Immune Suppression by PD-L2 Against Spontaneous and Treatment-Related Antitumor Immunity

Published OnlineFirst May 10, 2019; DOI: 10.1158/1078-0432.CCR-18-3991

Translational Cancer Mechanisms and Therapy Clinical Cancer Research Immune Suppression by PD-L2 against Spontaneous and Treatment-Related Antitumor Immunity Tokiyoshi Tanegashima1,2, Yosuke Togashi1, Koichi Azuma3, Akihiko Kawahara3, Ko Ideguchi4, Daisuke Sugiyama4, Fumio Kinoshita2,5, Jun Akiba6, Eiji Kashiwagi2, Ario Takeuchi2,TakumaIrie1, Katsunori Tatsugami2,Tomoaki Hoshino3, Masatoshi Eto2, and Hiroyoshi Nishikawa1,4

Abstract

Purpose: To evaluate the detailed immunosuppressive in tumor cells significantly suppressed antitumor immune þ role(s) of PD-L2 given that its detailed role(s) remains unclear responses, such as tumor antigen–specificCD8 Tcells, in PD-1 signal blockade therapy in animal models and and was involved in the resistance to treatment with humans. anti-PD-L1 mAb alone. This resistance was overcome by Experimental Design: We generated mouse cell lines har- anti-PD-1 mAb or combined treatment with anti-PD-L2 boring various status of PD-L1/PD-L2 and evaluated the tumor mAb. In clinical settings, antitumor immune responses growth and phenotypes of tumor-infiltrated lymphocytes were significantly correlated with PD-L2 expression in using several PD-1 signal blockades in animal models. In the tumor microenvironment in renal cell carcinoma humans, the correlation between immune-related gene expres- (RCC) and lung squamous cell carcinoma (LUSC). sion and CD274 (encoding PD-L1) or PDCD1LG2 (encoding Conclusions: We propose that PD-L2 as well as PD-L1 play PD-L2) was investigated using The Cancer Genome Atlas important roles in evading antitumor immunity, suggesting (TCGA) datasets. In addition, PD-L1 or PD-L2 expression in that PD-1/PD-L2 blockade must be considered for optimal þ tumor cells and CD8 T-cell infiltration were assessed by IHC. immunotherapy in PD-L2–expressing cancers, such as RCC Results: In animal models, we showed that PD-L2 and LUSC. expression alone or simultaneously expressed with PD-L1

Introduction ders from nonresponders are urgently needed. Tumors employ the PD-1 pathway to evade antitumor immunity, particularly Immune checkpoint blockade (ICB), including PD-1 blockade, þ CD8 T cells against tumor antigens (5, 6). PD-1 is mainly has been approved to treat various cancers, such as malignant expressed by activated T cells and binds to its ligands, PD-L1 and melanoma, non–small cell lung cancer (NSCLC), and renal cell PD-L2 (5, 6), resulting in immunosuppression. PD-L1 is carcinoma (RCC), leading to a paradigm shift in cancer thera- expressed by both antigen-presenting cells (APC) and tumor py (1–4). However, as the clinical efficacy is limited, more cells (7, 8). PD-1 signal blockade unleashes antitumor T-cell effective therapies and predictive biomarkers stratifying respon- responses by augmenting signals from the T-cell receptor and CD28 (9–11), an essential costimulatory molecule. Consistent with the importance of the PD-1/PD-L1 interaction, several stud- 1Division of Cancer Immunology, Research Institute/Exploratory Oncology Research and Clinical Trial Center (EPOC), National Cancer Center, Tokyo/ ies revealed favorable clinical courses using treatment with anti- Kashiwa, Japan. 2Department of Urology, Graduate School of Medical Sciences, PD-1/PD-L1 mAbs in patients harboring PD-L1–expressing Kyushu University, Fukuoka, Japan. 3Department of Respiratory Medicine, tumors (12, 13). However, some patients harboring PD-L1– Kurume University Hospital, Kurume, Japan. 4Department of Immunology, expressing tumors do not respond to PD-1 signal blockade 5 Nagoya University Graduate School of Medicine, Nagoya, Japan. Department treatment. In addition, patients with PD-L1–negative tumors of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu Univer- fi – 6 occasionally experience clinical ef cacy (4, 12 14). In large phase sity, Fukuoka, Japan. Department of Diagnostic Pathology, Kurume University fi Hospital, Kurume, Japan. III trials, the clinical bene t from anti-PD-1 mAbs appears to be independent of PD-L1 expression in some cancers, such as RCC Note: Supplementary data for this article are available at Clinical Cancer and lung squamous cell carcinoma (LUSC; refs. 4, 14–16). Research Online (http://clincancerres.aacrjournals.org/). The expression of another PD-1 ligand, PD-L2, was initially Corresponding Authors: Hiroyoshi Nishikawa, National Cancer Center, 6-5-1 thought to be restricted in APCs (6). Recently, several studies Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. Phone: 81-4-7133-1111, ext. 91385 showed that PD-L2 is expressed by both various immune cells and or 2157; Fax: 81-4-7130-0022; E-mail: [email protected]; and Yosuke Togashi, – [email protected] tumor cells, depending on microenvironmental stimuli (17 19). PD-L2 expression in tumor cells is a predictive factor for the Clin Cancer Res 2019;XX:XX–XX clinical efficacy of anti-PD-1 mAb in some studies (20). In this doi: 10.1158/1078-0432.CCR-18-3991 study, we evaluated the detailed immunosuppressive role(s) of 2019 American Association for Cancer Research. PD-L2 in animal models and humans. The Cancer Genome Atlas

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(CT26-NY-ESO-1), Renca (mouse kidney adenocarcinoma), Translational Relevance CMS5a (mouse fibrosarcoma), and A20 (mouse reticulum cell Although PD-L1, a ligand of PD-1, has mainly been studied sarcoma) cell lines were maintained in RPMI medium (FUJIFILM as a therapeutic target and predictive biomarker in PD-1 Wako Pure Chemical Corporation) supplemented with 10% FCS blockade therapy, the detailed role(s) of PD-L2, another (Biosera). CT26-NY-ESO-1 is a cell line derived from CT26 stably PD-1 ligand, remains unclear. Using preclinical animal mod- transfected with NY-ESO-1 (21). CT26, Renca, B16-F10, and A20 els, we showed that PD-L2 expression alone or simultaneously cell lines were obtained from the ATCC. MC-38 cell line was expressed with PD-L1 by tumor cells significantly suppressed obtained from Kerafast. CMS5a cell line was provided by the late antitumor immune responses, such as tumor antigen–specific Dr. Lloyd J. Old, Memorial Sloan Kettering Cancer Center (New þ CD8 T cells, and was involved in the resistance to treatment York, NY; ref. 22). All cell lines were used after confirming with anti-PD-L1 mAb. This resistance was overcome by anti- Mycoplasma ()byMycoplasma testing with a PCR Mycoplasma PD-1 mAb or combined treatment with anti-PD-L2 mAb. In Detection Kit (Takara Bio) according to the manufacturer's clinical settings, antitumor immune responses were signifi- instructions. Murine IFNg, IL2, IL4, TNFa, and granulocyte mac- cantly correlated with PD-L2 expression in the tumor micro- rophage colony stimulating factor (GM-CSF) were obtained from environment in renal cell carcinoma (RCC) and lung squa- PeproTech. Murine IFNa, IFNb, and tumor growth factor-b1 were mous cell carcinoma (LUSC). We propose that PD-L2 plays obtained from R&D Systems, Inc. Rat anti-mouse PD-1 (RMP1- an important role in evading antitumor immunity as well as 14) mAb, anti-PD-L1 (10F.9G2) mAb, anti-PD-L2 (TY25) mAbs, PD-L1, suggesting that PD-1/PD-L2 blockade must be consid- and control rat IgG2a (RTK2758) mAb used in the in vivo study ered for optimal immunotherapy in PD-L2–expressing can- were obtained from BioLegend. Anti-CD4 (GK1.5) mAb and anti- cers, such as RCC and LUSC. CD8b (Lyt 3.2) mAb were obtained from BioXCell.

Constructs, viral production, and transfection Mouse PDCD1LG2 cDNA was subcloned into pMXs-IRES-GFP, (TCGA) datasets revealed a strong correlation between decreased which was transfected into the packaged cell line using Lipofec- antitumor immune responses and PD-L2 rather than PD-L1 in the tamine 3000 Reagent (Thermo Fisher Scientific). After 48 hours of tumor microenvironment (TME) in several cancers, including retroviral production, the supernatant was concentrated and RCC and LUSC, which was confirmed by our clinical samples. dissolved in water. Subsequently, the viral lysate was transfected Therefore, we propose that PD-L2 should be considered as crucial with MC-38 and CT26-NY-ESO-1, and the PD-L2–expressing cell immunosuppressive mechanisms in clinical settings in addition lines were named MC-38-L2 and CT26-NY-ESO-1-L2, respective- to PD-L1. ly. GFP-expressing cell lines were also created for the controls and named MC-38-GFP and CT26-NY-ESO-1-GFP. To generate the PD-L1–knockout (KO) MC-38 cell line, we used a CRISPR/Cas9 Materials and Methods system. Briefly, guide RNA (gRNA)-targeting mouse CD274 (50- Patients GCTTGCGTTAGTGGTGTACT-30) was made using the GeneArt Twenty-nine patients with clear-cell and non–clear-cell RCC Precision gRNA Synthesis Kit (Thermo Fisher Scientific). The Cas9 who underwent surgical resection at Kyushu University Hospital protein (Thermo Fisher Scientific) and gRNA were electroporated (Fukuoka, Japan) from September 2017 to May 2018 were into MC-38 cells using the Neon Transfection System (Thermo enrolled in this study. Twenty-seven patients with LUSC who Fisher Scientific) per the manufacturer's instructions. The knock- underwent surgical resection at Kurume University Hospital from out cell line was named MC-38-L1KO. Either GFP or PD-L2 was January 2008 to December 2012 were enrolled in this study. The introduced into the MC-38-L1KO in the same manner, and these patients' clinical information was obtained from their medical cell lines were named MC-38-L1KO-GFP and MC-38-L1KO-L2, records. All patients provided written informed consent before respectively. undergoing the study procedures. The clinical protocol for this study was approved by the appropriate institutional review In vivo mouse studies boards and ethics committees at Kyushu University Hospital and Female C57BL/6, BALB/c and BALB/c nude mice (6–8 week) Kurume University Hospital (Kurume, Japan). This study was were purchased from CLEA Japan and used at 7–9 weeks of age. conducted in accordance with the Declaration of Helsinki. Tumor cells (1 106) were injected subcutaneously, and tumor size was monitored twice weekly. The mean of the long and short IHC diameters was applied for the tumor growth curves. Mice were Sections were immunostained using the Ventana Benchmark grouped when tumor volume reached approximately 100 mm3, XT Automated Staining System (Ventana Medical System) accord- and anti-PD-1/PD-L1/PD-L2 mAbs were administered intraperi- þ ing to the manufacturer's protocol. Tumor PD-L1 and PD-L2 toneally three times every 3 days thereafter. For CD4 T-cell or þ membrane expression was assessed; 1% was defined as positive CD8 T-cell depletion, anti-CD4 mAb (100 mg/mouse) or anti- þ and <1% as negative (4, 12, 14). The intertumoral CD8 T cells CD8b mAb (100 mg/mouse) was administered intraperitoneally were counted. Specifically, five areas [0.5 0.5 mm (0.25 mm2) on days 1, 0, and every 7 days after tumor injection. Tumors were fields] with the most abundant distribution were selected and harvested 14 days after tumor injection, and tumor-infiltrating counted in each case. lymphocytes (TIL) were analyzed with flow cytometry. All mouse experiments were approved by the Animals Committee for Ani- Cell lines and reagents mal Experimentation of the National Cancer Center Japan. All B16-F10 (mouse melanoma), MC-38 (mouse colon carcino- experiments met the U.S. Public Health Service Policy on Humane ma), CT26 (mouse colon carcinoma) expressing NY-ESO-1 Care and Use of Laboratory Animals.

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Flow cytometry Results Cells were washed using PBS with 2% FBS and stained with PD-L2 plays a limited role in immune suppression in murine mAbs specific for CD4, CD8, CD44, CD62L, PD-1, PD-L1, and tumor models PD-L2 as well as fixable viability dye (Thermo Fisher Scientific). To þ To elucidate the role of PD-L2 in tumor immunity, we first examine antigen-specific CD8 T cells, T-Select H-2Kb MuLV explored PD-L1 and PD-L2 expression in several murine tumor p15E Tetramer-KSPWFTTL-APC (MBL) was used per the manu- cell lines, including MC-38, CT26, B16-F10, Renca, CMS5a, and facturer's instructions. After tetramer staining, cells were stained A20. All tumor cell lines possessed variable PD-L1 expression that for cell surface markers (mAbs specific for CD4, CD8, and fixable was enhanced by stimulants, especially IFNa, b, and g. In contrast, viability dye). When intracellular cytokines were analyzed, cells PD-L2 was minimally detected by any cell lines even after stim- were stimulated for 5 hours with phorbol 12-myristate 13-acetate ulation (Fig. 1A; Supplementary Figs. S1 and S2). While PD-L2 is (PMA; 100 ng/mL)/ionomycin (2 mg/mL; Sigma Aldrich). Golgi- reportedly expressed by APCs (6), low expression in APCs Plug Reagent (1.3 mL/mL; BD Biosciences) was added for the last 4 þ þ þ þ (CD45 MHCII CD11c CD11b cells) was observed in animal hours of the culture. These cells were stained for surface markers models (ref. 23; Supplementary Fig. S3). In contrast to PD-L2, and then stained for intracellular cytokines (mAbs specific for APCs had considerable, although slightly lower than tumor cells, TNFa and IFNg). After washing, the cells were analyzed with an PD-L1 expression (Supplementary Fig. S3). We next examined LSR Fortessa Instrument (BD Biosciences) and FlowJo Software in vivo antitumor effects of anti-PD-1/PD-L1/PD-L2 mAbs using (Tree Star). MC-38 and CT26-NY-ESO-1 cells. Anti-PD-1 mAb and anti-PD-L1 mAb similarly inhibited MC-38 and CT26-NY-ESO-1 tumor Antibodies growth, whereas anti-PD-L2 mAb was ineffective against both Violet 500–conjugated anti-CD8 (53-6.7) mAb, Brilliant tumors (Fig. 1B). As neoantigen-specific T cells are reportedly – essential for antitumor effects by ICB (24), MuLV p15E (a neoan- Violet 786 (BV786) conjugated anti-CD4 (RM4-5) mAb, phyco- þ erythrin (PE)-and Cy7-conjugated anti-CD44 (IM7) mAb, BV421- tigen of MC-38–specific CD8 T cells) detected by MHC/peptide tetramers was examined in an MC-38 model. MuLV p15E tetra- conjugated anti-PD-L2 (TY25) mAb, and BV605-conjugated þ þ anti-CD11c (HL3) were purchased from BD Biosciences. mer CD8 T cells were significantly primed/augmented by treatment with anti-PD-1 mAb or anti-PD-L1 mAb but not with anti- BV421-conjugated anti-PD-1 (29F.1A12) mAb, peridinin chloro- þ PD-L2 mAb alone (Fig. 1C). Activated CD8 T cells, which were phyll protein complex, Cy5.5-conjugated anti-CD62L (MEL-14) þ assessed by the proportion of CD44 CD62L effector/memory mAb, BV421-conjugated anti-TNF-a (MP6-XT22) mAb, PE- þ þ þ conjugated anti-PD-L1 (10F.9G2) mAb, and AF488-conjugated CD8 T cells and the frequency of PD-1 CD8 T cells, were anti-MHC class II (M5/114.15.2) were purchased from Bio- significantly higher in TILs in mice treated with anti-PD-1 mAb Legend. FITC-conjugated anti-IFNg (XMG1.2) mAb were or anti-PD-L1 mAb compared with mice treated with isotype control or anti-PD-L2 mAb (Fig. 1D; Supplementary Fig. S4). In obtained from eBioscience. PE-conjugated anti-CD11b (M1/ þ þ þ 70) mAb were purchased from Thermo Fisher Scientific. The addition, TNFa IFNg CD8 T cells were significantly higher in antibodies used for IHC were PD-L1 (E1L3N, Cell Signaling mice treated with anti-PD-1 mAb or anti-PD-L1 mAb than in Technology), PD-L2 (#176611, R&D Systems, Inc.), and CD8 those treated with isotype control or anti-PD-L2 mAb (Fig. 1D; (4B11, Leica Biosystems). Supplementary Fig. S4). Therefore, PD-L1 plays a dominant immunosuppressive role compared with PD-L2 in animal models that are often used in tumor immunology studies. Real-time qRT-PCR RNA was extracted using RNeasy Mini Kit (Qiagen). cDNA was generated using SuperScript VILO (Thermo Fisher Scientific), and PD-L2 expression is a mechanism for evading antitumor real-time PCR reactions were performed with SYBR Green immunity Reagents (Thermo Fisher Scientific) using the QuantStudio 7 Flex Because PD-L2 was minimally expressed by the murine tumor Real-Time PCR System (Thermo Fisher Scientific). Gene expres- cell lines that are frequently used in preclinical studies for PD-1 sion changes relative to 18S ribosomal RNA as a housekeeping signal blockade although some human tumor cells highly express gene were calculated using the DDCT method. A forward primer PD-L2 (20, 25), we hypothesized that the lesser appreciation of fl (50-AGCCTGCTGTCACTTGCTAC-30) and reverse primer (50- PD-L2 in PD-1 signal blockade treatment may solely re ect the – TCCCAGTACACCACTAACGC-30) were used to detect CD274. lack of appropriate animal models using PD-L2 expressing tumor – A forward primer (50-CCTCAGCCTAGCAGAAACTTCA-30) and cell lines. The PD-L2 overexpressing cell lines MC-38 (MC-38-L2) reverse primer (50-CATCCGACTCAGAGGGTCAATG-30) were and CT26-NY-ESO-1 (CT26-NY-ESO-1-L2) were thus developed fl fi used to detect PDCD1LG2. A forward primer (50-TAGAGTGTT- to evaluate in uence of PD-L2 on PD-1 signal blockade ef cacies CAAAGCAGGCCC-30) and reverse primer (50-CCAACAAAATA- using anti-PD-1 mAb, anti-PD-L1 mAb, and anti-PD-L2 mAb. PD- fi GAACCGCGGT-30) were used to detect 18S. L2 expression was con rmed at both mRNA and protein levels using real-time qRT-PCR (Fig. 2A) and flow cytometry (Fig. 2B), respectively. The level of PD-L2 expression was equivalent to that Statistical analysis of endogenous PD-L1 expression after IFNg treatment (Fig. 2A GraphPad Prism7 (GraphPad Software) was used for statis- and B). PD-L2–expressing tumors grew rapidly compared with tical analysis. The overall survival was analyzed with Kaplan– those in control mice injected with parental tumors or GFP- Meier method and compared with log-rank test. The relation- expressing tumors (Fig. 2C) although these tumors exhibited ships between the groups were compared using Fisher exact comparable tumor growth in immunocompromised mice (Sup- þ test or Welch t test. P < 0.05 was considered statistically plementary Fig. S5A). Furthermore, to elucidate the role of CD4 þ significant. T cells and CD8 T cells, the tumor growth of MC-38/-GFP/-L2

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þ and CT-26-NY-ESO-1/-GFP/-L2 was evaluated in CD4 T-cell– or compared with anti-PD-L1 mAb or anti-PD-L2 mAb alone in þ þ þ CD8 T-cell–depleted mice. In CD8 T-cell–depleted mice, the MC-38-L2 tumor–bearing mice (Fig. 3B). Activated CD8 Tcells þ þ þ þ rapid tumor growth in PD-L2–expressing tumors was totally (CD44 CD62L effector/memory CD8 T cells and PD-1 CD8 þ þ þ abrogated although PD-L2–expressing tumors grew rapidly com- Tcells)andTNFa IFNg CD8 T cells in TILs were also significantly pared with the parental tumors or GFP-expressing tumors in higher in mice treated with the anti-PD-1 mAb or the combination þ CD4 T-cell–depleted mice (Supplementary Fig. S5B and 5C), of anti-PD-L1 mAb and anti-PD-L2 mAb compared with untreated þ indicating the importance of CD8 T-cell suppression by PD-L2. mice or mice treated either by anti-PD-L1 mAb or by anti-PD-L2 þ Accordingly, in the MC-38-L2 tumors, MuLV p15E tetramer mAb alone (Supplementary Fig. S9). Therefore, PD-L2 endows þ CD8 T cells were reduced compared with those of the control resistance to anti-PD-L1 mAb monotherapy, which can be overcome þ mice (Fig. 2D). In addition, activated CD8 T cells by combined treatment with anti-PD-L1 mAb and anti-PD-L2 mAb. þ þ þ þ (CD44 CD62L effector/memory CD8 T cells and PD-1 CD8 To further confirm the role of PD-L2, a PD-L1–knockout MC-38 þ þ þ T cells) and TNFa IFNg CD8 T cells in TILs were significantly cell line (MC-38-L1KO) was generated using a CRISPR/Cas9 lower in MC-38-L2 tumors than in the control parental tumors system, and the lack of PD-L1 protein expression was confirmed or GFP-expressing tumors (Fig. 2E; Supplementary Fig. S6). with flow cytometry (Supplementary Fig. S10). Either PD-L2 or Taken together, PD-L2 expressed by tumor cells functions as GFP was then expressed in MC-38-L1KO cells (MC-38-L1KO-L2 an immunosuppressive molecule against antitumor immunity, and MC-38-L1KO-GFP, respectively; Supplementary Fig. S10). þ particularly CD8 T cells. MC-38-L1KO tumors grew more slowly than the parental MC-38 tumors, whereas PD-L2 expression allowed MC-38-L1KO-L2 PD-L2 blockade is necessary for controlling PD-L2–expressing tumors to grow comparably with the parental MC-38 tumors tumors (Fig. 3C). In contrast, these tumors grew similarly in immuno- We next addressed how PD-L2 influenced the antitumor effects compromised mice (Supplementary Fig. S10). Anti-PD-1 mAb of anti-PD-1 mAb, anti-PD-L1 mAb, and anti-PD-L2 mAb. Anti- and anti-PD-L2 mAb significantly inhibited tumor growth, but PD-1 mAb significantly inhibited PD-L2–expressing tumor anti-PD-L1 mAb did not (Fig. 3D). Thus, PD-L2 alone sufficiently growth compared with anti-PD-L1 mAb or anti-PD-L2 mAb hampers antitumor immunity, allowing rapid tumor growth. alone. The combination of anti-PD-L1 mAb and anti-PD-L2 mAb showed a comparable tumor growth inhibition in PD-L2– PD-L2 expression is associated with antitumor immune expressing tumor growth as observed by anti-PD-1 mAb responses in various cancers (Fig. 3A). In addition, anti-PD-1 mAb did not show any addi- Because PD-L2 significantly suppressed antitumor immunity in tional antitumor effects against PD-L2–expressing tumors when animal models, we investigated the correlation between immune- combined with anti-PD-L2 mAb, indicating that anti-PD-1 mAb related gene expression and CD274 (encoding PD-L1) or alone can inhibit PD-1/PD-L2 interaction sufficiently to reinvig- PDCD1LG2 (encoding PD-L2) in humans using TCGA datasets. orate an effective antitumor immunity. (Supplementary Fig. S7). The expression levels of 43 genes were defined for the immune cell þ The tumor growth inhibition by these antibodies including anti- types, CD8 T cells, costimulatory APCs, costimulatory T cells, PD-L2 mAb was totally abrogated in immunocompromised mice coinhibitory APCs, coinhibitory T cells, and cytolytic activity þ or CD8 T-cell–depleted mice although they exhibited antitumor (ref. 26; Supplementary Table S1). We analyzed bladder cancer, þ effects against PD-L2–expressing tumors in CD4 T-cell–depleted gastric cancer, head and neck squamous cell carcinoma (HNSC), mice as well as in control mice (Supplementary Fig. S8A), indi- lung adenocarcinoma, LUSC, and RCC datasets because the cating that the antitumor efficacy against PD-L2–expressing efficacy of anti-PD-1 mAb or anti-PD-L1 mAb has been demon- þ tumors was attributed for unleashing CD8 T cells. In addition, strated in these cancers, and the published datasets were avail- anti-PD-L2 mAb did not exhibit antitumor immunity against able (4, 12, 14–16, 27–30). The correlation coefficient between parental tumors or GFP-expressing tumors in immunocompro- the expression level of each gene and that of CD274 or PDCD1LG2 mised mice (Supplementary Fig. S8B). Thus, the antitumor was calculated (Fig. 4A; Supplementary Fig. S11). Especially in efficacy of anti-PD-L2 mAb against PD-L2–expressing tumors RCC, PDCD1LG2 expression rather than CD274 expression was þ was dependent on the reactivation of CD8 T cells, but not significantly positively correlated with immune-related gene causedbyadirecteffectofPD-L2blockadeagainstPD-L2– expression (Fig. 4A). We further evaluated the correlation between þ þ expressing tumors. Accordingly, MuLV p15E tetramer CD8 CD274, PDCD1LG2,orPDCD1 expression and the INTERFER- Tcellsweresignificantly primed/augmented by anti-PD-1 mAb ON_GAMMA_RESPONSE gene signature (Fig. 4B; ref. 31). The or the combination of anti-PD-L1 mAb and anti-PD-L2 mAb heatmap highlights the quantitative differences in association

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-L2 expression 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 were subsequently subjected to flow cytometry. Gray, isotype control; blue, untreated; red, IFNg treated. B, In vivo efficacies of various ICBs including combinations against 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 growth was monitored twice weekly (n ¼ 8 per group). CT26-NY-ESO-1 cells (1.0 106) were injected subcutaneously on day 0, and ICB as indicated was administered on days 7, 10, and 13 (bottom). Tumor growth was monitored twice a week (n ¼ 5 per group). C, Tumor antigen (MuLV p15E)-specific CD8þ T cells in TILs (top). TILs were prepared from MC38 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 (n ¼ 6per group; bottom). D, Effector CD8þ T cells detected by CD44þCD62LCD8þ T cells, PD-1þCD8þ T cells, and cytokine-producing CD8þ T cells in TILs. TILs were prepared from MC38 tumors as in C, and CD44þCD62LCD8þ TILs, PD-1þCD8þ TILs, and TNFaþIFNgþCD8þ TILs were examined (n ¼ 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.

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between PD-1–related molecules and immune responses in the been fully elucidated. Using animal models, we showed that PD- TME across tumor types. In RCC and LUSC, PDCD1LG2 expres- L2 played an important role in evading antitumor immunity. In sion was strongly correlated with IFNg-related gene expression, addition, we revealed that PD-L2 was dominantly correlated with although CD274 expression was less significantly correlated. antitumor immune responses in the TME rather than PD-L1 in These data suggest that PD-L2 expression in RCC and LUSC RCC and LUSC using TCGA datasets and clinical samples. In strongly controls antitumor immune responses in the TME. preclinical animal models, anti-PD-1 mAb was effective against We next examined the relationship between CD274 or the PD-L2–expressing tumors, whereas PD-L2–expressing tumors PDCD1LG2 expression and survival from RCC and LUSC TCGA were resistant to anti-PD-L1 mAb alone. This limitation of anti- datasets. In RCC, no correlation between CD274 or PDCD1LG2 PD-L1 mAb was overcome by combined treatment with anti-PD- expressions and survival was observed. On the other hand, high L2 mAb. To our knowledge, this is the first report to directly show expression of CD274 or PDCD1LG2 equally correlated with a the importance of PD-L2 as a mechanism for evading antitumor better survival in LUSC, indicating the possible correlation immunity using preclinical animal models and clinical samples. þ between antitumor immune responses and a favorable prognosis CD8 T cells play major roles in antitumor immunity in the (Supplementary Fig. S12). TME by directly killing tumor cells (32). PD-L1 expressed by þ tumor cells inhibits antitumor effects of CD8 T cells (7). PD-L1 þ CD8 T-cell infiltration is accompanied by PD-L2 expression in expression in tumor cells is induced by two mechanisms: intrinsic RCC and LUSC induction by genetic alterations (innate expression) and stimu- þ Because the correlation between immune-related genes and lation by IFNg released from effector T cells, including CD8 T CD274 expression was relatively weak in RCC and LUSC, the cells (acquired expression; ref. 33). In many cancers, tumor cells expression of PD-L1 or PD-L2 in tumor cells and the number of express PD-L1 through the latter mechanism (acquired expres- þ infiltrated CD8 T cells were assessed by IHC in RCC (n ¼ 29) and sion; ref. 34, 35); that is, PD-L1 expression seems to be a surrogate LUSC (n ¼ 27) cohorts to address a role of PD-L2 expression for marker for the presence of antitumor immune responses, such as þ þ the suppression against antitumor immunity, particularly CD8 T CD8 T cell responses in the TME. PD-1 signal blockade unleashes cells. PD-L1- and PD-L2–expressing tumors (1% of tumor cells) suppressed antitumor T-cell immunity, resulting in tumor regres- were observed in 17.2% (5/29) and 72.4% (21/29) in RCC sion; thereby, PD-L1 expression becomes a predictive biomark- (Supplementary Table S2) and 55.6% (15/27) and 85.2% er (12, 36). However, in some cancers, including RCC and LUSC, (23/27) in LUSC (Supplementary Table S3), respectively. Corre- PD-L1 expression was not accompanied by clinical effectivity of lations between PD-L1 or PD-L2 expression and clinicopathologic PD-1 signal blockade treatment (4, 13, 14). Consistent with this features of RCC and LUSC are summarized in Supplementary finding, we found that PDCD1LG2 expression was more strongly Tables S2 and S3, respectively. In RCC, age, sex, Fuhrman nuclear correlated with immune-related gene expression than CD274 grade, pathological stage (pStage), and histology did not correlate expression, especially in RCC and LUSC, using TCGA datasets. with PD-L1 or PD-L2 expression (Supplementary Table S2). In Our cohort also revealed that PD-L2 expression in tumor cells was þ LUSC, age, pStage, smoking status, and driver gene status did not correlated with CD8 T-cell infiltration rather than PD-L1 expres- correlate with PD-L1 or -L2 expression (Supplementary Table S3). sion in RCC and LUSC, suggesting that PD-L2 expression can be a þ In RCC, CD8 T cells significantly highly infiltrated into PD-L2– more dominant immunosuppressive mechanism than PD-L1 in expressing tumors, such as RCC_12, compared with PD-L2– these cancer types. In addition, our in vivo animal models showed þ negative tumors, such as RCC_25. In LUSC, CD8 T cells showed that PD-L2 can be related to resistance to anti-PD-L1 mAb alone, higher infiltration into PD-L2–expressing tumors, such as which can be overcome by combined treatment with anti-PD-L2 LUSC_21, than PD-L2–negative tumors, such as LUSC_27. In mAb. Thus, the PD-1/PD-L2 interaction plays a more important þ contrast, CD8 T-cell infiltration was unrelated to PD-L1 expres- role in evading antitumor immunity than the PD-1/PD-L1 inter- sion in both tumors (Fig. 5; Supplementary Tables S2 and S3). In action in these cancers. accordance with our animal models, PD-L2 expression in Approximately 30% of patients with RCC possess locally immune cells was very low compared with tumor cells. Together advanced or metastatic RCC at diagnosis, and 40% of patients with TCGA dataset analyses, PD-L2 in tumor cells appears to play with localized RCC develop metastasis after primary surgical an important role in evading antitumor immunity in several treatment (37). Therefore, developing effective systemic therapies cancers, including RCC and LUSC. is critical in RCC treatment. Immunotherapeutic agents, such as IL2 and IFNa, have been used to treat advanced RCC with limited success (a 10%–22% response rate; refs. 38, 39). Along with Discussion understanding RCC biology, several molecularly targeted agents, Accumulating evidence shows that the important immunosup- including VEGF and mTOR, have been clinically introduced (40, pressive role of PD-L1 in the TME, while that of PD-L2 has not 41). While VEGF and mTOR inhibitors have provided marked

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 flow cytometry. 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 a week (n ¼ 6 per group). D, Tumor antigen (MuLV p15E)-specific CD8þ T cells in TILs (left). TILs were prepared from MC-38, MC-38-GFP, or MC-38-L2 tumors on day 14, and tumor antigen–specific CD8þ T cells were detected by MuLV p15E/H-2Kb tetramers. Summary of the MuLV p15E/H-2Kb tetramerþCD8þ T-cell frequencies (n ¼ 6 per group; right). E, Effector CD8þ T cells detected by CD44þCD62LCD8þ T cells, PD-1þCD8þ T cells, and cytokine-producing CD8þ T cells in TILs. TILs were prepared from each tumor as in C,and þ þ þ þ þ þ þ CD44 CD62L CD8 TILs, PD-1 CD8 TILs, and TNFa IFNg CD8 TILs were examined (n ¼ 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.

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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 (n ¼ 8 per group). CT26-NY-ESO-1-L2 cells (1.0 106) were injected subcutaneously on day 0, and ICB as indicated were administered on days 7, 10, and 13 (right). Tumor growth was monitored twice a week (n ¼ 5 per group). B, Tumor antigen (MuLV p15E)-specific CD8þ T cells in TILs (left). TILs were prepared from MC-38-L2 tumors treated with the indicated mAb on day 14, and tumor antigen–specific CD8þ T cells were detected by MuLV p15E/H-2Kb tetramers. Summary of the MuLV p15E/H-2Kb tetramerþCD8þ T-cell frequencies (n ¼ 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 growth was monitored twice a week (n ¼ 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, and ICB treatments as indicated were started on days 3, 6, and 9. Tumor growth was monitored twice a week (n ¼ 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.

clinical benefits in advanced RCC, some patients are inherently worldwide (43). Among NSCLCs, several molecular-targeted resistant to these therapies, and most patients acquire resistance to therapies have provided significant clinical benefits in lung ade- them (42). Lung cancers, in which approximately 80% are clas- nocarcinoma (44). However, systemic therapeutic options against sified as NSCLC, are leading causes of cancer-related mortality LUSC remain limited, resulting in poor prognoses compared with

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PD-L2 Is Important for Evading Antitumor Immunity

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, The scattergram and real numbers of the Pearson correlations between expression levels (RPKM) of CD274 or PDCD1LG2 and immune-related genes in RCC from TCGA datasets. Critical immune-related gene expression, including CD8A, GZMA, PRF1,andPDCD1, are shown in red. B, Heatmaps of correlation coefﬁcients between CD274, PDCD1LG2,orPDCD1 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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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 of RCC (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 Tcells and 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.

lung adenocarcinoma (44). Thus, more effective systemic thera- biomarker of PD-1 signal blockade in RCC or LUSC (4, 14). pies against these cancers are necessary, and PD-1 signal blockade Furthermore, in recent clinical trials, anti-PD-L1 mAb monother- treatment, which has been approved to treat these cancer types (4, apy was ineffective against RCC (45) and seemed less valuable 14), is thought to play a major role as a key therapeutic strategy. against LUSC compared with lung adenocarcinoma (28). These Yet, as the clinical efficacy is unsatisfactory, predictive biomarkers findings are consistent with our current findings showing that not are urgently needed. PD-L1 expression evaluated by IHC, which is only PD-1/PD-L1 but also PD-1/PD-L2 interaction could be used as a biomarker of PD-1 signal blockade, is not a predictive important in evading antitumor immunity in RCC and LUSC.

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PD-L2 Is Important for Evading Antitumor Immunity

While PD-L2 expression was initially thought to be restricted in proper use of PD-1 signal blockade treatment with reflective APCs, such as dendritic cells (6), that was negligible in our in vivo PD-L2 and PD-L1 expression in addition to minimizing irAEs is mouse models in contrast to PD-L1. In addition, we showed that an important issue for optimal cancer immunotherapy and our PD-L2 expression in several murine cancer cell lines was kept in low findings help clinicians to select optimal immunotherapy. level even with any stimulants, which can explain no efficacy of anti-PD-L2 mAb in sharp contrast to anti-PD-L1 mAb in our animal Disclosure of Potential Conflicts of Interest models. Thus, little attention has been paid to PD-L2 expression. It Y. Togashi reports receiving other commercial research support from Ono has been shown that PD-L2 is expressed by tumor cells similar to Pharmaceutical, Bristol-Myers Squibb, and AstraZeneca, and reports receiving our current human clinical sample study (20, 25), suggesting a speakers bureau honoraria from Ono Pharmaceutical and Chugai. K. Azuma discrepancy in PD-L2 expression between mouse models and reports receiving speakers bureau honoraria from Ono Pharmaceutical, – Bristol-Myers Squibb, AstraZeneca, and Chugai. M. Eto reports receiving com- humans. We therefore generate genetically engineered PD-L2 mercial research grants from Ono Pharmaceutical, Takeda, Pfizer, Astellas, expressing murine cancer cell lines harboring the comparable level and Kissei, and reports receiving speakers bureau honoraria from Ono of PD-L2 expression with endogenous PD-L1. The PD-L2–over- Pharmaceutical, Bristol-Myers Squibb, Pfizer, Novartis, Bayer, and Takeda. expressing tumors grew faster than the controls regardless of PD-L1 H. Nishikawa reports receiving speakers bureau honoraria and other commer- expression, although this was not observed in immunocompro- cial research support from Ono Pharmaceutical, Bristol-Myers Squibb, and fl mised mice, clearly indicating that PD-L2 expressed by tumor cells Chugai. No potential con icts of interest were disclosed by the other authors. can be involved in evading antitumor immunity. Accordingly, PD- L2–expressing tumors were resistant to anti-PD-L1 mAb alone, Authors' Contributions which was overcome bycombined treatment withanti-PD-L2 mAb, Conception and design: Y. Togashi, M. Eto, H. Nishikawa thus highlighting that more attention should be paid to PD-L2 Development of methodology: T. Tanegashima, Y. Togashi Acquisition of data (provided animals, acquired and managed patients, expression in clinical settings. However, why some cancers, includ- provided facilities, etc.): T. Tanegashima, K. Azuma, A. Kawahara, ing RCC and LUSC, exhibit PD-L2 expression rather than PD-L1 K. Ideguchi, D. Sugiyama, F. Kinoshita, J. Akiba, A. Takeuchi, K. Tatsugami, expression is unclear. A recent study revealed a difference between T. Hoshino, M. Eto the regulations of PD-L1 and PD-L2 expression (46). It was reported Analysis and interpretation of data (e.g., statistical analysis, biostatistics, that the IFNg–JAK1/JAK2–STAT1/STAT2/STAT3–IRF1 axis primar- computational analysis): T. Tanegashima, Y. Togashi, K. Azuma, A. Kawahara, ily regulated PD-L1 expression with IRF1 binding to its promoter. F. Kinoshita, J. Akiba, A. Takeuchi, T. Irie, K. Tatsugami, T. Hoshino, M. Eto, H. Nishikawa On the other hand, PD-L2 responded equally to IFNb and g and is Writing, review, and/or revision of the manuscript: T. Tanegashima, regulated through both IRF1 and STAT3, which bind to the PD-L2 Y. Togashi, H. Nishikawa promoter (46). In addition, genetic and/or epigenetic abnormal- Administrative, technical, or material support (i.e., reporting or organizing ities may inhibit PD-L1 expression, and PD-L2 compensates the data, constructing databases): E. Kashiwagi immunosuppressive role of PD-L1, or genetic and/or epigenetic Study supervision: H. Nishikawa abnormalities in the PD-L2 gene induce a more dominant expression than those of PD-L1 (47, 48). Acknowledgments In conclusion, we demonstrated that PD-L2 expression in The authors thank Ms. Tomoka Takaku, Miyuki Nakai, Konomi Onagawa, tumor cells appears to be strongly correlated with antitumor Megumi Takemura, Chie Haijima, Megumi Hoshino, Kumiko Yoshida, Eriko Gunshima, and Noriko Hakoda for their technical assistance. This study was immune responses in the TME especially in RCC and LUSC. supported by Grants-in-Aid for Scientific Research (S grant number 17H06162, Although little attention has been paid to PD-L2 expression in to H. Nishikawa; Challenging Exploratory Research grant number 16K15551, to animal models and clinical settings, mainly due to the lack of H. Nishikawa; Young Scientists number 17J09900, to Y. Togashi; and JSPS Research appropriate cell lines and examination methods for PD-L2 expres- Fellowship number 17K18388, to Y. Togashi) from the Ministry of Education, sion, respectively. PD-L2 clearly contributes to evading antitumor Culture, Sports, Science and Technology of Japan; the Project for Cancer Research, immunity, suggesting that the PD-1/PD-L2 interaction should be the Therapeutic Evolution (P-CREATE, number 16cm0106301h0002, to H. Nishi- – kawa) from the Japan Agency for Medical Research and Development; the National blocked when treating PD-L2 expressing tumors. Currently, both Cancer Center Research and Development Fund (number 28-A-7, to H. Nishi- anti-PD-1 mAb and anti-PD-L1 mAb are clinically available, kawa); the Naito Foundation (to Y. Togashi and H. Nishiskawa); the Takeda although the differences between these reagents are unclear. Foundation (to Y. Togashi); the Kobayashi Foundation for Cancer Research (to Y. Anti-PD-1 mAb may be clinically effective against broader types Togashi); the Novartis Research Grant (to Y. Togashi); the Bristol-Myers Squibb of cancer expressing either PD-L1 or PD-L2. Yet, as it is still an Research Grant (to Y. Togashi); theSGHFoundation (to Y. Togashi);andMitsui Life open question that PD-L1 may have other receptors, it is difficult Social Welfare Foundation (to M. Eto). to conclude that anti-PD-1 mAb is more useful in any types of The costs of publication of this article were defrayed in part by the payment of cancer (49). Furthermore, because anti-PD-L1 mAb does not page charges. This article must therefore be hereby marked advertisement in inhibit PD-L2, the frequencies of immune-related adverse events accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (irAE), such as endocrine system disorders and pneumonitis, are reported to be lower in patients treated with anti-PD-L1 mAb than Received December 6, 2018; revised April 8, 2019; accepted May 7, 2019; in those treated with anti-PD-1 mAb (50). Thus, considering the published first May 10, 2019.

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Immune Suppression by PD-L2 against Spontaneous and Treatment-Related Antitumor Immunity

Tokiyoshi Tanegashima, Yosuke Togashi, Koichi Azuma, et al.

Clin Cancer Res Published OnlineFirst May 10, 2019.

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