1, Joe-Marc Chauvin1, Cindy Sander1,John M. Kirkwood1, and Hassane M. Zarour1,2

Abstract

Immune checkpoint inhibitors show great promise as therapyfor advanced melanoma, heightening the need to determine themost effective use of these agents. Here, we report that pro-grammed death-1high (PD-1high) tumor antigen (TA)–specificCD8þ T cells present at periphery and at tumor sites in patientswith advanced melanoma upregulate IL10 receptor (IL10R)expression. Multiple subsets of peripheral blood mononucleo-cytes from melanoma patients produce IL10, which acts directlyon IL10Rþ TA-specific CD8þ T cells to limit their proliferation and

survival. PD-1 blockade augments expression of IL10R by TA-specific CD8þ T cells, thereby increasing their sensitivity to theimmunosuppressive effects of endogenous IL10.Conversely, IL10blockade strengthened the effects of PD-1 blockade in expandingTA-specific CD8þ T cells and reinforcing their function. Collec-tively, our findings offer a rationale to block both IL10 and PD-1to strengthen the counteraction of T-cell immunosuppression andto enhance the activity of TA-specific CD8þ T cell in advancedmelanoma patients. Cancer Res; 75(8); 1635–44. �2015 AACR.

IntroductionT cells recognize tumor antigens (TA) expressed by melanoma

cells and can induce tumor regression in animals and in humans(1). However, in the presence of high antigen load in chronic viralinfections and in cancer, antigen-specific CD8þ T cells becomedysfunctional/exhausted and lose their capacities to proliferate,produce cytokines, and lyse tumor cells (2, 3). These dysfunc-tional antigen-specific CD8þ T cells upregulate a number ofinhibitory receptors, including programmed death-1 (PD-1), andPD-1 blockade augments their expansion and functions in vitro(4–8). The capability of PD-1 blockade to provide persistentclinical benefit to approximately 30% to 40% of patients withadvanced melanoma has now been demonstrated in multipleclinical trials (9, 10). To further improve the clinical efficacyof PD-1 blockade, it appears critical to identify additional strategies tocounteract the major negative immunoregulatory pathwaysimpairing TA-specific CD8þ T cells in the tumor microenviron-ment (TME).

IL10 is a potent anti-inflammatory molecule produced byinnate and adaptive immune cells, including T cells, natural killercells, antigen-presenting cells (APC) as well as tumor cells, includ-ing melanoma (11–15). The immunosuppressive role of endog-enous IL10 in impeding APCs is supported by the demonstration

that neutralizing IL10 with anti-IL10R antibodies is required forthe stimulation of potent Th1 OVA-specific and TA-specific T-cellresponses in mice treated with Toll-like receptor ligands (16, 17).The role of IL10 role in cancer immunology remains controversial.In experimental tumormodels, IL10 appears to either promote orfacilitate tumor rejections (18–26). The effects of IL10 and IL10blockade on human TA-specific CD8þ T cells have not beenthoroughly evaluated yet. In chronic viral infections, IL10 andPD-1 pathways act synergistically through distinct pathways tosuppress T-cell functions, and dual IL10 and PD-1 blockadeappears more effective in restoring antiviral CD8þ and CD4þ

T-cell responses and viral clearance than either single blockadealone (27, 28). Whether IL10 added to PD-1 blockade furtherenhances TA-specific CD8þ T-cell functions inmelanomapatientsremains unknown.

Here, we report for the first time that PD-1high CD8þ T cellsdirected against the cancer-germline antigen NY-ESO-1 andPD-1high CD8þ tumor-infiltrating lymphocytes (TIL) isolatedfrom patients with advanced melanoma, upregulate IL10receptor (IL10R). Although PD-1 blockade in the presence ofcognate antigen increases the expansion and functions of NY-ESO-1–specific CD8þ T cells, it also augments IL10R expressionby TA-specific CD8þ T cells. We show that IL10 blockade addsto PD-1 blockade to increase the expansion and functions ofNY-ESO-1–specific CD8þ T cells, supporting the role of dualIL10 and PD-1 blockade to enhance TA-specific CTL responsesto melanoma.

Materials and MethodsSubjects

Blood samples and tumor specimen were obtained under theUniversity of Pittsburgh Cancer Institute Institutional ReviewBoard–approved protocols 00-079 and 05-140 from 12 HLA-A2þ patients with NY-ESO-1þ stage IV melanoma and sponta-neous NY-ESO-1–specific CD8þ T cells (Supplementary TableS1). The peripheral bloodmononuclear cells (PBMC) used in this

1Division of Hematology/Oncology, Department of Medicine, Univer-sity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.2Department of Immunology, University of Pittsburgh School of Med-icine, Pittsburgh, Pennsylvania.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Hassane Zarour, University of Pittsburgh, Suite 1.32a,5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3272; Fax: 412-623-7704; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-3016

�2015 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1635

on May 20, 2020. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 26, 2015; DOI: 10.1158/0008-5472.CAN-14-3016

http://cancerres.aacrjournals.org/

study were obtained from melanoma patients with no priorimmunotherapy. The same patients were used across all assays.

Phenotypic analysisCD8þ T lymphocytes were purified from PBMCs of patients

usingMACSColumnTechnology (Miltenyi Biotec). Alternatively,PBMCs were incubated for 6 days in culture medium containing50 IU/mL rhIL2 (PeproTech) with peptide NY-ESO-1 157–165 ormediumalone in the presence of 10mg/mL anti-IL10R (clone 3F9;BioLegend) or anti–PD-L1 (clone MIH1; eBioscience) or isotypecontrol antibodies and/or 20 ng/mL rhIL10 (PeproTech). Cellswere incubated either with HLA-A2/NY-ESO-1 157–165, HLA-A2/CMV 495–503, HLA-A2/EBV-BMLF-1 280–288, HLA-A2/Flu-M 58–66, or HLA-A2/MART-1 26–35 tetramers (TC metrix Ltd.)before staining with PD-1-PerCPCy5.5, IL10R-PE (BioLegend),and CD8-PE-Cy7, CD14-ECD, CD19-ECD, CD56-biotin (Beck-man Coulter), and streptavidin-ECD (Invitrogen)–conjugatedantibodies or reagent. Alternatively, after tetramer labeling, cellswere stained with PD-1-PECy7 (BioLegend), CD8-V500, CD69-FITC, or CD57-FITC, CD38-PerCp-Cy5.5 (BD Biosciences), HLA-DR-ECD, or CD25-ECD (Beckman Coulter). Alternatively,PBMCs were stained with CD11c-Alexa700 (eBioscience),CD19-APCCy7, CD56-FITC (BD Biosciences), CD8-PECy7,CD4-PerCPCy5.5 (BioLegend), CD14-ECD, and IL10R-PE. Aviolet amine reactive dye (Invitrogen) was used to assess theviability of the cells. p-STAT3-Alexa 488 (BD Biosciences) wasused to identify the phosphorylated form of STAT3 (Ser727). Atotal of 2.5 � 106 events were collected on a FACSAria machine(BD Biosciences) and analyzed with FlowJo software (Tree Star).

IL10 detectionThe concentrations of IL10 in supernatant or sera were deter-

mined using BDOptEIAHuman IL10 ELISA Set (BDBiosciences).To test IL10 production, CD8þ T cells were purified from PBMCs(MACS Column Technology), and labeled with tet-APC, CD8-PECy7, CD4-PE, and violet. A total of 6 � 104 FACS-sorted cellswere distributed into 96 wells with 200 mL medium containing50 IU/mL rhIL2, T2 cells (2:1 ratio) pulsedwithpeptideNY-ESO-1157–165 or control peptide HIVpol 476–484 (10 mg/mL). Super-natant was collected for ELISA assay after 48-hour incubation.Alternatively, CD4þ and CD8þ T cells were separated by MACSColumn Technology, the rest of the cells were labeled with CD14-ECD, CD11c-Alexa700, CD3-PerCPCy5.5, CD56-PE, and CD19-FITC, and sorted. Total RNA was extracted from each cell subset,and IL10 mRNA was detected using RT-QPCR as previouslydescribed (29).

Carboxyfluorescein diacetate succinimidyl ester proliferationassay

Proliferation assay was performed as described previously (8).CD8þ TILs were separated by MACS Column Technology andcultured inmediumcontaining50 IU/mL rhIL2 for 2days followedby carboxyfluorescein diacetate succinimidyl ester (CFSE) labelingand stimulation with anti-CD3 antibodies and autologous non-CD3 cells obtained from the same tumor for 6 days. Cells werestained and analyzed by flow cytometry.

Intracellular cytokine staining assay and apoptosis studiesIn vitro stimulation assays were performed as described previ-

ously (8). Briefly, PBMCs were stimulated for 6 days in thepresence of 10 mg/mL anti-IL10R and/or anti–PD-1 (clone

EH12.2H7; BioLegend) blocking mAb or isotype control anti-bodies before intracellular cytokine staining. Alternatively, cellswere harvested and then surface stainedwith APC-labeled A2/NY-ESO-1 tetramers, and subsequently with CD8-PECy7 andAnnexin V-FITC (ApoScreen Annexin V-FITC Apoptosis Kit; Beck-man Coulter).

Statistical analysisStatistical hypotheses were tested with the Wilcoxon signed

rank test (for paired results from the same patient) and unpaired ttest (for IL10 concentration) with GraphPad Prism statisticalanalysis program. Tests were two-sided and considered significantfor a P value of <0.05.

ResultsIL10R is upregulated by PD-1high NY-ESO-1–specific CD8þ Tcells

Using HLA-A2 (A2) tetramers (tet), we first investigated theexpression of IL10R and PD-1 on NY-ESO-1–specific, MART-1–specific, virus-specific [cytomegalovirus (CMV), Epstein-Barr virus(EBV), and influenza virus (Flu)] and total CD8þ T cells that aredetectable ex vivo in PBMCs of nine HLA-A� 0201þ (HLA-A2þ)stage IV melanoma patients. The percentages of IL10Rþ cellsamong NY-ESO-1–specific CD8þ T cells (mean 8.8% � SD2.8%) were significantly higher than those of MART-1–specific(2.4%� 1.5%), Flu-specific (1.6%� 1.3%), EBV-specific (3.4%�2.7%), and total tet� (1.7% � 1.0%) CD8þ T cells (Supplemen-tary Fig. S1A and S1B, left), albeit not significantly different fromthe percentages of IL10Rþ CMV-specific CD8þ T cells (5.1% �4.2%). Similar observations were made in terms of mean fluo-rescence intensity (MFI; Supplementary Fig. S1B, right). In agree-ment with previous findings (11, 30), IL10R was also constitu-tively expressed by multiple cell subsets of PBMCs with higherexpression detected on myeloid dendritic cells (DC) and B cells(P < 0.01, Supplementary Fig. S1C).

As previously shown, NY-ESO-1–specific CD8þ T cellsexpressed higher PD-1 levels (frequencies and MFI) thanMART-1–specific and virus-specific CD8þ T cells present inPBMCs of patients with advanced melanoma (6). Notably, PD-1high CD8þ T cells were detected in NY-ESO-1–specific CD8þ Tcells but not in MART-1–specific, virus-specific and total tet�

CD8þ T cells (Fig. 1A and Supplementary Fig. S1D). The percen-tages of IL10Rþ cells within PD-1high NY-ESO-1–specific CD8þ Tcells (mean 23.6% � SD 8.1%) were significantly higher thanwithin PD-1int (4.2% � 1.7%) and PD-1low (1.8% � 1.9%) NY-ESO-1–specific CD8þ T cells. Similar observations were made interms of MFI (Fig. 1B, right).

In agreement with previous studies (13, 31, 32), we detectedhigher levels of IL10 in sera of advanced melanoma patients thanin healthy donors (Supplementary Fig. S2A). Although multiplecellular subsets produced IL10,monocytes andDCs expressed thehighest IL10 levels in both melanoma patients and healthydonors (Supplementary Fig. S2B). Interestingly, in 1 patient withhigh NY-ESO-1–specific CD8þ T-cell frequency, ex vivo sorted PD-1þ NY-ESO-1 tetþ CD8þ T cells produced IL10 in the presence ofcognate peptide-pulsed APCs (Supplementary Fig. S2C), support-ing that dysfunctional PD-1high antigen-specific CD8þ T cells canproduce IL10 upon T-cell receptor (TCR) activation (33).

Collectively, our results show that circulating PD-1high NY-ESO-1–specific CD8þ T cells upregulate IL10R expression, and

Sun et al.

Cancer Res; 75(8) April 15, 2015 Cancer Research1636




that IL10 is produced at low levels by multiple cells in PBMCs ofpatients with advanced melanoma.

NY-ESO-1–specific CD8þ T cells upregulate IL10R along withPD-1 upon TCR activation

To determine the activation status of IL10Rþ PD-1high NY-ESO-1–specific CD8þ T cells, we evaluated their expression ofHLA-DR,CD38, andCD57. IL10Rþ PD-1high cells expressed higher levels ofHLA-DR,CD38, andCD57 than IL10R�PD-1high, IL10R�PD-1int,and IL10R� PD-1low NY-ESO-1–specific CD8þ T cells, suggestingthat they represent a highly activated NY-ESO-1–specific CD8þ T-cell subset (Fig. 2A).

To investigate whether IL10R upregulation occurs upon TCRactivation, we next assessed the sequential expression of IL10R

and PD-1 by spontaneous NY-ESO-1–specific CD8þ T cells pres-ent in PBMCs of patients with advanced melanoma over a 6-dayin vitro stimulation in the presence of cognate or irrelevantpeptide. We observed a significant increase in IL10R expression(percentage andMFI) by a fraction of NY-ESO-1–specific CD8þ Tcells following cognate antigen stimulation fromday 4 to day 6 ofin vitro stimulation (Fig. 2B). As previously reported, NY-ESO-1–specific CD8þ T cells also upregulated PD-1 expression uponstimulationwith cognate peptide (8), with amajority of the T cellsdisplaying a PD-1high phenotype starting at day 4 (SupplementaryFig. S3A and S3B). The percentages of IL10RþPD-1high amongNY-ESO-1–specific CD8þ T cells as well as the frequencies of IL10Rþ

PD-1high NY-ESO-1–specific CD8þ T cells among total CD8þ Tcells increased upon stimulation with cognate peptide (Fig. 2C).

Figure 1.IL10R is upregulated by PD-1high NY-ESO-1–specific CD8þ T cells. A and B, dot plots from one representative patient (A) and summary data for all 9 patientswith advanced melanoma (B) showing ex vivo IL10R expression by PD-1high and/or PD-1int and PD-1low subsets of A2/NY-ESO-1 157–165, A2/MART-1 26–35,A2/CMV495–503, A2/Flu-M 58–66, A2/EBVBMLF1 280–288 tetþ, and total tet�CD8þT cells; � ,P <0.05 and �� ,P <0.01. Horizontal bars depict themean percentageor MFI of IL10R expression. Data, representative of two independent experiments performed in duplicate.

Targeting IL10 and PD-1 in Melanoma

www.aacrjournals.org Cancer Res; 75(8) April 15, 2015 1637




Figure 2.NY-ESO-1–specific CD8þ T cellsupregulate IL10R and PD-1 upon TCRactivation. A, pooled data frommelanoma patients (n ¼ 8) showingthe percentage (left) and MFI (right)of ex vivo expression of HLA-DR,CD38, and CD57 on IL10Rþ and IL10R�

subsets in PD-1high and on IL10R�

subsets in PD-1int andPD-1lowNY-ESO-1–specific CD8þ T cells. B, IL10Rexpression on NY-ESO-1 tetþ CD8þ

T cells assessed ex vivo and afterindicated hours following in vitrostimulation with cognate peptide(NY-ESO-1 peptide) or irrelevantpeptide (HIV peptide; n ¼ 6). C, thepercentage of IL10RþPD-1high inNY-ESO-1 tetþ CD8þ T cells (left) andin total CD8þ T cells (right) tested in B.Data, representative of at least twoindependent experiments; � , P < 0.05and �� , P < 0.01. Horizontal bars depictthe mean percentage or MFI of cellsthat express the correspondingmolecule.

Sun et al.





Collectively, our findings show that IL10RþPD-1high NY-ESO-1–specific CD8þ T cells represent a highly activated T-cell subsetthat expands upon prolonged TCR activation.

NY-ESO-1–specific CD8þ T cells upregulate IL10R expressionupon PD-1 blockade

To investigate the effects of PD-1 blockade on IL10R expres-sion by total NY-ESO-1–specific CD8þ T cells, PBMCs ofmelanoma patients were stimulated with NY-ESO-1 peptidefor 6 days in the presence of blocking mAbs against PD-L1,IL10R, or isotype control antibodies, before tetramer and IL10Ror PD-1 labeling. We observed an increase in IL10R expressionby total NY-ESO-1 tetþ, but not tet� CD8þ T cells after incu-bation in the presence of cognate peptide and anti–PD-L1antibodies, as compared with cognate peptide and IgG controlantibodies (mean percentage of IL10Rþ: 35.3% � SD 12.7% vs.25.0% � 9.8% and mean MFI IL10R: 537 � SD 220 vs. 394 �156, respectively; Fig. 3A and B). In contrast, IL10R blockadedid not increase PD-1 expression by NY-ESO-1–specific CD8þ Tcells (Supplementary Fig. S4A and S4B).

Collectively, ourfindings show thatNY-ESO-1–specificCD8þTcells upregulate IL10R upon PD-1 blockade and prolonged anti-gen stimulation, whereas IL10R blockade has no effect on PD-1expression.

IL10 impedes the expansion of NY-ESO-1–specific CD8þ T cellsupon antigen stimulation

Becausewe have shown that IL10R expression is upregulated byPD-1high NY-ESO-1–specific CD8þ T cells and increases followingTCR activation, we next investigated the effect of IL10 on theexpansion and survival of NY-ESO-1–specifc CD8þ T cells uponstimulation with cognate antigen. CFSE-labeled PBMCs frommelanoma patients were stimulated for 6 days with NY-ESO-1157–165peptide in the presence or absence of rhIL10 and/or anti-IL10R mAbs. The frequencies of proliferating CFSElo NY-ESO-1–specific CD8þ T cells significantly decreased after in vitro stimu-lation in the presence of IL10 as compared with stimulation withpeptide only (0.5-fold change) and were restored in the presenceof blocking anti-IL10R mAbs (Fig. 4A–C). The percentages ofAnnexin Vþ NY-ESO-1–specific CD8þ T cells increased after a 6-day in vitro stimulation with cognate peptide in the presence ofrhIL10 (1.3-fold change), but not rhIL10 in combination withanti-IL10R mAbs, as compared with in vitro stimulation withpeptide only (Fig. 4D). Notably, in the presence of rhIL10, theexpression of phosphorylated STAT-3 (p-STAT3) in NY-ESO-1–specific CD8þ T cells was significantly higher than in EBV-specificCD8þ T cells, which express lower levels of IL10R, as comparedwith untreated cells (3- and 1.6-fold increase, respectively). Theeffect of IL10 on p-STAT3 expression was abolished in the pres-ence of blocking anti-IL10R mAbs, suggesting that IL10 actsdirectly on T cells through IL10R activation (Fig. 4E).

Collectively, our findings show that IL10 impedes the expansionofNY-ESO-1–specificCD8þ T cells by decreasing their proliferativecapacity and promoting their apoptosis. We also observed thatIL10 acts directly on IL10Rþ NY-ESO-1–specific CD8þ T cells.

IL10R blockade adds to PD-1 blockade to increase theexpansion and functions of NY-ESO-1–specific CD8þ T cells

We then investigated whether IL10R blockade alone or incombination with PD-1 blockade increased NY-ESO-1–specificCD8þ T-cell expansion and functions in response to cognateantigen. We observed a modest increase in the frequencies ofproliferating (CFSElo) and total NY-ESO-1 tetþ CD8þ T cells inthe presence of cognate peptide and anti-IL10RmAbs as comparedwith cognate peptide and IgG control antibodies (1.5- and 1.3-foldchanges, respectively; Fig. 5A and B and Supplementary Fig. S5Aand S5B). In line with previous findings (6, 7), PD-1 blockadesignificantly increased the frequencies of CFSElo and total NY-ESO-1–specific CD8þ T cells (1.7- and 1.4-fold changes, respectively).There was no significant difference between single IL10R blockadeand single PD-1 blockade on NY-ESO-1–specific CD8þ T cellsproliferation. Interestingly, dual IL10R and PD-1 blockade furtherincreased the frequencies of proliferating and total NY-ESO-1–specificCD8þ T cells as comparedwith incubationwith IgGcontrolantibodies, anti-IL10R mAbs alone or anti–PD-1 mAbs alone,resulting in the highest fold changes in the frequencies of CFSElo

and total NY-ESO-1 tetþ CD8þ T cells (2.5- and 1.9-fold changes,respectively; Fig. 5A and B and Supplementary Fig. S5A and S5B).

The frequencies of NY-ESO-1–specific CD8þ T cells that pro-duced IFNg , but not TNF, increased after a 6-day in vitro stimu-lation in the presence of cognate peptide and anti-IL10R mAbs ascompared with incubation with IgG control antibodies (1.3-foldchange; Fig. 5C andD, top and Supplementary Fig. S5C and S5D).PD-1 blockade alone increased the frequencies of IFNg- and TNF-producing NY-ESO-1–specific CD8þ T cells (1.6- and 1.5-foldchanges, respectively). Dual IL10 and PD-1 blockade further

Figure 3.NY-ESO-1–specific CD8þ T cells upregulate IL10R expression upon PD-1pathway blockade. A andB, dot plots fromone representative patient (A) andsummary data for 8 patients withmelanoma showing the percentage (B, left)and MFI (B, right) of IL10R expression by total A2/NY-ESO-1 157–165 tetþ andtotal tet� CD8þ T cells after culture. PBMCs of melanoma patients werestimulated with NY-ESO-1 peptide in the presence of blocking mAbs againstPD-L1 or isotype control antibodies before tetramer and IL10R labeling;� , P <0.05 and �� , P <0.01. Horizontal bars depict themean percentage orMFIof IL10R expression. Data, representative of two independent experimentsperformed in duplicate. IVS, in vitro stimulation.






increased the frequencies of IFNg- and TNF-producing NY-ESO-1–specific CD8þ T cells (2.1- and 1.9-fold changes,respectively; Fig. 5C and D, top and Supplementary Fig. S5C andS5D). In addition, we observed an increase in the frequencies ofIFNgþ TNFþ NY-ESO-1–specific CD8þ T cells (1.9-foldchange, Fig. 5C and D, bottom), suggesting that dual IL10R andPD-1 blockade expands polyfunctional TA-specific CD8þ T cells.

Collectively, ourfindings show that IL10blockade adds toPD-1blockade to enhance the expansion and functions of NY-ESO-1–specific CD8þ T cells.

CD8þ TILs present inmetastaticmelanoma coupregulate IL10Rand PD-1

To determine whether our findings on circulating TA-specificCD8þ T cells were relevant to CD8þ TILs present in the TME, weassessed IL10R and PD-1 expression by CD8þ TILs obtained frommetastatic lesions of nine melanoma patients. The percentages ofIL10Rþ cells were significantly higher in CD8þ TILs (mean 30.0%� SD19.6%) than in total NY-ESO-1 tetþ (8.8%� 2.8%) and tet�

(1.7% � 1.0%) CD8þ T cells present in PBMCs of melanomapatients (Fig. 6A and B left). Similar observations were made interms of MFI (Fig. 6B right). CD8þ TILs also upregulated PD-1expression and the percentages of IL10Rþ cells within PD-1high

CD8þ TILs (mean 39.8% � SD 24.4%) were significantly higherthan within PD-1int (29.2% � 27.6%) and PD-1low (20.7% �23.4%) CD8þ TILs. Similar observations were made in terms ofMFI (Fig. 6C and D).

To investigate whether IL10 blockade alone or in combinationwith PD-1 blockade increases the expansion of CD8þ TILs, CD8þ

T cells isolated from one metastatic tumor single cell suspensionwere incubated in vitro for 6 days in the presence of anti-CD3antibodies and autologous non-CD3 cells obtained from thesame tumor. We observed an increase in the proliferation ofCD8þ TILs in the presence of anti-IL10R or anti–PD-1 antibodiesas compared with IgG control antibodies. Dual IL10R and PD-1blockade further increased the frequencies of proliferating CD8þ

TILs (Fig. 6E).Collectively, our findings show that CD8þ TILs present in

metastaticmelanoma coupregulate PD-1 and IL10R, and that dualIL10 and PD-1 blockade promotes the expansion of CD8þ TILs.

DiscussionIn this article, we report that PD-1high TA-specific CD8þ T cells

in PBMCs and TILs of patients with advanced melanoma upre-gulate IL10R expression. Among PD-1þ antigen-specific CD8þ T

Figure 4.IL10 inhibits NY-ESO-1–specific CD8þ T-cell proliferation after prolonged antigen stimulation. A and B, dot plots from a representative patient (A) and summaryfor 7 patients with melanoma (B) showing the variation in the frequencies of CFSElo NY-ESO-1–specific CD8þ T cells for 106 CD8þ T cells. CFSE-labeled PBMCs frompatients were incubated with NY-ESO-1 157–165 peptide and rhIL10 or blocking mAb against IL10R (aIL10R) plus rhIL10 or an isotype control antibody (IgG)before the evaluation of A2/NY-ESO-1 157–165 tetþ CD8þ T-cell proliferation by flow cytometry. IVS, in vitro stimulation. C, fold change of the frequencies ofCFSElo NY-ESO-1–specific CD8þ T cells after in vitro stimulation with cognate peptide and rhIL10 or aIL10R plus rhIL10. The ratios of the percentages of CFSElo

NY-ESO-1–specific CD8þ T cells in the presence of rhIL10 or in the presence of aIL10R plus rhIL10 and IgG isotype control are shown. D, pooled data showingthe percentage of Annexin Vþ determined by flow cytometry for A2/NY-ESO-1 157–165 tetþ CD8þ T cells (n¼ 7) or A2/EBVBMLF1 280–288 tetþ CD8þ T cells (n¼ 4),which were incubated for 6 days with NY-ESO-1 157–165 peptide or BMLF1 280–288 peptide and rhIL10 or aIL10R plus rhIL10 or IgG. E, p-STAT3 expressionmeasured by flow cytometry in A2/NY-ESO-1 157–165 tetþ CD8þ T cells or A2/EBV BMLF1 280–288 tetþ CD8þ T cells from melanoma patients (n¼ 5) treated or notwith aIL10R and then stimulated or not with IL10. � , P < 0.05 and �� , P < 0.01.

Sun et al.





cells, it is now well established that PD-1high CD8þ T cellsrepresent a dysfunctional T-cell subset occurring upon persistentantigen stimulation in chronic infections and in cancer (4,5, 8, 34, 35). TA-specific dysfunctional CD8þ T cells present atperiphery and at tumor sites upregulate a number of inhibitoryreceptors, including PD-1, BTLA, and Tim-3, that bind to theirrespective ligands expressed by APCs and tumor cells in the TME(7, 8, 36). Here, we show that TA-specific CD8þ T cells increaseIL10R expression upon TCR activation and PD-1highIL10Rþ cellsupregulate HLA-DR and CD38, supporting that IL10R is a T-cellactivation marker expressed by chronically activated TA-specificCD8þ T cells in patients with advanced melanoma.

IL10 is an immunoregulatory cytokine, which is produced bymultiple innate and adaptive immune cells as well as melanomacells (11). IL10 inhibits antigen-specific T-cell responses byimpeding antigen presentation and costimulation, inducinghuman CD4þ T-cell anergy (37, 38). A number of observationshave also suggested that IL10 may exert immunostimulatoryeffects on CD8þ T cells, depending on their state of activation(39). Elevated IL10 levels have been reported in patients withHCV, HBV, and HIV infections, and correlate with T-cell dysfunc-tion and uncontrolled viral replication (17, 40–42).

The role of IL10 in cancer immunology remains controversial.IL10 transgenic mice, which produce moderate and transient

Figure 5.IL10R blockade adds to PD-1 blockade to increase the expansion and functions of NY-ESO-1–specific CD8þ T cells. A, representative flow-cytometry analysis fromone melanoma patient showing the percentages of CFSElo NY-ESO-1–specific CD8þ T cells among total CD8þ T cells in CFSE-based proliferation assay(n¼ 9). B, fold change of the frequencies of CFSElo (left) and total (right) NY-ESO-1–specific CD8þ T cells after in vitro stimulation with cognate peptide and aPD-1and/or aIL10R. The ratio of the percentages of CFSElo and total NY-ESO-1–specific CD8þ T cells in the presence of indicated antibody treatment and isotypecontrol antibody is shown. C, representative flow-cytometry analysis fromonemelanomapatient showing the percentages of IFNg- and TNF-producingNY0-ESO-1–specific CD8þ T cells among total CD8þ T cells (top) and the percentages of IFNgþTNFþ NY-ESO-1–specific CD8þ T cells among NY-ESO-1–specific CD8þ

T cells (bottom). PBMCs were incubated with NY-ESO-1 157–165 peptide or with HIVpol 476–484 peptide and aPD-1 and/or aIL10R or an isotype control antibody(IgG) before evaluating intracellular cytokine production of NY-ESO-1–specific CD8þ T cells upon stimulation with cognate peptide (n ¼ 8). D, fold changeof the frequencies of IFNgþ, TNFþ, and IFNgþTNFþ NY-ESO-1–specific CD8þ T cells after in vitro stimulation with cognate peptide and aPD-1 and/or aIL10R. Theratio of the frequency of cytokine-producing NY-ESO-1–specific T cells from melanoma patients in the presence of indicated antibody treatment and isotypecontrol antibody is shown. �, P < 0.05 and �� , P < 0.01. Data, representative of two independent experiments performed in duplicate. IVS, in vitro stimulation.






levels of IL10 upon TCR activation, are unable to control tumorgrowth (18, 19), supporting the immunosuppressive and protu-moral effects of IL10. Paradoxically, the administration of high-dose IL10 andpegylated IL10appears topromote tumor regressionin animals, and enhances the expansion and functions ofCD8þ TILs that express elevated levels of IL10R, resulting in tumorregression (20, 25, 26). In addition, chemically induced trans-planted tumors appear to grow better in IL10�/� mice (24). Incombinationwith cancer vaccines, and depending on the scheduleof injection, high-dose IL10 either promotes T-cell–mediatedtumor rejectionor tumorprogression, illustrating the twooppositeeffects of IL10 on T cells in vivo depending on their cell activationstate (23). In sharp contrast with the experimental models sup-porting the antitumoral activity of high-dose IL10, we measuredlow-level circulating IL10 in melanoma patients' sera. Althoughmultiple immune cells in PBMCs of patients with advancedmelanoma produce low-level IL10, we found that the highest IL10levels are produced by circulating CD11cþ and CD14þ cells inagreement with a previous study (41). In one patient with a highfrequency of NY-ESO-1–specific CD8þ T cells that upregulate PD-1, we observed that TA-specific CD8þ T cells produce IL10. Thesefindings are in line with previous reports of IL10 production byexhausted/dysfunctional LCMV-specific CD8þ T cells that upregu-late inhibitory receptors in chronic viral infections (33, 42).

In the present study, we show that IL10 decreases TA-specificCD8þ T-cell proliferation upon prolonged stimulation with cog-nate antigen and increases TA-specific CD8þ T-cell apoptosis.Furthermore, upon IL10 exposure, p-STAT3 expression in NY-

ESO-1–specific CD8þ T cells was significantly higher than in EBV-specific CD8þ T cells that do not upregulate PD-1 and IL10R,suggesting that IL10 exerts its immunosuppressive effects byacting directly on IL10Rþ TA-specific CD8þ T cells. Collectively,our data support that endogenous IL10 produced at low levels bymultiple innate and adaptive immune cells impedes the prolif-eration and survival of chronically activated TA-specific CD8þ Tcells in patients with advanced melanoma. Notably, IL10 pro-duction by circulating CD14þ cells of patients with advancedmelanoma appears to correlate with poor clinical outcome (41).

The immunosuppressive role of endogenous IL10 onT cells hasserved as a rationale for neutralizing IL10 to stimulate potentantigen–specific T-cell responses in experimental models. Neu-tralizationof IL10with anti-IL10RmAbs in combinationwith LPSrenders soluble antigen capable of stimulating potent Th1 type T-cell responses in vivo (16). IL10 blockade improves virus-specificT-cell function in mice chronically infected by LCMV virus (43),and dual IL10 and PD-1 blockade appears more effective inrestoring virus-specific T-cell function and controlling persistentviral infections (27). Monocytes isolated fromHIV patients upre-gulate PD-1 expression, produce IL10 upon PD-1 ligation in vitroto impair CD4þ T-cell activation, and dual IL10 and PD-1 block-ade restores T-cell responses to HIV in vitro (28). In cancerimmunology, IL10 blockade in combination with intratumoralinjection of CPG reverses tumor-infiltrating DC dysfunction toprime potent antitumor T-cell responses leading to tumor regres-sion in mice (17). One novel finding in this article is that IL10blockade adds to PD-1 blockade to increase the proliferation and

Figure 6.IL10R is highly upregulated by PD-1þ CD8þ TILs. A and B, dot plots from one representative patient (A) and summary data (B) showing ex vivo IL10R expressionby NY-ESO-1 tet� CD8þ T cells from PBMCs of healthy donors (n ¼ 9) and by CD8þ TILs from melanoma patients (n ¼ 9). C and D, dot plots from onerepresentative patient (C) and summarydata for all 9 patientswith advancedmelanoma (D) showing ex vivo IL10Rexpression byPD-1high, PD-1int, andPD-1low subsetsof CD8þ TILs. E, flow-cytometry analysis from one melanoma patient showing the percentages of CFSElo CD8þ TILs among total CD8þ TILs. CFSE-labeledCD8þ TILs were incubated with anti–CD3-pulsed non–T-cell fraction of one melanoma tumor single cell suspension in the presence of anti-IL10R and/or anti–PD-1or IgG control antibodies before the evaluation of the CD8þ T cells proliferation by flow cytometry; �� , P < 0.01. Horizontal bars depict the mean percentageor MFI of IL10R expression by NY-ESO-1 tet� CD8þ T cells or CD8þ TILs. Data, representative of two independent experiments performed in duplicate.


Sun et al.




functions of TA-specific CD8þ T cells isolated from PBMCs andTILs.Most interestingly, we observed that TA-specific CD8þ T cellsfurther upregulate IL10R upon PD-1 blockade. It is, therefore,tempting to speculate that upon PD-1 blockade, activated TA-specific CD8þ T cells may be rendered more sensitive to theimmunosuppressive effects of endogenous IL10. Therefore, IL10blockade may represent a potent therapeutic strategy to counter-act the direct negative regulatory effects of IL10 on T cells in theTME, which could further increase antitumor T-cell responses incombination with PD-1 blockade.

In summary, our findings demonstrate the upregulation ofIL10R by PD-1high TA-specific CD8þ T cells at periphery and attumor sites in patients with advancedmelanoma. They also showthat IL10 blockade adds to PD-1 blockade to further enhance theexpansion and functions of TA-specific CD8þ T cells. Such find-ings may have significant implications in terms of potent immu-notherapy of melanoma to further improve the clinical efficacy ofPD-1 blockade in the clinic.

Disclosure of Potential Conflicts of InterestH.M. Zarour reports receiving commercial research grants from Bristol Myers

Squibb andMerck. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: Z. Sun, J. Fourcade, J.M. Kirkwood, H.M. ZarourDevelopment of methodology: Z. Sun, O. Pagliano, H.M. ZarourAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Sun, J.-M. Chauvin, C. SanderAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Sun, J.M. Kirkwood, H.M. ZarourWriting, review, and/or revision of the manuscript: Z. Sun, J. Fourcade,J.-M. Chauvin, J.M. Kirkwood, H.M. ZarourAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Sun, J. Fourcade, O. PaglianoStudy supervision: J.M. Kirkwood, H.M. Zarour

Grant SupportThis study was supported by NIH grants R01CA112198 and R01CA157467

(H.M. Zarour) and P50CA121973 (J.M. Kirkwood).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 10, 2014; revised January 9, 2015; accepted January 29,2015; published OnlineFirst February 26, 2015.

References1. Boon T, Coulie PG, Van den Eynde BJ, van der Bruggen P. Human T-cell

responses against melanoma. Annu Rev Immunol 2006;24:175–208.2. Wherry EJ, Ahmed R. Memory CD8 T-cell differentiation during viral

infection. J Virol 2004;78:5535–45.3. Wherry EJ. T cell exhaustion. Nat Immunol 2011;12:492–9.4. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, et al.

Restoring function in exhausted CD8 T cells during chronic viral infection.Nature 2006;439:682–7.

5. Blackburn SD, Shin H, Haining WN, Zou T, Workman CJ, Polley A, et al.Coregulation of CD8þ T-cell exhaustion by multiple inhibitory receptorsduring chronic viral infection. Nat Immunol 2009;10:29–37.

6. Fourcade J, Kudela P, Sun Z, Shen H, Land SR, Lenzner D, et al. PD-1 is aregulator of NY-ESO-1-specific CD8þ T-cell expansion in melanomapatients. J Immunol 2009;182:5240–9.

7. Fourcade J, Sun Z, BenallaouaM, Guillaume P, Luescher IF, Sander C, et al.Upregulation of Tim-3 and PD-1 expression is associated with tumorantigen-specific CD8þ T-cell dysfunction inmelanoma patients. J ExpMed2010;207:2175–86.

8. Fourcade J, Sun Z, Pagliano O, Guillaume P, Luescher IF, Sander C, et al.CD8(þ) T cells specific for tumor antigens can be rendered dysfunctionalby the tumor microenvironment through upregulation of the inhibitoryreceptors BTLA and PD-1. Cancer Res 2012;72:887–96.

9. Topalian SL,Hodi FS, Brahmer JR,Gettinger SN, SmithDC,McDermottDF,et al. Safety, activity, and immune correlates of anti–PD-1 antibody incancer. N Engl J Med 2012;366:2443–54.

10. Hamid O, Robert C, Daud A, Hodi FS, HwuWJ, Kefford R, et al. Safety andtumor responses with lambrolizumab (Anti-PD-1) in melanoma. N Engl JMed 2013;369:134–44.

11. Moore KW, deWaal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 andthe interleukin-10 receptor. Annu Rev Immunol 2001;19:683–765.

12. Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. Regulation andfunctions of the IL-10 family of cytokines in inflammation and disease.Annu Rev Immunol 2011;29:71–109.

13. Sato T, McCue P, Masuoka K, Salwen S, Lattime EC, Mastrangelo MJ, et al.Interleukin 10 production by human melanoma. Clin Cancer Res1996;2:1383–90.

14. ChenQ,Daniel V,MaherDW,Hersey P. Production of IL-10 bymelanomacells: examination of its role in immunosuppression mediated by mela-noma. Int J Cancer 1994;56:755–60.

15. Dummer W, Bastian BC, Ernst N, Schanzle C, Schwaaf A, Brocker EB.Interleukin-10 production in malignant melanoma: preferential detection

of IL-10-secreting tumor cells in metastatic lesions. Int J Cancer 1996;66:607–10.

16. Castro AG,NeighborsM,Hurst SD, Zonin F, Silva RA,Murphy E, et al. Anti-interleukin 10 receptor monoclonal antibody is an adjuvant for T helpercell type 1 responses to soluble antigen only in the presence of lipopoly-saccharide. J Exp Med 2000;192:1529–34.

17. Vicari AP, Chiodoni C, Vaure C, Ait-Yahia S, Dercamp C, Matsos F, et al.Reversal of tumor-induced dendritic cell paralysis by CpG immunostimu-latory oligonucleotide and anti-interleukin 10 receptor antibody. J ExpMed 2002;196:541–9.

18. Sharma S, Stolina M, Lin Y, Gardner B, Miller PW, Kronenberg M, et al. Tcell-derived IL-10 promotes lung cancer growth by suppressing both T-celland APC function. J Immunol 1999;163:5020–8.

19. Hagenbaugh A, Sharma S, Dubinett SM, Wei SH, Aranda R, Cheroutre H,et al. Altered immune responses in interleukin 10 transgenic mice. J ExpMed 1997;185:2101–10.

20. Berman RM, Suzuki T, Tahara H, Robbins PD, Narula SK, Lotze MT.Systemic administration of cellular IL-10 induces an effective, specific,and long-lived immune response against established tumors in mice.J Immunol 1996;157:231–8.

21. Giovarelli M, Musiani P, Modesti A, Dellabona P, Casorati G, Allione A,et al. Local release of IL-10 by transfected mouse mammary adenocarci-noma cells does not suppress but enhances antitumor reaction and elicits astrong cytotoxic lymphocyte and antibody-dependent immune memory.J Immunol 1995;155:3112–23.

22. Richter G, Kruger-Krasagakes S, Hein G,Huls C, Schmitt E, Diamantstein T,et al. Interleukin 10 transfected into Chinese hamster ovary cells preventstumor growth and macrophage infiltration. Cancer Res 1993;53:4134–7.

23. Fujii S, Shimizu K, Shimizu T, Lotze MT. Interleukin-10 promotes themaintenance of antitumor CD8(þ) T-cell effector function in situ. Blood2001;98:2143–51.

24. Tanikawa T, Wilke CM, Kryczek I, Chen GY, Kao J, Nunez G, et al.Interleukin-10 ablation promotes tumor development, growth, andmetas-tasis. Cancer Res 2012;72:420–9.

25. Emmerich J,Mumm JB, Chan IH, LaFaceD, TruongH,McClanahan T, et al.IL-10directly activates and expands tumor-residentCD8(þ) T cellswithoutde novo infiltration from secondary lymphoid organs. Cancer Res2012;72:3570–81.

26. Mumm JB, Emmerich J, Zhang X, Chan I, Wu L, Mauze S, et al. IL-10 elicitsIFNgamma-dependent tumor immune surveillance. Cancer Cell 2011;20:781–96.






27. Brooks DG, Ha SJ, Elsaesser H, Sharpe AH, Freeman GJ, Oldstone MB.IL-10 and PD-L1 operate through distinct pathways to suppress T-cellactivity during persistent viral infection. Proc Natl Acad Sci U S A2008;105:20428–33.

28. Said EA, Dupuy FP, Trautmann L, Zhang Y, Shi Y, El-Far M, et al. Pro-grammed death-1-induced interleukin-10 production by monocytesimpairs CD4þ T-cell activation during HIV infection. Nat Med 2010;16:452–9.

29. Stordeur P, Poulin LF, Craciun L, Zhou L, Schandene L, de Lavareille A, et al.Cytokine mRNA quantification by real-time PCR (vol 259, pg 55, 2002).J Immunol Methods 2002;262:229.

30. Liu Y, Wei SH, Ho AS, de Waal Malefyt R, Moore KW. Expression cloningand characterization of a human IL-10 receptor. J Immunol 1994;152:1821–9.

31. Fortis C, Foppoli M, Gianotti L, Galli L, Citterio G, Consogno G, et al.Increased interleukin-10 serum levels in patients with solid tumours.Cancer Lett 1996;104:1–5.

32. Nemunaitis J, Fong T, Shabe P, Martineau D, Ando D. Comparison ofserum interleukin-10 (IL-10) levels between normal volunteers andpatients with advanced melanoma. Cancer Invest 2001;19:239–47.

33. JinHT, AndersonAC, TanWG,West EE,Ha SJ, Araki K, et al. Cooperation ofTim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection.Proc Natl Acad Sci U S A 2010;107:14733–8.

34. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, et al.PD-1 expression onHIV-specific T cells is associated with T-cell exhaustionand disease progression. Nature 2006;443:350–4.

35. Trautmann L, Janbazian L,ChomontN, Said EA,Gimmig S, Bessette B, et al.Upregulation of PD-1 expression on HIV-specific CD8þ T cells leads toreversible immune dysfunction. Nat Med 2006;12:1198–202.

36. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC.Targeting Tim-3 andPD-1 pathways to reverse T-cell exhaustion and restoreanti-tumor immunity. J Exp Med 2010;207:2187–94.

37. Groux H, Bigler M, de Vries JE, Roncarolo MG. Interleukin-10 induces along-term antigen-specific anergic state in human CD4þ T cells. J Exp Med1996;184:19–29.

38. O'Garra A, Barrat FJ,Castro AG,Vicari A,HawrylowiczC. Strategies for useofIL-10 or its antagonists in human disease. Immunol Rev 2008;223:114–31.

39. Groux H, Bigler M, de Vries JE, Roncarolo MG. Inhibitory and stimulatoryeffects of IL-10 on human CD8þ T cells. J Immunol 1998;160:3188–93.

40. Clerici M, Wynn TA, Berzofsky JA, Blatt SP, Hendrix CW, Sher A, et al. Roleof interleukin-10 in T helper cell dysfunction in asymptomatic individualsinfected with the human immunodeficiency virus. J Clin Invest 1994;93:768–75.

41. Torisu-Itakura H, Lee JH, Huynh Y, Ye X, Essner R, Morton DL. Monocyte-derived IL-10 expression predicts prognosis of stage IVmelanoma patients.J Immunother 2007;30:831–8.

42. Wherry EJ, Ha SJ, Kaech SM,HainingWN, Sarkar S, Kalia V, et al. Molecularsignature of CD8þ T-cell exhaustion during chronic viral infection. Immu-nity 2007;27:670–84.

43. Ejrnaes M, Filippi CM, Martinic MM, Ling EM, Togher LM, Crotty S, et al.Resolution of a chronic viral infection after interleukin-10 receptor block-ade. J Exp Med 2006;203:2461–72.


Sun et al.




2015;75:1635-1644. Published OnlineFirst February 26, 2015.Cancer Res Zhaojun Sun, Julien Fourcade, Ornella Pagliano, et al.

T Cells+CD8IL10 and PD-1 Cooperate to Limit the Activity of Tumor-Specific

Updated version

10.1158/0008-5472.CAN-14-3016doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2015/02/27/0008-5472.CAN-14-3016.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/75/8/1635.full#ref-list-1

This article cites 43 articles, 22 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/75/8/1635.full#related-urls

This article has been cited by 6 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/75/8/1635To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-14-3016

http://cancerres.aacrjournals.org/content/suppl/2015/02/27/0008-5472.CAN-14-3016.DC1

http://cancerres.aacrjournals.org/content/75/8/1635.full#ref-list-1

http://cancerres.aacrjournals.org/content/75/8/1635.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/75/8/1635


IL10 and PD-1 Cooperate to Limit the Activity of Tumor ... · Microenvironment and Immunology IL10...

Documents

Transcript of IL10 and PD-1 Cooperate to Limit the Activity of Tumor ... · Microenvironment and Immunology IL10...