Immune Suppression by PD-L2 against Spontaneous and ... · (Biosera). CT26-NY-ESO-1isacelllinederived fromCT26stably transfected with NY-ESO-1 (21). CT26, Renca, B16-F10, and A20

Translational Cancer Mechanisms and Therapy

Immune Suppression by PD-L2 againstSpontaneous and Treatment-Related AntitumorImmunityTokiyoshi Tanegashima1,2, Yosuke Togashi1, Koichi Azuma3, Akihiko Kawahara3,Ko Ideguchi4, Daisuke Sugiyama4, Fumio Kinoshita2,5, Jun Akiba6, Eiji Kashiwagi2,Ario Takeuchi2,Takuma Irie1, Katsunori Tatsugami2,TomoakiHoshino3,Masatoshi Eto2, andHiroyoshi Nishikawa1,4

Abstract

Purpose: To evaluate the detailed immunosuppressiverole(s) of PD-L2 given that its detailed role(s) remains unclearin PD-1 signal blockade therapy in animal models andhumans.

Experimental Design: We generated mouse cell lines har-boring various status of PD-L1/PD-L2 and evaluated the tumorgrowth and phenotypes of tumor-infiltrated lymphocytesusing several PD-1 signal blockades in animal models. Inhumans, the correlation between immune-related gene expres-sion and CD274 (encoding PD-L1) or PDCD1LG2 (encodingPD-L2) was investigated using The Cancer Genome Atlas(TCGA) datasets. In addition, PD-L1 or PD-L2 expression intumor cells and CD8þ T-cell infiltration were assessed by IHC.

Results: In animal models, we showed that PD-L2expression alone or simultaneously expressed with

PD-L1 in tumor cells significantly suppressed antitumorimmune responses, such as tumor antigen–specificCD8þ T cells, and was involved in the resistance totreatment with anti-PD-L1 mAb alone. This resistancewas overcome by anti-PD-1 mAb or combined treat-ment with anti-PD-L2 mAb. In clinical settings, antitu-mor immune responses were significantly correlatedwith PD-L2 expression in the tumor microenvironmentin renal cell carcinoma (RCC) and lung squamous cellcarcinoma (LUSC).

Conclusions:We propose that PD-L2 as well as PD-L1 playimportant roles in evading antitumor immunity, suggestingthat PD-1/PD-L2 blockade must be considered for optimalimmunotherapy in PD-L2–expressing cancers, such as RCCand LUSC.

IntroductionImmune checkpoint blockade (ICB), including PD-1 blockade,

has been approved to treat various cancers, such as malignantmelanoma, non–small cell lung cancer (NSCLC), and renal cellcarcinoma (RCC), leading to a paradigm shift in cancer thera-py (1–4). However, as the clinical efficacy is limited, moreeffective therapies and predictive biomarkers stratifying respon-

ders from nonresponders are urgently needed. Tumors employthe PD-1 pathway to evade antitumor immunity, particularlyCD8þ T cells against tumor antigens (5, 6). PD-1 is mainlyexpressed by activated T cells and binds to its ligands, PD-L1 andPD-L2 (5, 6), resulting in immunosuppression. PD-L1 isexpressed by both antigen-presenting cells (APC) and tumorcells (7, 8). PD-1 signal blockade unleashes antitumor T-cellresponses by augmenting signals from the T-cell receptor andCD28 (9–11), an essential costimulatory molecule. Consistentwith the importance of the PD-1/PD-L1 interaction, several stud-ies revealed favorable clinical courses using treatment with anti-PD-1/PD-L1 mAbs in patients harboring PD-L1–expressingtumors (12, 13). However, some patients harboring PD-L1–expressing tumors do not respond to PD-1 signal blockadetreatment. In addition, patients with PD-L1–negative tumorsoccasionally experience clinical efficacy (4, 12–14). In large phaseIII trials, the clinical benefit from anti-PD-1 mAbs appears to beindependent of PD-L1 expression in some cancers, such as RCCand lung squamous cell carcinoma (LUSC; refs. 4, 14–16).

The expression of another PD-1 ligand, PD-L2, was initiallythought to be restricted in APCs (6). Recently, several studiesshowed that PD-L2 is expressed by both various immune cells andtumor cells, depending on microenvironmental stimuli (17–19).PD-L2 expression in tumor cells is a predictive factor for theclinical efficacy of anti-PD-1 mAb in some studies (20). In thisstudy, we evaluated the detailed immunosuppressive role(s) ofPD-L2 in animal models and humans. The Cancer Genome Atlas

1Division of Cancer Immunology, Research Institute/Exploratory OncologyResearch and Clinical Trial Center (EPOC), National Cancer Center, Tokyo/Kashiwa, Japan. 2Department of Urology, Graduate School of Medical Sciences,Kyushu University, Fukuoka, Japan. 3Department of Respiratory Medicine,Kurume University Hospital, Kurume, Japan. 4Department of Immunology,Nagoya University Graduate School of Medicine, Nagoya, Japan. 5Departmentof Anatomic Pathology, Graduate School of Medical Sciences, Kyushu Univer-sity, Fukuoka, Japan. 6Department of Diagnostic Pathology, Kurume UniversityHospital, Kurume, Japan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Hiroyoshi Nishikawa, National Cancer Center, 6-5-1Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. Phone: 81-4-7133-1111, ext. 91385or 2157; Fax: 81-4-7130-0022; E-mail: [email protected]; and Yosuke Togashi,[email protected]

Clin Cancer Res 2019;25:4808–19

doi: 10.1158/1078-0432.CCR-18-3991

�2019 American Association for Cancer Research.

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(TCGA) datasets revealed a strong correlation between decreasedantitumor immune responses and PD-L2 rather than PD-L1 in thetumor microenvironment (TME) in several cancers, includingRCC and LUSC, which was confirmed by our clinical samples.Therefore, we propose that PD-L2 should be considered as crucialimmunosuppressive mechanisms in clinical settings in additionto PD-L1.

Materials and MethodsPatients

Twenty-nine patients with clear-cell and non–clear-cell RCCwho underwent surgical resection at Kyushu University Hospital(Fukuoka, Japan) from September 2017 to May 2018 wereenrolled in this study. Twenty-seven patients with LUSC whounderwent surgical resection at Kurume University Hospital fromJanuary 2008 to December 2012 were enrolled in this study. Thepatients' clinical information was obtained from their medicalrecords. All patients provided written informed consent beforeundergoing the study procedures. The clinical protocol for thisstudy was approved by the appropriate institutional reviewboards and ethics committees at Kyushu University Hospital andKurume University Hospital (Kurume, Japan). This study wasconducted in accordance with the Declaration of Helsinki.

IHCSections were immunostained using the Ventana Benchmark

XTAutomated Staining System (VentanaMedical System) accord-ing to the manufacturer's protocol. Tumor PD-L1 and PD-L2membrane expression was assessed;�1%was defined as positiveand <1% as negative (4, 12, 14). The intertumoral CD8þ T cellswere counted. Specifically, five areas [0.5 � 0.5 mm (0.25 mm2)fields] with the most abundant distribution were selected andcounted in each case.

Cell lines and reagentsB16-F10 (mouse melanoma), MC-38 (mouse colon carcino-

ma), CT26 (mouse colon carcinoma) expressing NY-ESO-1

(CT26-NY-ESO-1), Renca (mouse kidney adenocarcinoma),CMS5a (mouse fibrosarcoma), and A20 (mouse reticulum cellsarcoma) cell lines were maintained in RPMI medium (FUJIFILMWako Pure Chemical Corporation) supplemented with 10% FCS(Biosera). CT26-NY-ESO-1 is a cell line derived from CT26 stablytransfected with NY-ESO-1 (21). CT26, Renca, B16-F10, and A20cell lines were obtained from the ATCC. MC-38 cell line wasobtained from Kerafast. CMS5a cell line was provided by the lateDr. Lloyd J. Old, Memorial Sloan Kettering Cancer Center (NewYork, NY; ref. 22). All cell lines were used after confirmingMycoplasma (�) by Mycoplasma testing with a PCR MycoplasmaDetection Kit (Takara Bio) according to the manufacturer'sinstructions. Murine IFNg , IL2, IL4, TNFa, and granulocyte mac-rophage colony stimulating factor (GM-CSF) were obtained fromPeproTech. Murine IFNa, IFNb, and tumor growth factor-b1wereobtained from R&D Systems, Inc. Rat anti-mouse PD-1 (RMP1-14) mAb, anti-PD-L1 (10F.9G2) mAb, anti-PD-L2 (TY25) mAbs,and control rat IgG2a (RTK2758) mAb used in the in vivo studywere obtained fromBioLegend. Anti-CD4 (GK1.5)mAb and anti-CD8b (Lyt 3.2) mAb were obtained from BioXCell.

Constructs, viral production, and transfectionMouse PDCD1LG2 cDNAwas subcloned into pMXs-IRES-GFP,

which was transfected into the packaged cell line using Lipofec-tamine 3000 Reagent (Thermo Fisher Scientific). After 48 hours ofretroviral production, the supernatant was concentrated anddissolved in water. Subsequently, the viral lysate was transfectedwith MC-38 and CT26-NY-ESO-1, and the PD-L2–expressing celllines were named MC-38-L2 and CT26-NY-ESO-1-L2, respective-ly. GFP-expressing cell lines were also created for the controls andnamed MC-38-GFP and CT26-NY-ESO-1-GFP. To generate thePD-L1–knockout (KO) MC-38 cell line, we used a CRISPR/Cas9system. Briefly, guide RNA (gRNA)-targeting mouse CD274 (50-GCTTGCGTTAGTGGTGTACT-30) was made using the GeneArtPrecision gRNA Synthesis Kit (Thermo Fisher Scientific). The Cas9protein (Thermo Fisher Scientific) and gRNA were electroporatedinto MC-38 cells using the Neon Transfection System (ThermoFisher Scientific) per the manufacturer's instructions. The knock-out cell line was named MC-38-L1KO. Either GFP or PD-L2 wasintroduced into the MC-38-L1KO in the same manner, and thesecell lines were named MC-38-L1KO-GFP and MC-38-L1KO-L2,respectively.

In vivo mouse studiesFemale C57BL/6, BALB/c and BALB/c nude mice (6–8 week)

were purchased from CLEA Japan and used at 7–9 weeks of age.Tumor cells (1 � 106) were injected subcutaneously, and tumorsize was monitored twice weekly. The mean of the long and shortdiameters was applied for the tumor growth curves. Mice weregrouped when tumor volume reached approximately 100 mm3,and anti-PD-1/PD-L1/PD-L2 mAbs were administered intraperi-toneally three times every 3 days thereafter. For CD4þ T-cell orCD8þ T-cell depletion, anti-CD4 mAb (100 mg/mouse) or anti-CD8b mAb (100 mg/mouse) was administered intraperitoneallyondays�1, 0, and every 7days after tumor injection. Tumorswereharvested 14 days after tumor injection, and tumor-infiltratinglymphocytes (TIL) were analyzed with flow cytometry. All mouseexperiments were approved by the Animals Committee for Ani-mal Experimentation of the National Cancer Center Japan. Allexperimentsmet theU.S. PublicHealth Service Policy onHumaneCare and Use of Laboratory Animals.

Translational Relevance

Although PD-L1, a ligand of PD-1, has mainly been studiedas a therapeutic target and predictive biomarker in PD-1blockade therapy, the detailed role(s) of PD-L2, anotherPD-1 ligand, remains unclear. Using preclinical animal mod-els, we showed that PD-L2 expression alone or simultaneouslyexpressed with PD-L1 by tumor cells significantly suppressedantitumor immune responses, such as tumor antigen–specificCD8þ T cells, and was involved in the resistance to treatmentwith anti-PD-L1 mAb. This resistance was overcome by anti-PD-1 mAb or combined treatment with anti-PD-L2 mAb. Inclinical settings, antitumor immune responses were signifi-cantly correlated with PD-L2 expression in the tumor micro-environment in renal cell carcinoma (RCC) and lung squa-mous cell carcinoma (LUSC). We propose that PD-L2 playsan important role in evading antitumor immunity as well asPD-L1, suggesting that PD-1/PD-L2 blockade must be consid-ered for optimal immunotherapy in PD-L2–expressing can-cers, such as RCC and LUSC.

PD-L2 Is Important for Evading Antitumor Immunity

www.aacrjournals.org Clin Cancer Res; 25(15) August 1, 2019 4809




Tanegashima et al.

Clin Cancer Res; 25(15) August 1, 2019 Clinical Cancer Research4810




Flow cytometryCells were washed using PBS with 2% FBS and stained with

mAbs specific for CD4, CD8, CD44, CD62L, PD-1, PD-L1, andPD-L2 aswell asfixable viability dye (ThermoFisher Scientific). Toexamine antigen-specific CD8þ T cells, T-Select H-2Kb MuLVp15E Tetramer-KSPWFTTL-APC (MBL) was used per the manu-facturer's instructions. After tetramer staining, cells were stainedfor cell surface markers (mAbs specific for CD4, CD8, and fixableviability dye). When intracellular cytokines were analyzed, cellswere stimulated for 5 hours with phorbol 12-myristate 13-acetate(PMA; 100 ng/mL)/ionomycin (2 mg/mL; Sigma Aldrich). Golgi-Plug Reagent (1.3 mL/mL; BDBiosciences) was added for the last 4hours of the culture. These cells were stained for surface markersand then stained for intracellular cytokines (mAbs specific forTNFa and IFNg). After washing, the cells were analyzed with anLSR Fortessa Instrument (BD Biosciences) and FlowJo Software(Tree Star).

AntibodiesViolet 500–conjugated anti-CD8 (53-6.7) mAb, Brilliant

Violet 786 (BV786)–conjugated anti-CD4 (RM4-5) mAb, phyco-erythrin (PE)-andCy7-conjugated anti-CD44 (IM7)mAb, BV421-conjugated anti-PD-L2 (TY25) mAb, and BV605-conjugatedanti-CD11c (HL3) were purchased from BD Biosciences.BV421-conjugated anti-PD-1 (29F.1A12) mAb, peridinin chloro-phyll protein complex, Cy5.5-conjugated anti-CD62L (MEL-14)mAb, BV421-conjugated anti-TNF-a (MP6-XT22) mAb, PE-conjugated anti-PD-L1 (10F.9G2) mAb, and AF488-conjugatedanti-MHC class II (M5/114.15.2) were purchased from Bio-Legend. FITC-conjugated anti-IFNg (XMG1.2) mAb wereobtained from eBioscience. PE-conjugated anti-CD11b (M1/70) mAb were purchased from Thermo Fisher Scientific. Theantibodies used for IHC were PD-L1 (E1L3N, Cell SignalingTechnology), PD-L2 (#176611, R&D Systems, Inc.), and CD8(4B11, Leica Biosystems).

Real-time qRT-PCRRNA was extracted using RNeasy Mini Kit (Qiagen). cDNA was

generated using SuperScript VILO (Thermo Fisher Scientific), andreal-time PCR reactions were performed with SYBR GreenReagents (Thermo Fisher Scientific) using the QuantStudio 7 FlexReal-Time PCR System (Thermo Fisher Scientific). Gene expres-sion changes relative to 18S ribosomal RNA as a housekeepinggene were calculated using the DDCT method. A forward primer(50-AGCCTGCTGTCACTTGCTAC-30) and reverse primer (50-TCCCAGTACACCACTAACGC-30) were used to detect CD274.A forward primer (50-CCTCAGCCTAGCAGAAACTTCA-30) andreverse primer (50-CATCCGACTCAGAGGGTCAATG-30) were

used to detect PDCD1LG2. A forward primer (50-TAGAGTGTT-CAAAGCAGGCCC-30) and reverse primer (50-CCAACAAAATA-GAACCGCGGT-30) were used to detect 18S.

Statistical analysisGraphPad Prism7 (GraphPad Software) was used for statis-

tical analysis. The overall survival was analyzed with Kaplan–Meier method and compared with log-rank test. The relation-ships between the groups were compared using Fisher exacttest or Welch t test. P < 0.05 was considered statisticallysignificant.

ResultsPD-L2 plays a limited role in immune suppression in murinetumor models

To elucidate the role of PD-L2 in tumor immunity, we firstexplored PD-L1 and PD-L2 expression in several murine tumorcell lines, including MC-38, CT26, B16-F10, Renca, CMS5a, andA20. All tumor cell lines possessed variable PD-L1 expression thatwas enhanced by stimulants, especially IFNa,b, and g . In contrast,PD-L2 was minimally detected by any cell lines even after stim-ulation (Fig. 1A; Supplementary Figs. S1 and S2). While PD-L2 isreportedly expressed by APCs (6), low expression in APCs(CD45þMHCIIþCD11cþCD11bþ cells) was observed in animalmodels (ref. 23; Supplementary Fig. S3). In contrast to PD-L2,APCs had considerable, although slightly lower than tumor cells,PD-L1 expression (Supplementary Fig. S3). We next examinedin vivo antitumor effects of anti-PD-1/PD-L1/PD-L2 mAbs usingMC-38 andCT26-NY-ESO-1 cells. Anti-PD-1mAbandanti-PD-L1mAb similarly inhibited MC-38 and CT26-NY-ESO-1 tumorgrowth, whereas anti-PD-L2 mAb was ineffective against bothtumors (Fig. 1B). As neoantigen-specific T cells are reportedlyessential for antitumor effects by ICB (24), MuLV p15E (a neoan-tigen of MC-38–specific CD8þ T cells) detected by MHC/peptidetetramers was examined in an MC-38 model. MuLV p15E tetra-merþCD8þ T cells were significantly primed/augmented by treat-ment with anti-PD-1 mAb or anti-PD-L1 mAb but not with anti-PD-L2 mAb alone (Fig. 1C). Activated CD8þ T cells, which wereassessed by the proportion of CD44þCD62L� effector/memoryCD8þ T cells and the frequency of PD-1þCD8þ T cells, weresignificantly higher in TILs in mice treated with anti-PD-1 mAbor anti-PD-L1 mAb compared with mice treated with isotypecontrol or anti-PD-L2 mAb (Fig. 1D; Supplementary Fig. S4). Inaddition, TNFaþIFNgþCD8þ T cells were significantly higher inmice treated with anti-PD-1 mAb or anti-PD-L1 mAb than inthose treated with isotype control or anti-PD-L2 mAb (Fig. 1D;Supplementary Fig. S4). Therefore, PD-L1 plays a dominant

Figure 1.Anti-PD-1 mAb and anti-PD-L1 mAb are able to effectively treat MC-38 and CT26-NY-ESO-1 tumors, whereas anti-PD-L2 mAb is not. A, PD-L1 and PD-L2expression in MC-38 and CT26-NY-ESO-1 treated with or without IFNg . Cell lines were treated with or without IFNg (1,000 IU/mL) for 24 hours and weresubsequently subjected to flow cytometry. Gray, isotype control; blue, untreated; red, IFNg treated. B, In vivo efficacies of various ICBs including combinationsagainst MC38 and CT26-NY-ESO-1 tumors. MC-38 cells (1.0� 106) were injected subcutaneously on day 0, and ICB treatment as indicated was started on days 3,6, and 9 (top). Tumor growthwas monitored twice weekly (n¼ 8 per group). CT26-NY-ESO-1 cells (1.0� 106) were injected subcutaneously on day 0, and ICB asindicated was administered on days 7, 10, and 13 (bottom). Tumor growthwas monitored twice a week (n¼ 5 per group). C, Tumor antigen (MuLV p15E)-specificCD8þ T cells in TILs (top). TILs were prepared fromMC38 tumors on day 14, and tumor antigen–specific CD8þ T cells were detected by MuLV p15E/H-2Kb

tetramers. b-galactosidase/H-2Kb tetramer staining served as the control. Summary of the MuLV p15E/H-2Kb tetramerþCD8þ T-cell frequencies (n¼ 6 pergroup; bottom).D, Effector CD8þ T cells detected by CD44þCD62L�CD8þ T cells, PD-1þCD8þ T cells, and cytokine-producing CD8þ T cells in TILs. TILs wereprepared fromMC38 tumors as in C, and CD44þCD62L�CD8þ TILs, PD-1þCD8þ TILs, and TNFaþIFNgþCD8þ TILs were examined (n¼ 6 per group). N.S., notsignificant; � , P < 0.05; �� , P < 0.01; �� , P < 0.001. In vivo experiments were performed at least twice. Representative data are shown from two to threeindependent experiments.






Tanegashima et al.





immunosuppressive role comparedwith PD-L2 in animalmodelsthat are often used in tumor immunology studies.

PD-L2 expression is a mechanism for evading antitumorimmunity

Because PD-L2 was minimally expressed by the murine tumorcell lines that are frequently used in preclinical studies for PD-1signal blockade although some human tumor cells highly expressPD-L2 (20, 25), we hypothesized that the lesser appreciation ofPD-L2 in PD-1 signal blockade treatment may solely reflect thelack of appropriate animalmodels usingPD-L2–expressing tumorcell lines. ThePD-L2–overexpressing cell linesMC-38 (MC-38-L2)and CT26-NY-ESO-1 (CT26-NY-ESO-1-L2) were thus developedto evaluate influence of PD-L2 on PD-1 signal blockade efficaciesusing anti-PD-1mAb, anti-PD-L1mAb, and anti-PD-L2mAb. PD-L2 expression was confirmed at both mRNA and protein levelsusing real-time qRT-PCR (Fig. 2A) and flow cytometry (Fig. 2B),respectively. The level of PD-L2 expression was equivalent to thatof endogenous PD-L1 expression after IFNg treatment (Fig. 2Aand B). PD-L2–expressing tumors grew rapidly compared withthose in control mice injected with parental tumors or GFP-expressing tumors (Fig. 2C) although these tumors exhibitedcomparable tumor growth in immunocompromised mice (Sup-plementary Fig. S5A). Furthermore, to elucidate the role of CD4þ

T cells and CD8þ T cells, the tumor growth of MC-38/-GFP/-L2and CT-26-NY-ESO-1/-GFP/-L2 was evaluated in CD4þ T-cell– orCD8þ T-cell–depleted mice. In CD8þ T-cell–depleted mice, therapid tumor growth in PD-L2–expressing tumors was totallyabrogated although PD-L2–expressing tumors grew rapidly com-pared with the parental tumors or GFP-expressing tumors inCD4þ T-cell–depleted mice (Supplementary Fig. S5B and 5C),indicating the importance of CD8þ T-cell suppression by PD-L2.Accordingly, in the MC-38-L2 tumors, MuLV p15E tetramerþ

CD8þ T cells were reduced compared with those of the controlmice (Fig. 2D). In addition, activated CD8þ T cells(CD44þCD62L� effector/memory CD8þ T cells and PD-1þCD8þ

T cells) and TNFaþIFNgþCD8þ T cells in TILs were significantlylower in MC-38-L2 tumors than in the control parental tumorsor GFP-expressing tumors (Fig. 2E; Supplementary Fig. S6).Taken together, PD-L2 expressed by tumor cells functions asan immunosuppressive molecule against antitumor immunity,particularly CD8þ T cells.

PD-L2 blockade is necessary for controlling PD-L2–expressingtumors

We next addressed how PD-L2 influenced the antitumor effectsof anti-PD-1 mAb, anti-PD-L1 mAb, and anti-PD-L2 mAb. Anti-PD-1 mAb significantly inhibited PD-L2–expressing tumorgrowth compared with anti-PD-L1 mAb or anti-PD-L2 mAbalone. The combination of anti-PD-L1 mAb and anti-PD-L2 mAbshowed a comparable tumor growth inhibition in PD-L2–

expressing tumor growth as observed by anti-PD-1 mAb(Fig. 3A). In addition, anti-PD-1 mAb did not show any addi-tional antitumor effects against PD-L2–expressing tumors whencombined with anti-PD-L2 mAb, indicating that anti-PD-1 mAbalone can inhibit PD-1/PD-L2 interaction sufficiently to reinvig-orate an effective antitumor immunity. (Supplementary Fig. S7).The tumor growth inhibition by these antibodies including anti-PD-L2mAbwas totally abrogated in immunocompromised miceor CD8þ T-cell–depletedmice although they exhibited antitumoreffects against PD-L2–expressing tumors in CD4þ T-cell–depletedmice as well as in control mice (Supplementary Fig. S8A), indi-cating that the antitumor efficacy against PD-L2–expressingtumors was attributed for unleashing CD8þ T cells. In addition,anti-PD-L2 mAb did not exhibit antitumor immunity againstparental tumors or GFP-expressing tumors in immunocompro-mised mice (Supplementary Fig. S8B). Thus, the antitumorefficacy of anti-PD-L2 mAb against PD-L2–expressing tumorswas dependent on the reactivation of CD8þ T cells, but notcaused by a direct effect of PD-L2 blockade against PD-L2–expressing tumors. Accordingly, MuLV p15E tetramerþCD8þ

T cells were significantly primed/augmented by anti-PD-1 mAbor the combination of anti-PD-L1 mAb and anti-PD-L2 mAbcompared with anti-PD-L1 mAb or anti-PD-L2 mAb alone inMC-38-L2 tumor–bearing mice (Fig. 3B). Activated CD8þ T cells(CD44þCD62L� effector/memory CD8þ T cells and PD-1þCD8þ

T cells) and TNFaþIFNgþCD8þ T cells in TILswere also significantlyhigher in mice treated with the anti-PD-1 mAb or the combinationof anti-PD-L1 mAb and anti-PD-L2 mAb compared with untreatedmice or mice treated either by anti-PD-L1 mAb or by anti-PD-L2mAb alone (Supplementary Fig. S9). Therefore, PD-L2 endowsresistance to anti-PD-L1mAbmonotherapy,which canbeovercomeby combined treatment with anti-PD-L1mAb and anti-PD-L2mAb.

To further confirm the role of PD-L2, a PD-L1–knockoutMC-38cell line (MC-38-L1KO) was generated using a CRISPR/Cas9system, and the lack of PD-L1 protein expression was confirmedwith flow cytometry (Supplementary Fig. S10). Either PD-L2 orGFP was then expressed in MC-38-L1KO cells (MC-38-L1KO-L2and MC-38-L1KO-GFP, respectively; Supplementary Fig. S10).MC-38-L1KO tumors grew more slowly than the parental MC-38tumors, whereas PD-L2 expression allowed MC-38-L1KO-L2tumors to grow comparably with the parental MC-38 tumors(Fig. 3C). In contrast, these tumors grew similarly in immuno-compromised mice (Supplementary Fig. S10). Anti-PD-1 mAband anti-PD-L2 mAb significantly inhibited tumor growth, butanti-PD-L1mAb did not (Fig. 3D). Thus, PD-L2 alone sufficientlyhampers antitumor immunity, allowing rapid tumor growth.

PD-L2 expression is associated with antitumor immuneresponses in various cancers

Because PD-L2 significantly suppressed antitumor immunity inanimalmodels, we investigated the correlation between immune-

Figure 2.PD-L2 expression in tumor cells attenuated antitumor immune responses in the TME. PD-L1 and PD-L2 expression in tumor cell lines with real-time qRT-PCR (A)and flow cytometry (B). Cell lines were treated with or without IFNg (1,000 IU/mL) for 24 hours and were subsequently subjected to real-time qRT-PCR and flowcytometry. Gray, isotype control; blue, untreated; red, IFNg treated. C, In vivo tumor growth of MC-38-L2 tumors (left) and CT26-NY-ESO-1-L2 (right). Tumor cells(1.0� 106) were injected subcutaneously on day 0, and tumor growth was monitored twice aweek (n¼ 6 per group).D, Tumor antigen (MuLV p15E)-specificCD8þ T cells in TILs (left). TILs were prepared fromMC-38, MC-38-GFP, or MC-38-L2 tumors on day 14, and tumor antigen–specific CD8þ T cells were detected byMuLV p15E/H-2Kb tetramers. Summary of the MuLV p15E/H-2Kb tetramerþCD8þ T-cell frequencies (n¼ 6 per group; right). E, Effector CD8þ T cells detected byCD44þCD62L�CD8þ T cells, PD-1þCD8þ T cells, and cytokine-producing CD8þ T cells in TILs. TILs were prepared from each tumor as in C, andCD44þCD62L�CD8þ TILs, PD-1þCD8þ TILs, and TNFaþIFNgþCD8þ TILs were examined (n¼ 6 per group). N.S., not significant; � , P < 0.05; �� , P < 0.01;�� , P < 0.001. In vivo experiments were performed at least twice. Representative data are shown from two to three independent experiments.






related gene expression and CD274 (encoding PD-L1) orPDCD1LG2 (encoding PD-L2) in humans using TCGA datasets.The expression levels of 43 geneswere defined for the immune celltypes, CD8þ T cells, costimulatory APCs, costimulatory T cells,coinhibitory APCs, coinhibitory T cells, and cytolytic activity(ref. 26; Supplementary Table S1). We analyzed bladder cancer,gastric cancer, head and neck squamous cell carcinoma (HNSC),lung adenocarcinoma, LUSC, and RCC datasets because theefficacy of anti-PD-1 mAb or anti-PD-L1 mAb has been demon-strated in these cancers, and the published datasets were avail-able (4, 12, 14–16, 27–30). The correlation coefficient betweenthe expression level of each gene and that ofCD274orPDCD1LG2was calculated (Fig. 4A; Supplementary Fig. S11). Especially inRCC, PDCD1LG2 expression rather than CD274 expression wassignificantly positively correlated with immune-related geneexpression (Fig. 4A).We further evaluated the correlation betweenCD274, PDCD1LG2, or PDCD1 expression and the INTERFER-ON_GAMMA_RESPONSE gene signature (Fig. 4B; ref. 31). Theheatmap highlights the quantitative differences in associationbetween PD-1–related molecules and immune responses in theTME across tumor types. In RCC and LUSC, PDCD1LG2 expres-sion was strongly correlated with IFNg-related gene expression,although CD274 expression was less significantly correlated.These data suggest that PD-L2 expression in RCC and LUSCstrongly controls antitumor immune responses in the TME.

We next examined the relationship between CD274 orPDCD1LG2 expression and survival from RCC and LUSC TCGAdatasets. In RCC, no correlation between CD274 or PDCD1LG2expressions and survival was observed. On the other hand, highexpression of CD274 or PDCD1LG2 equally correlated with abetter survival in LUSC, indicating the possible correlationbetween antitumor immune responses and a favorable prognosis(Supplementary Fig. S12).

CD8þ T-cell infiltration is accompanied by PD-L2 expression inRCC and LUSC

Because the correlation between immune-related genes andCD274 expression was relatively weak in RCC and LUSC, theexpression of PD-L1 or PD-L2 in tumor cells and the number ofinfiltrated CD8þ T cells were assessed by IHC in RCC (n¼ 29) andLUSC (n ¼ 27) cohorts to address a role of PD-L2 expression forthe suppression against antitumor immunity, particularly CD8þ Tcells. PD-L1- and PD-L2–expressing tumors (�1% of tumor cells)were observed in 17.2% (5/29) and 72.4% (21/29) in RCC(Supplementary Table S2) and 55.6% (15/27) and 85.2%(23/27) in LUSC (Supplementary Table S3), respectively. Corre-lations betweenPD-L1or PD-L2 expression and clinicopathologicfeatures of RCC and LUSC are summarized in SupplementaryTables S2 and S3, respectively. In RCC, age, sex, Fuhrman nucleargrade, pathological stage (pStage), and histology did not correlatewith PD-L1 or PD-L2 expression (Supplementary Table S2). InLUSC, age, pStage, smoking status, and driver gene status did notcorrelate with PD-L1 or -L2 expression (Supplementary Table S3).In RCC, CD8þ T cells significantly highly infiltrated into PD-L2–expressing tumors, such as RCC_12, compared with PD-L2–negative tumors, such as RCC_25. In LUSC, CD8þ T cells showedhigher infiltration into PD-L2–expressing tumors, such asLUSC_21, than PD-L2–negative tumors, such as LUSC_27. Incontrast, CD8þ T-cell infiltration was unrelated to PD-L1 expres-sion in both tumors (Fig. 5; Supplementary Tables S2 and S3). Inaccordance with our animal models, PD-L2 expression in

immune cells was very low compared with tumor cells. Togetherwith TCGA dataset analyses, PD-L2 in tumor cells appears to playan important role in evading antitumor immunity in severalcancers, including RCC and LUSC.

DiscussionAccumulating evidence shows that the important immunosup-

pressive role of PD-L1 in the TME, while that of PD-L2 has notbeen fully elucidated. Using animal models, we showed that PD-L2 played an important role in evading antitumor immunity. Inaddition, we revealed that PD-L2 was dominantly correlated withantitumor immune responses in the TME rather than PD-L1 inRCC and LUSC using TCGA datasets and clinical samples. Inpreclinical animal models, anti-PD-1 mAb was effective againstthe PD-L2–expressing tumors, whereas PD-L2–expressing tumorswere resistant to anti-PD-L1 mAb alone. This limitation of anti-PD-L1 mAb was overcome by combined treatment with anti-PD-L2mAb. To our knowledge, this is the first report to directly showthe importance of PD-L2 as a mechanism for evading antitumorimmunity using preclinical animal models and clinical samples.

CD8þ T cells play major roles in antitumor immunity in theTME by directly killing tumor cells (32). PD-L1 expressed bytumor cells inhibits antitumor effects of CD8þ T cells (7). PD-L1expression in tumor cells is induced by twomechanisms: intrinsicinduction by genetic alterations (innate expression) and stimu-lation by IFNg released from effector T cells, including CD8þ Tcells (acquired expression; ref. 33). In many cancers, tumor cellsexpress PD-L1 through the latter mechanism (acquired expres-sion; ref. 34, 35); that is, PD-L1 expression seems to be a surrogatemarker for the presence of antitumor immune responses, such asCD8þT cell responses in the TME. PD-1 signal blockade unleashessuppressed antitumor T-cell immunity, resulting in tumor regres-sion; thereby, PD-L1 expression becomes a predictive biomark-er (12, 36). However, in some cancers, including RCC and LUSC,PD-L1 expression was not accompanied by clinical effectivity ofPD-1 signal blockade treatment (4, 13, 14). Consistent with thisfinding, we found that PDCD1LG2 expression was more stronglycorrelated with immune-related gene expression than CD274expression, especially in RCC and LUSC, using TCGA datasets.Our cohort also revealed that PD-L2 expression in tumor cells wascorrelated with CD8þ T-cell infiltration rather than PD-L1 expres-sion in RCC and LUSC, suggesting that PD-L2 expression can be amore dominant immunosuppressive mechanism than PD-L1 inthese cancer types. In addition, our in vivo animal models showedthat PD-L2 can be related to resistance to anti-PD-L1 mAb alone,which can be overcome by combined treatment with anti-PD-L2mAb. Thus, the PD-1/PD-L2 interaction plays a more importantrole in evading antitumor immunity than the PD-1/PD-L1 inter-action in these cancers.

Approximately 30% of patients with RCC possess locallyadvanced or metastatic RCC at diagnosis, and 40% of patientswith localized RCC develop metastasis after primary surgicaltreatment (37). Therefore, developing effective systemic therapiesis critical in RCC treatment. Immunotherapeutic agents, such asIL2 and IFNa, have been used to treat advanced RCCwith limitedsuccess (a �10%–22% response rate; refs. 38, 39). Along withunderstanding RCC biology, several molecularly targeted agents,including VEGF and mTOR, have been clinically introduced (40,41). While VEGF and mTOR inhibitors have provided markedclinical benefits in advanced RCC, some patients are inherently

Tanegashima et al.





resistant to these therapies, andmost patients acquire resistance tothem (42). Lung cancers, in which approximately 80% are clas-sified as NSCLC, are leading causes of cancer-related mortalityworldwide (43). Among NSCLCs, several molecular-targeted

therapies have provided significant clinical benefits in lung ade-nocarcinoma (44).However, systemic therapeutic options againstLUSC remain limited, resulting in poor prognoses compared withlung adenocarcinoma (44). Thus, more effective systemic

Figure 3.

PD-L2 expressed by tumor cells is involved in anti-PD-L1 mAb resistance.A, In vivo efficacies of various ICBs including combinations against MC-38-L2 and CT26-NY-ESO-1-L2 tumors. MC-38-L2 cells (1.0� 106) were injected subcutaneously on day 0, and ICB treatments as indicated were started on days 3, 6, and 9 (left).Tumor growth was monitored twice a week (n¼ 8 per group). CT26-NY-ESO-1-L2 cells (1.0� 106) were injected subcutaneously on day 0, and ICB as indicatedwere administered on days 7, 10, and 13 (right). Tumor growth was monitored twice a week (n¼ 5 per group). B, Tumor antigen (MuLV p15E)-specific CD8þ Tcells in TILs (left). TILs were prepared fromMC-38-L2 tumors treated with the indicated mAb on day 14, and tumor antigen–specific CD8þ T cells were detectedby MuLV p15E/H-2Kb tetramers. Summary of the MuLV p15E/H-2Kb tetramerþCD8þ T-cell frequencies (n¼ 8 per group; right). C, In vivo tumor growth of MC-38-,MC-38-L1KO, MC-38-L1KO-GFP, or MC-38-L1KO-L2 tumors. Tumor cells (1.0� 106) were injected subcutaneously, and tumor growthwas monitored twice a week(n¼ 8 per group).D, In vivo efficacies of various ICBs against MC38-L1KO-L2 tumors. MC38-L1KO-L2 cells (1.0� 106) were injected subcutaneously on day 0, andICB treatments as indicated were started on days 3, 6, and 9. Tumor growth was monitored twice a week (n¼ 8 per group). N.S., not significant; � , P < 0.05;�� , P < 0.01; �� , P < 0.001. In vivo experiments were performed at least twice. Representative data are shown from two to three independent experiments.






Figure 4.

PDCD1LG2 (encoding PD-L2) expression rather than CD274 (encoding PD-L1) is correlated with immune-related gene expression in RCC and LUSC. A, Thescattergram and real numbers of the Pearson correlations between expression levels (RPKM) of CD274 or PDCD1LG2 and immune-related genes in RCC fromTCGA datasets. Critical immune-related gene expression, including CD8A, GZMA, PRF1, and PDCD1, are shown in red. B, Heatmaps of correlation coefficientsbetween CD274, PDCD1LG2, or PDCD1 expression and the hallmark gene sets of the INTERFERON_GAMMA_RESPONSE signatures in bladder cancer (BLCA),gastric cancer (GC), HNSC, lung adenocarcinoma (LUAD), LUSC, and RCC.

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therapies against these cancers are necessary, and PD-1 signalblockade treatment, which has been approved to treat thesecancer types (4, 14), is thought to play a major role as a keytherapeutic strategy. Yet, as the clinical efficacy is unsatisfactory,predictive biomarkers are urgently needed. PD-L1 expressionevaluated by IHC, which is used as a biomarker of PD-1 signalblockade, is not a predictive biomarker of PD-1 signal blockade

in RCC or LUSC (4, 14). Furthermore, in recent clinical trials,anti-PD-L1 mAbmonotherapy was ineffective against RCC (45)and seemed less valuable against LUSC compared with lungadenocarcinoma (28). These findings are consistent with ourcurrent findings showing that not only PD-1/PD-L1 butalso PD-1/PD-L2 interaction could be important in evadingantitumor immunity in RCC and LUSC.

Figure 5.

PD-L2 expression is accompanied by CD8þ T-cell infiltration in the TMEs of RCC and LUSC. PD-L1, PD-L2 expression, and CD8þ T-cell infiltration in the TME ofRCC (A) and LUSC (B) were examined by IHC. Representative staining of PD-L1, PD-L2, and CD8 (top). Correlation between the number of infiltrated CD8þ T cellsand PD-L1 expression (left) and PD-L2 expression (right; bottom). RCC_12 and LUSC_21, PD-L2–expressing tumor; RCC_25 and LUSC_27, PD-L2–negative tumor;Scale bar, 100 mm; N.S., not significant; �� , P < 0.001.






While PD-L2 expression was initially thought to be restricted inAPCs, such as dendritic cells (6), that was negligible in our in vivomouse models in contrast to PD-L1. In addition, we showed thatPD-L2 expression in severalmurine cancer cell lineswas kept in lowlevel even with any stimulants, which can explain no efficacy ofanti-PD-L2mAb in sharp contrast to anti-PD-L1mAb inour animalmodels. Thus, little attention has been paid to PD-L2 expression. Ithas been shown that PD-L2 is expressed by tumor cells similar toour current human clinical sample study (20, 25), suggesting adiscrepancy in PD-L2 expression between mouse models andhumans. We therefore generate genetically engineered PD-L2–expressing murine cancer cell lines harboring the comparable levelof PD-L2 expression with endogenous PD-L1. The PD-L2–over-expressing tumors grew faster than the controls regardless of PD-L1expression, although this was not observed in immunocompro-mised mice, clearly indicating that PD-L2 expressed by tumor cellscan be involved in evading antitumor immunity. Accordingly, PD-L2–expressing tumors were resistant to anti-PD-L1 mAb alone,whichwasovercomebycombined treatmentwithanti-PD-L2mAb,thus highlighting that more attention should be paid to PD-L2expression in clinical settings. However, why some cancers, includ-ing RCC and LUSC, exhibit PD-L2 expression rather than PD-L1expression is unclear. A recent study revealed a difference betweenthe regulationsof PD-L1andPD-L2expression (46). Itwas reportedthat the IFNg–JAK1/JAK2–STAT1/STAT2/STAT3–IRF1 axis primar-ily regulated PD-L1 expression with IRF1 binding to its promoter.On the other hand, PD-L2 responded equally to IFNb and g and isregulated through both IRF1 and STAT3, which bind to the PD-L2promoter (46). In addition, genetic and/or epigenetic abnormal-ities may inhibit PD-L1 expression, and PD-L2 compensates theimmunosuppressive role of PD-L1, or genetic and/or epigeneticabnormalities in the PD-L2 gene induce a more dominant expres-sion than those of PD-L1 (47, 48).

In conclusion, we demonstrated that PD-L2 expression intumor cells appears to be strongly correlated with antitumorimmune responses in the TME especially in RCC and LUSC.Although little attention has been paid to PD-L2 expression inanimal models and clinical settings, mainly due to the lack ofappropriate cell lines and examinationmethods for PD-L2 expres-sion, respectively. PD-L2 clearly contributes to evading antitumorimmunity, suggesting that the PD-1/PD-L2 interaction should beblocked when treating PD-L2–expressing tumors. Currently, bothanti-PD-1 mAb and anti-PD-L1 mAb are clinically available,although the differences between these reagents are unclear.Anti-PD-1 mAb may be clinically effective against broader typesof cancer expressing either PD-L1 or PD-L2. Yet, as it is still anopen question that PD-L1 may have other receptors, it is difficultto conclude that anti-PD-1 mAb is more useful in any types ofcancer (49). Furthermore, because anti-PD-L1 mAb does notinhibit PD-L2, the frequencies of immune-related adverse events(irAE), such as endocrine system disorders and pneumonitis, arereported to be lower in patients treatedwith anti-PD-L1mAb thanin those treated with anti-PD-1 mAb (50). Thus, considering the

proper use of PD-1 signal blockade treatment with reflectivePD-L2 and PD-L1 expression in addition to minimizing irAEs isan important issue for optimal cancer immunotherapy and ourfindings help clinicians to select optimal immunotherapy.

Disclosure of Potential Conflicts of InterestY. Togashi reports receiving other commercial research support from Ono

Pharmaceutical, Bristol-Myers Squibb, and AstraZeneca, and reports receivingspeakers bureau honoraria from Ono Pharmaceutical and Chugai. K. Azumareports receiving speakers bureau honoraria from Ono Pharmaceutical,Bristol-Myers Squibb, AstraZeneca, and Chugai. M. Eto reports receiving com-mercial research grants from Ono Pharmaceutical, Takeda, Pfizer, Astellas,and Kissei, and reports receiving speakers bureau honoraria from OnoPharmaceutical, Bristol-Myers Squibb, Pfizer, Novartis, Bayer, and Takeda.H. Nishikawa reports receiving speakers bureau honoraria and other commer-cial research support from Ono Pharmaceutical, Bristol-Myers Squibb, andChugai. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Y. Togashi, M. Eto, H. NishikawaDevelopment of methodology: T. Tanegashima, Y. TogashiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Tanegashima, K. Azuma, A. Kawahara,K. Ideguchi, D. Sugiyama, F. Kinoshita, J. Akiba, A. Takeuchi, K. Tatsugami,T. Hoshino, M. EtoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Tanegashima, Y. Togashi, K. Azuma, A. Kawahara,F. Kinoshita, J. Akiba, A. Takeuchi, T. Irie, K. Tatsugami, T. Hoshino, M. Eto,H. NishikawaWriting, review, and/or revision of the manuscript: T. Tanegashima,Y. Togashi, H. NishikawaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. KashiwagiStudy supervision: H. Nishikawa

AcknowledgmentsThe authors thank Ms. Tomoka Takaku, Miyuki Nakai, Konomi Onagawa,

Megumi Takemura, Chie Haijima, Megumi Hoshino, Kumiko Yoshida, ErikoGunshima, and Noriko Hakoda for their technical assistance. This study wassupported by Grants-in-Aid for Scientific Research (S grant number 17H06162,to H. Nishikawa; Challenging Exploratory Research grant number 16K15551, toH.Nishikawa; YoungScientists number 17J09900, to Y. Togashi; and JSPSResearchFellowship number 17K18388, to Y. Togashi) from the Ministry of Education,Culture, Sports, Science and Technology of Japan; the Project for Cancer Research,the Therapeutic Evolution (P-CREATE, number 16cm0106301h0002, to H. Nishi-kawa) from the JapanAgency forMedical Research andDevelopment; theNationalCancer Center Research and Development Fund (number 28-A-7, to H. Nishi-kawa); the Naito Foundation (to Y. Togashi and H. Nishiskawa); the TakedaFoundation (to Y. Togashi); the Kobayashi Foundation for Cancer Research (to Y.Togashi); the Novartis Research Grant (to Y. Togashi); the Bristol-Myers SquibbResearchGrant (toY. Togashi); theSGHFoundation (toY. Togashi); andMitsui LifeSocial Welfare Foundation (to M. Eto).

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 December 6, 2018; revised April 8, 2019; accepted May 7, 2019;published first May 10, 2019.

References1. Hodi FS,O'Day SJ,McDermott DF,Weber RW, Sosman JA,Haanen JB, et al.

Improved survivalwith ipilimumab in patients withmetastaticmelanoma.N Engl J Med 2010;363:711–23.

2. 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.

3. Brahmer JR, Tykodi SS, ChowLQ,HwuWJ, Topalian SL,HwuP, et al. Safetyand activity of anti-PD-L1 antibody in patients with advanced cancer.N Engl J Med 2012;366:2455–65.

4. Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S,et al. Nivolumab versus everolimus in advanced renal-cell carcinoma.N Engl J Med 2015;373:1803–13.

Tanegashima et al.





5. Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al.Engagement of the PD-1 immunoinhibitory receptor by a novel B7 familymember leads to negative regulation of lymphocyte activation. J Exp Med2000;192:1027–34.

6. Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I,et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation.Nat Immunol 2001;2:261–8.

7. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al.Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mecha-nism of immune evasion. Nat Med 2002;8:793–800.

8. Curiel TJ, Wei S, Dong H, Alvarez X, Cheng P, Mottram P, et al. Blockade ofB7-H1 improves myeloid dendritic cell-mediated antitumor immunity.Nat Med 2003;9:562–7.

9. Yokosuka T, Takamatsu M, Kobayashi-Imanishi W, Hashimoto-Tane A,Azuma M, Saito T. Programmed cell death 1 forms negative costimulatorymicroclusters that directly inhibit T cell receptor signaling by recruitingphosphatase SHP2. J Exp Med 2012;209:1201–17.

10. Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, et al. T cellcostimulatory receptor CD28 is a primary target for PD-1–mediatedinhibition. Science 2017;355:1428–33.

11. Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, et al.Rescue of exhausted CD8 T cells by PD-1–targeted therapies is CD28-dependent. Science 2017;355:1423–7.

12. Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al.Nivolumab versus docetaxel in advanced nonsquamous non-small-celllung cancer. N Engl J Med 2015;373:1627–39.

13. Carbone DP, Reck M, Paz-Ares L, Creelan B, Horn L, Steins M, et al. First-line nivolumab in stage IV or recurrent non-small-cell lung cancer. N Engl JMed 2017;376:2415–26.

14. Brahmer J, Reckamp KL, Baas P, Crino L, Eberhardt WE, Poddubskaya E,et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med 2015;373:123–35.

15. Bellmunt J, de Wit R, Vaughn DJ, Fradet Y, Lee JL, Fong L, et al. Pem-brolizumab as second-line therapy for advanced urothelial carcinoma.N Engl J Med 2017;376:1015–26.

16. Kang Y-K, Boku N, Satoh T, Ryu M-H, Chao Y, Kato K, et al. Nivolumab inpatients with advanced gastric or gastro-oesophageal junction cancerrefractory to, or intolerant of, at least two previous chemotherapy regimens(ONO-4538-12, ATTRACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 2017;390:2461–71.

17. Okazaki T, Honjo T. PD-1 and PD-1 ligands: from discovery to clinicalapplication. Int Immunol 2007;19:813–24.

18. Rozali EN, Hato SV, Robinson BW, Lake RA, Lesterhuis WJ. Programmeddeath ligand 2 in cancer-induced immune suppression. ClinDev Immunol2012;656340.

19. ZhongX, Tumang JR,GaoW,BaiC, Rothstein TL. PD-L2 expression extendsbeyond dendritic cells:macrophages to B1 cells enriched for VH11:VH12and phosphatidylcholine binding. Eur J Immunol 2007;37:2405–10.

20. Yearley JH, Gibson C, Yu N, Moon C, Murphy E, Juco J, et al. PD-L2expression in human tumors: relevance to anti-PD-1 therapy in cancer.Clin Cancer Res 2017;23:3158–67.

21. Muraoka D, Kato T, Wang L, Maeda Y, Noguchi T, Harada N, et al. Peptidevaccine induces enhanced tumor growth associated with apoptosis induc-tion in CD8þ T cells. J Immunol 2010;185:3768–76.

22. Ikeda H, Ohta N, Fukuhara K, Miyazaki H, Wang L, Fukuhara K, et al.Mutated mitogen-activated protein kinase- a tumor rejection antigen ofmouse sarcoma. Proc Natl Acad Sci U S A 1997;94:6375–9.

23. Spranger S, Dai D, Horton B, Gajewski TF. Tumor-residing Batf3 dendriticcells are required for effector T cell trafficking and adoptive T cell therapy.Cancer Cell 2017;31:711–23.

24. Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy.Science 2015;348:69–74.

25. Shin S-J, Jeon YK, Kim P-J, Cho YM, Koh J, Chung DH, et al. Clinicopath-ologic analysis of PD-L1 and PD-L2 expression in renal cell carcinoma-Association with oncogenic proteins status. Ann Surg Oncol 2016;23:694–702.

26. Rooney MS, Shukla SA, Wu CJ, Getz G, Hacohen N. Molecular and geneticproperties of tumors associated with local immune cytolytic activity. Cell2015;160:48–61.

27. Ferris RL, BlumenscheinG Jr, Fayette J, Guigay J, Colevas AD, Licitra L, et al.Nivolumab for recurrent squamous-cell carcinoma of the head and neck.N Engl J Med 2016;375:1856–67.

28. Achim R, Fabrice B, Daniel W, Keunchil P, Fortunato C, Joachim VP, et al.Atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer (OAK): a phase 3, open-label, multicentre rando-mised controlled trial. Lancet 2017;389:255–65.

29. Cerami E,Gao J,DogrusozU,Gross BE, Sumer SO, Aksoy BA, et al. The cBiocancer genomics portal: an open platform for exploring multidimensionalcancer genomics data. Cancer Discov 2012;2:401–4.

30. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al.Integrative analysis of complex cancer genomics and clinical profiles usingthe cBioPortal. Sci Signal 2013;6:pl1.

31. Liberzon A, Birger C, Thorvaldsdottir H, Ghandi M, Mesirov JP, Tamayo P.TheMolecular SignaturesDatabase (MSigDB) hallmark gene set collection.Cell Syst 2015;1:417–25.

32. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunitycycle. Immunity 2013;39:1–10.

33. Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: acommon denominator approach to cancer therapy. Cancer Cell 2015;27:450–61.

34. Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, et al. MPDL3280A(anti-PD-L1) treatment leads to clinical activity in metastatic bladdercancer. Nature 2014;515:558–62.

35. Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, et al.Predictive correlates of response to the anti-PD-L1 antibody MPDL3280Ain cancer patients. Nature 2014;515:563–7.

36. Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fulop A, et al.Pembrolizumab versus chemotherapy for PD-L1-positive non-small-celllung cancer. N Engl J Med 2016;375:1823–33.

37. Fisher R, GoreM, Larkin J. Current and future systemic treatments for renalcell carcinoma. Semin Cancer Biol 2013;23:38–45.

38. Unverzagt S,Moldenhauer I, CoppinC,Greco F, Seliger B. Immunotherapyfor metastatic renal cell carcinoma [Protocol]. Cochrane Database SystRev 2015:CD011673.

39. Negrier S, Escudier B, Lasset C, Douillard J-Y, Savary J, Chevreau C, et al.Recombinant human interleukin-2, recombinant human interferon alfa-2a, or both in metastatic renal-cell carcinoma. Groupe Francais d'Immu-notherapie. N Engl J Med 1998;338:1272–8.

40. Dancey J. mTOR signaling and drug development in cancer. Nat Rev ClinOncol 2010;7:209–19.

41. Vachhani P, George S. VEGF inhibitors in renal cell carcinoma. Clin AdvHematol Oncol 2016;14:1016–28.

42. Rini BI, Atkins MB. Resistance to targeted therapy in renal-cell carcinoma.Lancet Oncol 2009;10:992–1000.

43. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin2018;68:7–30.

44. Herbst RS, Morgensztern D, Boshoff C. The biology and management ofnon-small cell lung cancer. Nature 2018;553:446–54.

45. McDermott DF,HuseniMA, AtkinsMB,Motzer RJ, Rini BI, Escudier B, et al.Clinical activity and molecular correlates of response to atezolizumabalone or in combination with bevacizumab versus sunitinib in renal cellcarcinoma. Nat Med 2018;24:749–57.

46. Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, RodriguezGA, et al. Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Cell Rep 2017;19:1189–201.

47. Kataoka K, Shiraishi Y, Takeda Y, Sakata S, Matsumoto M, Nagano S, et al.Aberrant PD-L1 expression through 30-UTR disruption inmultiple cancers.Nature 2016;534:402–6.

48. Ikeda S, Okamoto T, Okano S, Umemoto Y, Tagawa T, Morodomi Y,et al. PD-L1 is upregulated by simultaneous amplification of the PD-L1and JAK2 genes in non-small cell lung cancer. J Thorac Oncol 2016;11:62–71.

49. Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ. Programmeddeath-1 ligand 1 interacts specificallywith the B7-1 costimulatorymoleculeto inhibit T cell responses. Immunity 2007;27:111–22.

50. Baxi S, Yang A, Gennarelli RL, Khan N, Wang Z, Boyce L, et al. Immune-related adverse events for anti-PD-1 and anti-PD-L1 drugs: systematicreview and meta-analysis. BMJ 2018;360:k793.






2019;25:4808-4819. Published OnlineFirst May 10, 2019.Clin Cancer Res Tokiyoshi Tanegashima, Yosuke Togashi, Koichi Azuma, et al. Treatment-Related Antitumor ImmunityImmune Suppression by PD-L2 against Spontaneous and

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