PD-1/SHP-2 Inhibits Tc1/Th1 Phenotypic Responses and the … · dampened Tc1/Th1 skewing and...

Microenvironment and Immunology

PD-1/SHP-2 Inhibits Tc1/Th1 PhenotypicResponses and the Activation of T Cells in theTumor MicroenvironmentJing Li1, Hyun-Bae Jie2, Yu Lei2, Neil Gildener-Leapman2, Sumita Trivedi2,Tony Green3, Lawrence P. Kane4, and Robert L. Ferris2,4,5

Abstract

Immune rejection of tumors is mediated by IFNg productionand T-cell cytolytic activity. These processes are impeded by PD-1,a coinhibitory molecule expressed on T cells that is elevated intumor-infiltrating lymphocytes (TIL). PD-1 elevation may reflectT-cell exhaustion marked by decreased proliferation, productionof type I cytokines, and poor cytolytic activity. Although anti–PD-1 antibodies enhance IFNg secretion after stimulation of theT-cell receptor (TCR), the mechanistic link between PD-1 and itseffects on T-cell help (Tc1/Th1 skewing) remains unclear. Inprospectively collected cancer tissues, we found that TIL exhibiteddampened Tc1/Th1 skewing and activation compared withperipheral blood lymphocytes (PBL). When PD-1 bound its

ligand PD-L1, we observed a marked suppression of critical TCRtarget genes and Th1 cytokines. Conversely, PD-1 blockade re-versed these suppressive effects of PD-1:PD-L1 ligation. We alsofound that the TCR-regulated phosphatase SHP-2 was expressedhigher in TIL than in PBL, tightly correlating with PD-1 expressionand negative regulation of TCR target genes. Overall, these resultsdefined a PD-1/SHP-2/STAT1/T-bet signaling axis mediating thesuppressive effects of PD-1 on Th1 immunity at tumor sites. Ourfindings argue that PD-1 or SHP-2 blockade will be sufficient torestore robust Th1 immunity and T-cell activation and therebyreverse immunosuppression in the tumor microenvironment.Cancer Res; 75(3); 508–18. �2014 AACR.

IntroductionAlthough recent advances in surgery, chemotherapy, and radio-

therapy have been developed, the overall 5-year survival rate forhead and neck squamous cell carcinoma (HNSCC) remains atabout 50%. The tumor microenvironment in patients withHNSCC is highly immunosuppressive, suggesting a potential touse immunotherapies to improve survival of patients withHNSCC (1). One of the most important immune resistancemechanisms involves coinhibitory pathways mediated by im-mune checkpoint receptors (ICR), such as CTLA-4, PD-1, BTLA,and LAG-3. These ICRs and their ligands are commonly over-expressed in the tumor microenvironment (2–6), suggesting apromising approach to activate antitumor immune response of

T cells by blockade of ICRs (7). CTLA-4 antagonistic mAb(ipilimumab) has significant activity in patients with metastaticmelanoma (8) and was approved by FDA in 2010 for melanoma.Anti–PD-1mAbs have also demonstrated clinical efficacy in early-stage clinical trials for various tumor types and may providedurable antitumor responses (9). However, despite the clinicalbenefit of ICR antagonist antibodies, themechanism of improvedimmune response is still poorly understood.

Optimal T-cell–based antitumor immunity requires both Tc1-biased CD8þ T cells acting as cytolytic effector cells and CD4þ

Th1 cells, to enhance the potency and duration of antitumorresponse. In response to IFNg and IL12, STAT1 and STAT4 bindto the Tbx21 (encoding T-bet) enhancer and induce a T-bet–dependent Tc1 response (including IFNg production and cytolyticdevelopment) in CD8þ T cells (10). Development of Th1 cellsrequires a multistep mechanism in which the transcriptionfactors STAT1, T-bet, and STAT4 are sequentially activated (11,12). Sustained tumor regression that results from antitumortherapies such as cancer vaccines is dependent on a strong typeI immune response. In contrast, CD4þ Th2 cells induceM2-biasedtumor-associated macrophages and suppress CD8þ antitumorresponse, driving a more tumor-permissive microenvironment(1). Therefore, skewing to a type I–dominant tumor microenvi-ronment is indispensable to enhance efficacy of antitumorimmunotherapy.

Although blockade of the PD-1 pathway (PD-1/PD-L1) en-hances production of IFNg (a hallmark Th1 cytokine) and cyto-lytic activity of tumor-infiltrating T cells in both tumor-bearingmice and patientswith cancer (13, 14), the link between PD-1 andtype I immunity remains vague. After clustering with the T-cellreceptor (TCR) for antigen during inflammatory conditions, PD-1

1Department of Pharmacology and Pharmaceutical Sciences, Schoolof Medicine, Tsinghua University, Beijing, China. 2Department of Oto-laryngology, University of Pittsburgh, Pittsburgh, Pennsylvania. 3Uni-versity of Pittsburgh Health Sciences, Pittsburgh, Pennsylvania.4Department of Immunology, University of Pittsburgh, Pittsburgh,Pennsylvania. 5Cancer Immunology Program, University of PittsburghCancer Institute, Pittsburgh, Pennsylvania.

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

H.-B. Jie and Y. Lei contributed equally to this article.

Corresponding Author: Robert L. Ferris, University of Pittsburgh Cancer Immu-nology Program, Hillman Cancer Center Research Pavilion, 5117 Centre Avenue,Room2.26b, Pittsburgh, PA 15213-1863. Phone: 412-623-0327; Fax: 412-623-4840;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-1215

�2014 American Association for Cancer Research.

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can recruit the phosphatase SHP-2 (15, 16) and relieve SHP-2from its autoinhibited state (17). In contrast, blockade of PD-1signaling inhibits phosphorylation of SHP-2 (18). We thereforehypothesized that PD-1 suppresses beneficial type I–dominantimmune responses in the tumor microenvironment through thePD-1/SHP-2/p-STAT1/T-bet axis. Stimulation of the TCR andCD28 would lead to activation of the PI3K/Akt/mTOR/p-S6pathway, which is important for sustaining T-cell survival andexpansion (19). Thus, PD-1 might interfere with TCR/CD28signaling to mediate the suppression of T-cell survival and pro-liferation in patients with cancer. In addition, PD-1 can turn offTCR/CD28 signals by inhibiting TCR-proximal kinases in T cells.In this study, we used unique, paired, freshly isolated tumor-infiltrating lymphocytes (TIL) and peripheral blood lymphocytes(PBL) specimens to investigate the in vivo phenotypic and func-tional impact of PD-1 expression on TCR signaling and Thskewing directly in the tumor microenvironment of patients withcancer, since these TILs have appeared to bemore suppressed thanin the peripheral circulation (2).

Patients and MethodsPatients and specimens

Peripheral blood samples and fresh tumor specimens wereobtained from 41 patients with HNSCC. All patients were seenin the Department of Otolaryngology at the University of Pitts-burgh Medical Center, and all subjects signed an informed con-sent approved by the Institutional Review Board of the Universityof Pittsburgh (IRB# 99-06; Pittsburgh, PA). The clinicopatholog-ical features of the patients with HNSCC in this study are shownin Table 1. The patient cohort included 7 females and 34 maleswith a mean age of 58 years (range: 26–74 years).

Collection of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes

Venous blood from patients with HNSCC was drawn intoheparinized tubes and centrifuged on Ficoll–Hypaque gradients(GE Healthcare Life Sciences). Peripheral blood mononuclearcells (PBMC) were recovered, washed in RPMI-1640 medium(Sigma), and either used immediately for experiments or resus-pended in freezing media containing 10% DMSO, transferred toMr. Frosty containers (Thermo Scientific), and stored at �80�Cuntil flow-cytometric analysis. For TIL isolation, fresh tumorsfrom patients with HNSCC were minced into small pieces man-ually or using a gentleMACS Dissociator (Miltenyi Biotec), thentransferred to 70-mm cell strainers (BD) and mechanically sepa-rated using the plunger of a 5-mL syringe. The cells passingthrough the cell strainer were collected and subjected to Ficoll–Hypaque gradient centrifugation. After centrifugation, mononu-clear cells were recovered and stored at �80�C until flow-cyto-metric analysis or immediately used for experiments.

Antibodies and flow cytometryThe following anti-human antibodies were used for staining:

CD3-Alexa Fluor 700, FOXP3-PerCP/Cy5.5, phospho-STAT1(pY701)-PE, phospho-STAT4 (pY693)-Alexa Fluor 488, and T-bet-BV711 purchased from BD Biosciences, CD3-PE-Cy7, PD-1-PerCP/Cy5.5, and CD25-PE-Cy7 purchased from Biolegend,CD8-PE-TR and CD4-PE-TR purchased from Life Technologies,PD-1-APC (Clone: MIH-4, eBioscience), phospho-S6 (Ser235/236)-Alexa Fluor 488 (Cell Signaling Technology), SH-PTP2

(C-18; Santa Cruz Biotechnology), and APC-conjugated F(ab')2fragment goat anti-rabbit IgG (Jackson ImmunoResearch). Intra-cellular staining of FOXP3, SHP-2, p-STAT1, T-bet, p-STAT4, andp-S6 was performed as follows: PBMCs or TILs were stained withsurface marker antibodies, fixed with fixation/permeabilizationbuffer (eBioscience), washed, and stained for intracellular anti-gens in 1� permeabilization buffer. Cells were analyzed on anLSRFortessa (BD) or CyAn (Dako) flow cytometer, and dataanalyzed using FlowJo (Tree Star) or Summit V4.3 software(Dako), respectively. The acquisition and analysis gates wererestricted to the lymphocyte gate based on characteristic proper-ties of the cells in the forward and side scatter. Dead cells wereexcluded on the basis of viability dye staining (Zombie AquaFixable Viability Dye, Biolegend).

ImmunohistochemistryFormalin-fixed paraffin-embedded tissue sections were

deparaffinized and dehydrated in xylene and graded ethanolsolutions. Antigen retrieval was conducted in Tris-EDTA buffer.Immunoperoxidase stains were performed according to a stan-dard protocol on the Ventana Benchmark Ultra platform.PD-L1 antibody was provided by Dr. Gordon Freeman atthe Dana Farber Cancer Institute (Boston, MA). PD-1 wasstained using a mAb NAT105 at 1:500 titration. IFNg antibodywas purchased from Abcam and incubated at 1:500 titration.Staining was interpreted by an oral and maxillofacial patho-logist. Both the intensity and percentage of area of stainingwere evaluated. Representative pictures of matching areas weretaken at �400.

Restimulation of TIL using anti-CD3/-CD28/hIgG1 or anti-CD3/-CD28/PD-L1 beads

LEAF purified anti-human CD3 (clone UCHT1, Biolegend),LEAF purified anti-human CD28 (clone CD28.2, Biolegend) plusPD-L1-hIgG1 Fc fusion protein (R&D Systems) or control humanIgG1 (Southern Biotech) was covalently coupled to DynabeadsM-450 Epoxy beads according to the manufacturer's protocol(Life Technologies).We kept constant the total amount of proteinat 5 mg per 107 beads as previously described (20). Generally, 107

beads were coated with 1 mg of anti-CD3 (20%of total protein), 1mg of anti-CD28, and 60% of either PD-L1-hIGg1 Fc fusionprotein or control human IgG1. Covalent coupling of the proteinsto the beads was performed in 0.1 mol/L sodium phosphatebuffer for 24 hours at room temperature with gentle tilting androtation.

TILs were freshly isolated from tumor specimens and sub-jected to restimulation experiments. Total TILs were culturedwith beads at a fixed cell:bead ratio of 1:10. Briefly, 0.5 � 106

TILs were plated in a 96-well U-bottom tissue culture plate withbeads in 200 mL RPMI-1640 complete media in the presence of100 mg/mL anti–PD-1 (BMS-936558) or hIgG4 isotype controlprovided by Bristol–Myers–Squibb, or 50 mmol/L fusaruside(6) as indicated. After 48-hour incubation at 37�C with 5%CO2, supernatants were collected and cells were stained andsubjected to flow-cytometric analysis.

Western blot analysisWestern blot analysis was performed with phospho-SHP-2

(Tyr580) antibody (Cell Signaling Technology), SH-PTP2 Anti-body (C-18; Santa Cruz Biotechnology), and monoclonal anti–b-actin antibody (Sigma).

PD-1/SHP-2 Inhibits Type I Response and TCR Activation in TME

www.aacrjournals.org Cancer Res; 75(3) February 1, 2015 509




Luminex assayTIL culture supernatant levels of IFNg , TNFa, IL2, IL4, IL5, and

IL10 were tested using a human magnetic cytokine/chemokinepanel 6-plex kit (Millipore) and analyzed by the UPCI LuminexCore Facility.

Statistical analysisAverages were calculated as means. For nonparametric distri-

bution of samples, P values were calculated by Wilcoxon–Mann–Whitney tests using GraphPad Prism (GraphPad). P values of<0.05were considered tobe significant. �,P<0.05; ��,P<0.01; ��,P < 0.001.

ResultsTILs have dampened Tc1/Th1 phenotypic responses andactivation status compared with PBL

To determine the status of Tc1/Th1 activation of T cellsin the tumor microenvironment, we analyzed expression ofp-STAT1, T-bet, and p-STAT4 in T cells from paired PBLs andTILs from patients with HNSCC. Interestingly, CD8þ TIL had

significantly lower p-STAT1, T-bet, and p-STAT4 expressionat baseline (Fig. 1A and B, p-STAT1: P ¼ 0.005; T-bet: P ¼0.0003, and p-STAT4: P ¼ 0.02), lower p-STAT1 and T-betafter TCR stimulation (Fig. 1C and D, p-STAT1 and T-bet:P ¼ 0.03) compared with PBL, which also correlates withdeficient expression of perforin in CD8þ TIL (unpublisheddata). CD4þ TIL possessed similar p-STAT1 and slightly higherT-bet but dramatically lower p-STAT4 at baseline (Fig. 2Aand B, T-bet: P ¼ 0.002 and p-STAT4: P ¼ 0.02), lowerp-STAT1 and T-bet after TCR stimulation (Fig. 2C and D,p-STAT1 and T-bet: P ¼ 0.03) compared with PBL. Foxp3-CD4þ T cells also manifested a similar abortive Th1 differen-tiation program in TIL.

Next, we investigated the activation status of TIL marked byexpression of phosphorylated ribosomal protein S6 (p-S6), adownstream target of the PI3K pathway (21). As expected,expression of p-S6 was significantly lower in CD8þ and CD4þ

TIL than in PBL at baseline (Figs. 1A and B and 2A and B, CD8:P¼ 0.0001 and CD4: P ¼ 0.005) and poststimulation (Figs. 1Cand D and Fig. 2C and D, CD8, CD4: P ¼ 0.03), suggesting adampened activation status of tumor-infiltrating T cells,

Table 1. Clinicopathological features of the patients with head and neck cancer in this study

Patient Gender Age, y Tumor site T-stage, path N-stage, path M-stage, path

1 Female 62 OC T4A N0 MX2 Male 71 L T4A N0 MX3 Male 54 OC T2 N0 MX4 Female 68 OC T1 N0 MX5 Male 72 OC T4A N0 MX6 Male 50 L T4A N2C MX7 Male 57 OC T4A N3 M08 Male 63 OC T2 N0 M09 Male 61 L T3 N0 MX10 Male 59 OP T2 N2B MX11 Female 53 L T3 N2B MX12 Female 69 OC T4 N2C MX13 Male 58 OC T4A N2B MX14 Male 54 OC T2 N2C MX15 Male 49 L T4A N2B MX16 Male 68 OP T4A N1 MX17 Male 52 L T4A N1 MX18 Male 26 OC T2 N2B MX19 Male 63 OC T2 N0 M020 Female 56 L TX N0 MX21 Male 54 OC T4A N2C MX22 Male 68 OC T4A N0 MX23 Male 59 L T3 N0 M024 Male 52 OC T4A N0 MX25 Male 58 OP TX NX MX26 Male 49 L T3 N0 MX27 Female 69 OP TX N1B MX28 Male 74 OP T1 N2B MX29 Male 69 OC T4A N1 MX30 Male 64 OP T1 N0 MX31 Male 46 OP T2 N2B MX32 Male 54 OP T2 N2A MX33 Male 47 L T2 N2A MX34 Male 56 OC T3 N0 M035 Male 60 OC T2 N0 M036 Male 61 OC N/A N/A N/A37 Male 44 OP T1 N2B M038 Female 72 OC T4A N0 MX39 Male 71 OC T4A N1 MX40 Male 51 OP N/A N/A N/A41 Male 42 n/a N/A N/A N/A

Abbreviations: L, larynx; OC, oral cavity; OP, oropharynx.

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Cancer Res; 75(3) February 1, 2015 Cancer Research510




despite the presence of multiple tumor antigenic stimulus inthe tumor microenvironment from which they were freshlyisolated.

PD-1 suppresses TCR-stimulated upregulation of p-STAT1,T-bet, and p-S6

Despite the fact that PD-L1, the major ligand for PD-1, isvariably and heterogeneously expressed on HNSCC tumor cells(4 and Supplementary Fig. S1), we observed colocalization ofPD-1þ TIL with PD-L1þ tumor cells in vivo in the tumormicroenvironment (Fig. 3), indicating that the PD-1 inhibitorysignaling is relevant and functional in the tumor-infiltratingT cells. Having demonstrated impaired Tc1/Th1 responses andactivation of TIL, we investigated whether PD-1 signaling coulddirectly regulate p-STAT1 and T-bet, which are important reg-

ulators of the Th1 phenotype, in the tumor microenvironment.To explore this possibility, we generated anti-CD3/-CD28/PD-L1 or anti-CD3/-CD28/hIgG1 (control Ab)-coated beads.Total TILs isolated from patients with HNSCC were stimulatedwith these beads in the presence or absence of anti–PD-1 block-ade (BMS-936558). Interestingly, TIL that highly expressedPD-1 showed lower p-STAT1 and T-bet, when stimulated withanti-CD3/-CD28/PD-L1 beads, than when stimulated withanti-CD3/-CD28/hIgG1 beads. This result indicates that PD-1ligation with polyvalent PD-L1 suppresses upregulation ofp-STAT1 and T-bet due to TCR stimulation. In addition,anti–PD-1 blockade can restore p-STAT1 and T-bet expressionin TIL stimulated with anti-CD3/-CD28/PD-L1 beads (Fig. 4A–C, P ¼ 0.02 and Supplementary Fig. S2A and S2B), whichsuggests that inhibition of PD-1 signaling using a clinically

Figure 1.CD8þ TILs have dampened Tc1phenotypic responses and activationcomparedwith PBL. p-STAT1, T-bet, p-STAT4, and p-S6 levels in CD8þ PBLand TIL from patients with HNSCCwere analyzed by intracellular flowcytometry. Representative figures(A) and summary data (B) showpercentage of p-STAT1 (Y701)þ

(n ¼ 15), T-betþ (n ¼ 16), p-STAT4(Y693)þ (n ¼ 7), and p-S6 (S235/236)þ (n ¼ 13) cells in CD8þ TILcompared with paired PBL at baseline.Total PBL and TIL were stimulatedwith anti-CD3/-CD28/hIgG1 beads(bead: cell ¼ 10:1) for 48 hours andthen p-STAT1, T-bet, and p-S6 weretested by flow cytometry.Representative figures (C) andsummary data (D) of percentage ofp-STAT1 (Y701)þ, T-betþ, and p-S6(S235/236)þ (n ¼ 6) cells in CD8þ

TIL compared with paired PBLpoststimulation are shown. Statisticalsignificance was determined by theWilcoxon (nonparametric paired) test.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






effective blocking mAb could reverse the suppressive effectsof PD-1 on Th1 phenotypic responses. However, anti–PD-1blockade did not increase p-STAT1 or T-bet expression in TILstimulated with anti-CD3/-CD28/hIgG1 (isotype control mAb)beads.

Next, we investigated whether PD-1 signaling interfereswith signals downstream of TCR activation. Of interest,p-S6, which can be upregulated by TCR signaling, wasdecreased by the ligation of PD-1 using PD-L1–coated beads.Consequently, blockade of PD-1 by anti–PD-1 Ab (BMS-936558) restored upregulation of p-S6 (Fig. 4A and D, P ¼0.02 and Supplementary Fig. S2C). These findings suggestthat PD-1 signaling interferes with activation of downstreamT-cell activation molecules (such as p-S6) induced by TCRstimulation, promoting dysfunction of T cells in the tumormicroenvironment.

PD-1 suppresses secretion of Th1 cytokines but not Th2cytokines by TIL upon TCR stimulation

Because we observed that PD-1 could suppress p-STAT1 andT-bet, the transcription factors regulating production of Th1cytokines by CD8þ and CD4þ T cells, we next investigatedwhether production of Th1 cytokines upon TCR stimulation isinfluenced by PD-1 ligation or anti–PD-1 blockade. Super-natants of TIL cultured with anti-CD3/-CD28/hIgG1 or anti-CD3/-CD28/PD-L1 beads for 48 hours, with or without anti–PD-1 blockade (BMS-936558), were analyzed by Luminexfor Th1/Th2 cytokines secretion. As expected, secretion ofthe Th1 cytokines IFNg (P ¼ 0.008), TNFa (data not shown),and IL2 (P ¼ 0.02, P ¼ 0.04) was lower in TIL withanti-CD3/-CD28/PD-L1 stimulation, compared with thosestimulated with anti-CD3/-CD28/hIgG1, whereas PD-1 block-ade Ab could reverse the inhibitory effects of PD-1:PD-L1

Figure 2.CD4þ TILs show abortive Th1differentiation and low activationcomparedwith PBL. p-STAT1, T-bet, p-STAT4, and p-S6 levels in CD4þ PBLand TIL from patients with HNSCCwere analyzed by intracellular flowcytometry. Representative figures(A) and summary data (B) showpercentage of p-STAT1 (Y701)þ (n ¼15), T-betþ (n¼ 16), p-STAT4 (Y693)þ

(n¼ 7), and p-S6 (S235/236)þ (n¼ 13)cells in CD4þ TIL compared withpaired PBL at baseline. Total PBL andTIL were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell ¼ 10:1)for 48 hours and then p-STAT1, T-bet,and p-S6 were tested by flowcytometry. Representative figures (C)and summary data (D) of percentageof p-STAT1 (Y701)þ, T-betþ, andp-S6 (S235/236)þ (n ¼ 6) cells inCD4þ TIL compared with paired PBLpoststimulation are shown. Statisticalsignificance was determined by theWilcoxon (nonparametric paired) test.� , P < 0.05; �� , P < 0.01. P > 0.05 wasconsidered to be not significant (n.s.).

Li et al.





ligation. Production of the Th2 cytokine IL10 was not alteredby PD-1 ligation or PD-1 blockade (Fig. 4E). To validate our exvivo findings in vivo, we conducted immunohistochemistryanalysis of PD-1 and IFNg in serial sections of the originaltumor tissues. When expression of PD-1 on TIL was high, theamount of IFNg in the tumor microenvironment was low(Tumor 1), and vice versa (Tumor 2, Fig. 4F). Taken together,these findings suggest that PD-1 signaling negatively regulatesTc1/Th1 responses by suppressing activation of p-STAT1 and T-bet and secretion of Th1 cytokines.

SHP-2 is overexpressed in TIL and strongly correlates withPD-1 expression

After ligand binding, PD-1 clusters with the TCR and canrecruit SHP-2 to its immunoreceptor tyrosine-based switchmotif (ITSM), where SHP-2 is phosphorylated (22). SHP-2has been suggested to be a mediator of PD-1 inhibitory signals(23, 24). Because PD-1 is highly expressed in TIL (22, 24), weexamined whether expression of SHP-2 itself correlates withPD-1 expression on TIL from patients with HNSCC. Expres-sion of SHP-2 was tested in paired PBL and TIL from patients

with HNSCC by flow cytometry. Although SHP-2 is ubiqui-tously expressed in T cells, the MFI of SHP-2 was significant-ly higher in TIL, compared with PBL (Fig. 5A, CD8, CD4:P ¼ 0.002). In addition, the MFI of SHP-2 was even higher inPD-1þ TIL than in PD-1� TIL (Fig. 5B, CD8, CD4: P ¼ 0.002).We also observed that the levels of SHP-2 expression in tumor-infiltrating T cells from patients with HNSCC positivelycorrelated with PD-1 expression (Fig. 5C, R2 ¼ 0.8643,P ¼ 0.02). Together, these observations strongly suggest thatexpression of SHP-2 correlates with PD-1 expression, partic-ularly at the tumor sites.

SHP-2 activation by fusaruside suppresses p-STAT1/T-bet andproduction of Th1 cytokines

Because SHP-2 can function as a negative regulator ofp-STAT1 in tumor cells (15) and lymphocytes (16), we nextinvestigated whether activation of SHP-2 regulates the inhib-itory effects of PD-1 signaling on p-STAT1 and T-bet. Toinvestigate this possibility, we used fusaruside, a small-mole-cule compound that specifically induces phosphorylation ofSHP-2 (Supplementary Fig. S3; refs. 16, 25), to activate SHP-2directly in TIL, bypassing ligand engagement of PD-1. As shownin Fig. 6A, upregulation of p-STAT1 and T-bet by TCR/CD28stimulation was inhibited by fusaruside (50 mmol/L for48 hours), even when the PD-1 signaling pathway was blocked(P ¼ 0.03). These results indicate that activation of SHP-2suppresses p-STAT1 and T-bet in a fashion similar to PD-1signaling. We also tested Th1/Th2 cytokines in the supernatantsof TIL cultured in the presence or absence of fusaruside withTCR stimulation. Consistent with decreases in p-STAT1 andT-bet, activation of SHP-2 by fusaruside suppresses productionof the Th1 cytokines IFNg (P ¼ 0.02, P ¼ 0.008), TNFa (datanot shown) and IL2, but not the Th2 cytokine IL10 (Fig. 6B).Therefore, PD-1 appears to suppress Tc1/Th1 phenotypicresponses that are controlled by p-STAT1/T-bet in the tumormicroenvironment by recruiting and activating SHP-2 to skewaway from a Th1-biased antitumor response.

DiscussionIn this study, we provide a mechanistic explanation for

how PD-1 suppresses type I immunity and T-cell activationin the tumor microenvironment. First, we show that TILs thatexpress significantly more PD-1 manifest dampened Tc1/Th1phenotypic responses and activation status compared with Tcells in PBL. Second, ligation of PD-1 to PD-L1–coated beadssuppresses p-STAT1, T-bet, p-S6 and production of Th1 cyto-kines due to TCR stimulation, while an antagonist PD-1 mAb(BMS-936558) can reverse the negative effects of PD-1 signal-ing. Third, we demonstrate that SHP-2, the downstreammediator of PD-1, is increased in TIL and is tightly correlatedwith PD-1 expression. Furthermore, activation of SHP-2 byfusaruside can bypass PD-1 signaling to induce suppressionof Tc1/Th1 phenotypic responses marked by expression ofp-STAT1 and T-bet and secretion of Th1 cytokines. Takentogether, our study describes a novel function for PD-1 insuppressing type I immunity, through inhibition of p-STAT1/T-bet, via SHP-2 activation, and in antagonizing TCR/CD28signaling to decrease p-S6 expression.

Immune escape of tumors results from loss of tumor antigenexpression (due to loss of expression of strong rejection antigens

Figure 3.PD-1þ TILs colocalize with PD-L1þ HNSCC cells in the tumormicroenvironment. Hematoxylin and eosin (H&E; left) and PD-1/PD-L1double immunoperosidase stainings (right) were performed, andrepresentative matching areas in five (n ¼ 5) tumors are shown. PD-1–positive lymphocytes are labeled with a red chromogen, and PD-L1–positive HNSCC cells are labeled with a brown chromogen. Images weretaken at �200.






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D

E

Figure 4.PD-1 ligation with bead-coated PD-L1 suppresses p-STAT1, T-bet, p-S6, and production of Th1 cytokines upon TCR stimulation, while anti–PD-1 blockade couldreverse the suppressive effects of PD-1. Total TILs were stimulated with anti-CD3/-CD28/hIgG1 or anti-CD3/-CD28/PD-L1–coated beads (bead: cell ¼ 10:1) for48 hours in the presence of 100 mg/mL hIgG4 or anti–PD-1 (BMS-936558), then p-STAT1, T-bet, and p-S6 were analyzed by flow cytometry. Supernatantsfrom each condition were collected and stored at �80�C. Th1 (IFNg and IL2) and Th2 (IL10) cytokines in the supernatants were determined by Luminex. A,representative data showing p-STAT1 (Y701), T-bet, and p-S6 (S235/236) levels in CD8þ TIL under the described conditions. (Continued on the following page.)

Li et al.





or loss of MHC class I molecules; refs. 26, 27) and establish-ment of the immunosuppressive tumor microenvironment.Immunosuppression of effector T cells in the tumor microenvi-ronment is mediated by increased expression of coinhibitoryreceptors (such as PD-1 and CTLA-4) that inhibit activationof T cells, or by immunosuppressive cytokines (such as TGFband IL10) derived from both tumor cells and infiltrating Tregand MDSC (28–31). These inhibitory mechanisms are consis-tent with our observation of a dampened Th1/Tc1 phenotypicresponse in TIL (Figs. 1 and 2).

In the ongoing clinical trials of anti–PD-1/PD-L1 therapies,there is discussion of whether clinical responses to PD-1/PD-L1blockade correlate with PD-L1 expression on tumor cells orimmune cells. Recently, a report on the clinical trial of anti–PD-1 mAb (BMS-936558) therapy in patients with cancer showedthat pretreatment expression of PD-L1 on tumors was associ-ated with enhanced clinical responses (9). In our restimulationsystem, anti–PD-1 blockade did not increase expression of p-STAT1 and T-bet or production of Th1 cytokines in TIL stim-ulated with anti-CD3/-CD28 alone. This might be because

Figure 5.SHP-2 is overexpressed in TIL and correlates with PD-1þ expression. Expression level of SHP-2 in TIL and paired PBL from patients with HNSCC (n ¼ 10)was assessed by flow cytometry. A, representative figure (top) and summary data (bottom) showing MFI of SHP-2 in CD8þ and CD4þ TIL compared with PBL.B, representative figure (top) and summary data (bottom) showing MFI of SHP-2 in PD-1� and PD-1þ CD8þ and CD4þ TIL. Statistical significance wasdetermined by the Wilcoxon (nonparametric paired) test. �� , P < 0.01. C, expression levels of PD-1 and SHP-2 (shown by MFI) in tumor-infiltrating T cellsfrom 5 patients with HNSCC.

(Continued.) Summary data of frequency of p-STAT1 (Y701)þ (B), T-betþ (C), and p-S6 (S235/236)þ (D) in CD8þ and CD4þ TIL with indicated conditions areshown (n ¼ 7). E, summary data of amount of IFNg , IL2, and IL10 in the supernatants of TIL cultured under indicated conditions are shown. The graphs present themean � SEM from 8 patients with HNSCC. F, immunohistochemistry analysis of PD-1 and IFNg in serial sections of representative HNSCC tumors. Statisticalsignificance was determined by the Wilcoxon (nonparametric paired) test. � , P < 0.05; �� , P < 0.01. P > 0.05 was considered to be not significant (n.s.).






when we isolated TIL, PD-L1þ tumor cells interacting with PD-1þ TIL in the tumor microenvironment (Fig. 3) were depleted,so that PD-1 ligands were much less abundant than in thetumor sites. Therefore, we might only observe beneficial effectsof anti–PD-1 blockade on type I antitumor immunity whenPD-L1 is reintroduced into the culture to mimic the real tumormicroenvironment. Our findings also suggest an important rolefor PD-L1 expression on tumor cells in triggering PD-1 inhib-itory signaling in the interacting T cells (Fig. 4).

Type I–biased innate effector cells (such as IL12-producing DCand IFNg-producing NK/NKT cells) are crucial for inducing Th1CD4þ cells and Tc1 CD8þ cells with optimal cytotoxicity andeffector functions for tumor cell lysis. Adoptive transfer of Th1cells (32) and antigen-specific Tc1 cells (33) elicits strong anti-tumor activity. In contrast, type II–biased effector cells, which

produce IL4, IL10, and TGFb, negatively regulate type I antitumorimmunity and make the tumor microenvironment more tumorpermissive. Thus, Th1/Tc1-biased antitumor immunity is highlydesirable for rejection of tumors by the host immune system.Alteration of the Th1/Th2 balance should therefore be consideredas a strategy for cancer immunotherapy, too.

PD-1 blockade has been shown to augment Th1 and Th17responses (as evidenced by increased production of IFNg , IL2,TNFa, IL6, and IL17) and to suppress production of the Th2cytokines IL5 and IL13 in reactivated T cells fromperipheral bloodof patients with prostate and advanced melanoma cancer (34).However, in our system, PD-1 signaling inhibits production ofTh1 cytokines (IFNg , IL2, and TNFa) by TIL, without alteringproduction of the Th2 cytokine IL10 (Fig. 4E). In contrast, IL4and IL5, the other two Th2 cytokines, were below the limit of

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CD8 TIL CD4 TIL

p-STAT1

T-bet

% p

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+ ce

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Anti-CD3/CD28/lgG + DMSO

Anti-CD3/CD28/PD-L1 + anti–PD-1 + DMSOAnti-CD3/CD28/lgG + 50 mmol/L fusaruside

Anti-CD3/CD28/PD-L1 + anti–PD-1 + 50 mmol/L fusaruside

A

B

Figure 6.SHP-2 activation by fusaruside suppresses p-STAT1/T-bet and production of Th1 cytokines upon TCR stimulation. Total TILs were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell ¼ 10:1) or anti-CD3/-CD28/PD-L1 beads plus 100 mg/mL anti–PD-1 blockade (BMS-936558) for 48 hours in thepresence of 50 mmol/L fusaruside or DMSO. Then p-STAT1 and T-bet were analyzed by flow cytometry. Supernatants were collected and stored at �80�C.Th1 (IFNg and IL2) and Th2 (IL10) cytokines in the supernatants were determined by Luminex. A, summary data of frequency of p-STAT1þ and T-betþ

cells in CD8þ and CD4þ TIL at different conditions are shown (n ¼ 6). B, summary data of amount of IFNg (n ¼ 8), IL2 (n ¼ 4), and IL10 (n ¼ 8) in thesupernatants of TIL cultured under indicated conditions. The graphs present the mean � SEM from different patients with HNSCC. Statistical significancewas determined by the Wilcoxon (nonparametric paired) test. � , P < 0.05; �� , P < 0.01. P > 0.05 was considered to be not significant (n.s.).

Li et al.





detection, indicating that they might not be actively produced byTIL even with TCR stimulation. We think that the PD-1/SHP-2signaling interferes with the Th1 skewing but not directly acts onTh2 differentiation. Fully differentiated Th2 phenotypes wouldnot appear without some Th2-driving condition(s). Thus, PD-1blockade appears to enhance Th1 responses butmay not alter Th2responses in the tumor microenvironment. In addition, in CT26tumor-bearing mice, injection of anti–PD-L1 antibody induceshigher levels of T-bet in CD8þ TIL (35). What is more, phosphor-ylated SHP-2 can selectively sequester STAT1 from kinases thatmediate phosphorylation and thus suppress the STAT1-depen-dent Th1 immune responses (16). Taken together, these findingssuggest that Th1 immunity can be efficiently modulated by PD-1or SHP-2.

In conclusion, anti–PD-1 blockade, which is being activelyexplored as an immunotherapy agent in clinical trials and hasshown clinical efficacy in several solid tumors, can improve T-cell–based immunotherapy by restoring a robust type I antitumorimmunity and enhancing T-cell activation. Biomarkers of anti–PD-1 activity are needed to monitor the efficacy of this type ofimmunotherapy, which we suggest should include successfulrestoration of Th1 phenotypes. In addition, SHP-2 inhibitorystrategies might be a powerful tool for cancer immunotherapy.Thus, SHP-2 not only suppresses Tc1/Th1 skewing of tumor-infiltrating T cells, but also inhibits pSTAT1-dependent expressionofHLA/APM(elements of the antigenprocessingmachinery), andsecretion of T-cell attracting chemokines RANTES and IP10 (15)and the cytokine IL12 (unpublished data) by head and neckcancer cells. Therefore, development of a specific SHP-2 inhibitorwould be a promising strategy for cancer immunotherapy in thefuture.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Li, H.-B. Jie, R.L. FerrisDevelopment of methodology: J. Li, H.-B. Jie, R.L. FerrisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Li, H.-B. Jie, N. Gildener-Leapman, S. Trivedi, Y. LeiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Li, Y. Lei, L.P. KaneWriting, review, and/or revision of the manuscript: J. Li, S. Trivedi, L.P. Kane,R.L. FerrisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Li, T. Green, R.L. FerrisStudy supervision: R.L. Ferris

AcknowledgmentsThe authors thank Dr. Gordon Freeman (Dana-Farber Cancer Institute) for

PD-L1 Ab, Dr. Renxiang Tan (Nanjing University, China) for generating andproviding fusaruside (25), and Dr. Yang Sun, Qiang Xu (Nanjing University,China), and Gang Liu (Tsinghua University, China) for generous assistance.

Grant SupportThis work was supported by NIH grants R01 DE019727 and P50CA097190.

This project used the UPCI Cancer Biomarkers Facility: Luminex Core Labora-tory and Flow Cytometry Facility that are supported in part by award P30CA047904. J. Li was supported by the China Scholarship Council. Y. Lei wassupported by T32 CA060397 (R.L. Ferris) and NIH K99 DE024173.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 23, 2014; revised September 17, 2014; accepted October 31,2014; published OnlineFirst December 5, 2014.

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Li et al.




2015;75:508-518. Published OnlineFirst December 5, 2014.Cancer Res Jing Li, Hyun-Bae Jie, Yu Lei, et al. Activation of T Cells in the Tumor MicroenvironmentPD-1/SHP-2 Inhibits Tc1/Th1 Phenotypic Responses and the

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