Purpose:

To evaluate the detailed immunosuppressive role(s) of PD-L2 given that its detailed role(s) remains unclear in PD-1 signal blockade therapy in animal models and humans.

Experimental Design:

We generated mouse cell lines harboring various status of PD-L1/PD-L2 and evaluated the tumor growth and phenotypes of tumor-infiltrated lymphocytes using several PD-1 signal blockades in animal models. In humans, the correlation between immune-related gene expression and CD274 (encoding PD-L1) or PDCD1LG2 (encoding PD-L2) was investigated using The Cancer Genome Atlas (TCGA) datasets. In addition, PD-L1 or PD-L2 expression in tumor cells and CD8+ T-cell infiltration were assessed by IHC.

Results:

In animal models, we showed that PD-L2 expression alone or simultaneously expressed with PD-L1 in tumor cells significantly suppressed antitumor immune responses, such as tumor antigen–specific CD8+ T cells, and was involved in the resistance to treatment with anti-PD-L1 mAb alone. This resistance was overcome by anti-PD-1 mAb or combined treatment with anti-PD-L2 mAb. In clinical settings, antitumor immune responses were significantly correlated with PD-L2 expression in the tumor microenvironment in renal cell carcinoma (RCC) and lung squamous cell carcinoma (LUSC).

Conclusions:

We propose that PD-L2 as well as PD-L1 play important roles in evading antitumor immunity, suggesting that PD-1/PD-L2 blockade must be considered for optimal immunotherapy in PD-L2–expressing cancers, such as RCC and LUSC.

Although PD-L1, a ligand of PD-1, has mainly been studied as a therapeutic target and predictive biomarker in PD-1 blockade therapy, the detailed role(s) of PD-L2, another PD-1 ligand, remains unclear. Using preclinical animal models, we showed that PD-L2 expression alone or simultaneously expressed with PD-L1 by tumor cells significantly suppressed antitumor immune responses, such as tumor antigen–specific CD8+ T cells, and was involved in the resistance to treatment with anti-PD-L1 mAb. This resistance was overcome by anti-PD-1 mAb or combined treatment with anti-PD-L2 mAb. In clinical settings, antitumor immune responses were significantly correlated with PD-L2 expression in the tumor microenvironment in renal cell carcinoma (RCC) and lung squamous cell carcinoma (LUSC). We propose that PD-L2 plays an important role in evading antitumor immunity as well as PD-L1, suggesting that PD-1/PD-L2 blockade must be considered for optimal immunotherapy in PD-L2–expressing cancers, such as RCC and LUSC.

Immune checkpoint blockade (ICB), including PD-1 blockade, has been approved to treat various cancers, such as malignant melanoma, non–small cell lung cancer (NSCLC), and renal cell carcinoma (RCC), leading to a paradigm shift in cancer therapy (1–4). However, as the clinical efficacy is limited, more effective therapies and predictive biomarkers stratifying responders from nonresponders are urgently needed. Tumors employ the PD-1 pathway to evade antitumor immunity, particularly CD8+ T cells against tumor antigens (5, 6). PD-1 is mainly expressed by activated T cells and binds to its ligands, PD-L1 and PD-L2 (5, 6), resulting in immunosuppression. PD-L1 is expressed by both antigen-presenting cells (APC) and tumor cells (7, 8). PD-1 signal blockade unleashes antitumor T-cell 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 studies revealed favorable clinical courses using treatment with anti-PD-1/PD-L1 mAbs in patients harboring PD-L1–expressing tumors (12, 13). However, some patients harboring PD-L1–expressing tumors do not respond to PD-1 signal blockade treatment. In addition, patients with PD-L1–negative tumors occasionally experience clinical efficacy (4, 12–14). In large phase III trials, the clinical benefit from anti-PD-1 mAbs appears to be independent of PD-L1 expression in some cancers, such as RCC and lung squamous cell carcinoma (LUSC; refs. 4, 14–16).

The expression of another PD-1 ligand, PD-L2, was initially thought to be restricted in APCs (6). Recently, several studies showed that PD-L2 is expressed by both various immune cells and tumor cells, depending on microenvironmental stimuli (17–19). PD-L2 expression in tumor cells is a predictive factor for the clinical efficacy of anti-PD-1 mAb in some studies (20). In this study, we evaluated the detailed immunosuppressive role(s) of PD-L2 in animal models and humans. The Cancer Genome Atlas (TCGA) datasets revealed a strong correlation between decreased antitumor immune responses and PD-L2 rather than PD-L1 in the tumor microenvironment (TME) in several cancers, including RCC and LUSC, which was confirmed by our clinical samples. Therefore, we propose that PD-L2 should be considered as crucial immunosuppressive mechanisms in clinical settings in addition to PD-L1.

Patients

Twenty-nine patients with clear-cell and non–clear-cell RCC who underwent surgical resection at Kyushu University Hospital (Fukuoka, Japan) from September 2017 to May 2018 were enrolled in this study. Twenty-seven patients with LUSC who underwent surgical resection at Kurume University Hospital from January 2008 to December 2012 were enrolled in this study. The patients' clinical information was obtained from their medical records. All patients provided written informed consent before undergoing the study procedures. The clinical protocol for this study was approved by the appropriate institutional review boards and ethics committees at Kyushu University Hospital and Kurume University Hospital (Kurume, Japan). This study was conducted in accordance with the Declaration of Helsinki.

IHC

Sections were immunostained using the Ventana Benchmark XT Automated Staining System (Ventana Medical System) according to the manufacturer's protocol. Tumor PD-L1 and PD-L2 membrane expression was assessed; ≥1% was defined as positive and <1% as negative (4, 12, 14). The intertumoral CD8+ T cells were counted. Specifically, five areas [0.5 × 0.5 mm (0.25 mm2) fields] with the most abundant distribution were selected and counted in each case.

Cell lines and reagents

B16-F10 (mouse melanoma), MC-38 (mouse colon carcinoma), CT26 (mouse colon carcinoma) expressing NY-ESO-1 (CT26-NY-ESO-1), Renca (mouse kidney adenocarcinoma), CMS5a (mouse fibrosarcoma), and A20 (mouse reticulum cell sarcoma) cell lines were maintained in RPMI medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FCS (Biosera). CT26-NY-ESO-1 is a cell line derived from CT26 stably transfected with NY-ESO-1 (21). CT26, Renca, B16-F10, and A20 cell lines were obtained from the ATCC. MC-38 cell line was obtained from Kerafast. CMS5a cell line was provided by the late Dr. Lloyd J. Old, Memorial Sloan Kettering Cancer Center (New York, NY; ref. 22). All cell lines were used after confirming Mycoplasma (−) by Mycoplasma testing with a PCR Mycoplasma Detection Kit (Takara Bio) according to the manufacturer's instructions. Murine IFNγ, IL2, IL4, TNFα, and granulocyte macrophage colony stimulating factor (GM-CSF) were obtained from PeproTech. Murine IFNα, IFNβ, and tumor growth factor-β1 were obtained 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 study were obtained from BioLegend. Anti-CD4 (GK1.5) mAb and anti-CD8β (Lyt 3.2) mAb were obtained from BioXCell.

Constructs, viral production, and transfection

Mouse PDCD1LG2 cDNA was subcloned into pMXs-IRES-GFP, which was transfected into the packaged cell line using Lipofectamine 3000 Reagent (Thermo Fisher Scientific). After 48 hours of retroviral production, the supernatant was concentrated and dissolved in water. Subsequently, the viral lysate was transfected with MC-38 and CT26-NY-ESO-1, and the PD-L2–expressing cell lines were named MC-38-L2 and CT26-NY-ESO-1-L2, respectively. 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 system. Briefly, guide RNA (gRNA)-targeting mouse CD274 (5′-GCTTGCGTTAGTGGTGTACT-3′) was made using the GeneArt Precision gRNA Synthesis Kit (Thermo Fisher Scientific). The Cas9 protein (Thermo Fisher Scientific) and gRNA were electroporated into MC-38 cells using the Neon Transfection System (Thermo Fisher Scientific) per the manufacturer's instructions. The knockout cell line was named MC-38-L1KO. Either GFP or PD-L2 was introduced into the MC-38-L1KO in the same manner, and these cell lines were named MC-38-L1KO-GFP and MC-38-L1KO-L2, respectively.

In vivo mouse studies

Female 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 tumor size was monitored twice weekly. The mean of the long and short diameters was applied for the tumor growth curves. Mice were grouped when tumor volume reached approximately 100 mm3, and anti-PD-1/PD-L1/PD-L2 mAbs were administered intraperitoneally three times every 3 days thereafter. For CD4+ T-cell or CD8+ T-cell depletion, anti-CD4 mAb (100 μg/mouse) or anti-CD8β mAb (100 μg/mouse) was administered intraperitoneally on days −1, 0, and every 7 days after tumor injection. Tumors were harvested 14 days after tumor injection, and tumor-infiltrating lymphocytes (TIL) were analyzed with flow cytometry. All mouse experiments were approved by the Animals Committee for Animal Experimentation of the National Cancer Center Japan. All experiments met the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Flow cytometry

Cells were washed using PBS with 2% FBS and stained with mAbs specific for CD4, CD8, CD44, CD62L, PD-1, PD-L1, and PD-L2 as well as fixable viability dye (Thermo Fisher Scientific). To examine antigen-specific CD8+ T cells, T-Select H-2Kb MuLV p15E Tetramer-KSPWFTTL-APC (MBL) was used per the manufacturer's instructions. After tetramer staining, cells were stained for cell surface markers (mAbs specific for CD4, CD8, and fixable viability dye). When intracellular cytokines were analyzed, cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate (PMA; 100 ng/mL)/ionomycin (2 μg/mL; Sigma Aldrich). GolgiPlug Reagent (1.3 μL/mL; BD Biosciences) was added for the last 4 hours of the culture. These cells were stained for surface markers and then stained for intracellular cytokines (mAbs specific for TNFα and IFNγ). After washing, the cells were analyzed with an LSR Fortessa Instrument (BD Biosciences) and FlowJo Software (Tree Star).

Antibodies

Violet 500–conjugated anti-CD8 (53-6.7) mAb, Brilliant Violet 786 (BV786)–conjugated anti-CD4 (RM4-5) mAb, phycoerythrin (PE)-and Cy7-conjugated anti-CD44 (IM7) mAb, BV421-conjugated anti-PD-L2 (TY25) mAb, and BV605-conjugated anti-CD11c (HL3) were purchased from BD Biosciences. BV421-conjugated anti-PD-1 (29F.1A12) mAb, peridinin chlorophyll protein complex, Cy5.5-conjugated anti-CD62L (MEL-14) mAb, BV421-conjugated anti-TNF-α (MP6-XT22) mAb, PE-conjugated anti-PD-L1 (10F.9G2) mAb, and AF488-conjugated anti-MHC class II (M5/114.15.2) were purchased from BioLegend. FITC-conjugated anti-IFNγ (XMG1.2) mAb were obtained from eBioscience. PE-conjugated anti-CD11b (M1/70) mAb were purchased from Thermo Fisher Scientific. The antibodies used for IHC were PD-L1 (E1L3N, Cell Signaling Technology), PD-L2 (#176611, R&D Systems, Inc.), and CD8 (4B11, Leica Biosystems).

Real-time qRT-PCR

RNA was extracted using RNeasy Mini Kit (Qiagen). cDNA was generated using SuperScript VILO (Thermo Fisher Scientific), and real-time PCR reactions were performed with SYBR Green Reagents (Thermo Fisher Scientific) using the QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). Gene expression changes relative to 18S ribosomal RNA as a housekeeping gene were calculated using the DDCT method. A forward primer (5′-AGCCTGCTGTCACTTGCTAC-3′) and reverse primer (5′-TCCCAGTACACCACTAACGC-3′) were used to detect CD274. A forward primer (5′-CCTCAGCCTAGCAGAAACTTCA-3′) and reverse primer (5′-CATCCGACTCAGAGGGTCAATG-3′) were used to detect PDCD1LG2. A forward primer (5′-TAGAGTGTTCAAAGCAGGCCC-3′) and reverse primer (5′-CCAACAAAATAGAACCGCGGT-3′) were used to detect 18S.

Statistical analysis

GraphPad Prism7 (GraphPad Software) was used for statistical analysis. The overall survival was analyzed with Kaplan–Meier method and compared with log-rank test. The relationships between the groups were compared using Fisher exact test or Welch t test. P < 0.05 was considered statistically significant.

PD-L2 plays a limited role in immune suppression in murine tumor models

To elucidate the role of PD-L2 in tumor immunity, we first explored PD-L1 and PD-L2 expression in several murine tumor cell lines, including MC-38, CT26, B16-F10, Renca, CMS5a, and A20. All tumor cell lines possessed variable PD-L1 expression that was enhanced by stimulants, especially IFNα, β, and γ. In contrast, PD-L2 was minimally detected by any cell lines even after stimulation (Fig. 1A; Supplementary Figs. S1 and S2). While PD-L2 is reportedly expressed by APCs (6), low expression in APCs (CD45+MHCII+CD11c+CD11b+ cells) was observed in animal models (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 examined in vivo antitumor effects of anti-PD-1/PD-L1/PD-L2 mAbs using 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 growth, whereas anti-PD-L2 mAb was ineffective against both tumors (Fig. 1B). As neoantigen-specific T cells are reportedly essential for antitumor effects by ICB (24), MuLV p15E (a neoantigen of MC-38–specific CD8+ T cells) detected by MHC/peptide tetramers was examined in an MC-38 model. MuLV p15E tetramer+CD8+ T cells were significantly primed/augmented by treatment with anti-PD-1 mAb or anti-PD-L1 mAb but not with anti-PD-L2 mAb alone (Fig. 1C). Activated CD8+ T cells, which were assessed by the proportion of CD44+CD62L effector/memory CD8+ T cells and the frequency of PD-1+CD8+ T cells, were significantly higher in TILs in mice treated with anti-PD-1 mAb or anti-PD-L1 mAb compared with mice treated with isotype control or anti-PD-L2 mAb (Fig. 1D; Supplementary Fig. S4). In addition, TNFα+IFNγ+CD8+ T cells were significantly higher in mice treated with anti-PD-1 mAb or anti-PD-L1 mAb than in those treated with isotype control or anti-PD-L2 mAb (Fig. 1D; 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.

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 IFNγ. Cell lines were treated with or without IFNγ (1,000 IU/mL) for 24 hours and were subsequently subjected to flow cytometry. Gray, isotype control; blue, untreated; red, IFNγ 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 = 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 = 5 per group). C, Tumor antigen (MuLV p15E)-specific CD8+ T cells in TILs (top). TILs were prepared from MC38 tumors on day 14, and tumor antigen–specific CD8+ T cells were detected by MuLV p15E/H-2Kb tetramers. β-galactosidase/H-2Kb tetramer staining served as the control. Summary of the MuLV p15E/H-2Kb tetramer+CD8+ T-cell frequencies (n = 6 per group; bottom). D, Effector CD8+ T cells detected by CD44+CD62LCD8+ T cells, PD-1+CD8+ T cells, and cytokine-producing CD8+ T cells in TILs. TILs were prepared from MC38 tumors as in C, and CD44+CD62LCD8+ TILs, PD-1+CD8+ TILs, and TNFα+IFNγ+CD8+ TILs were examined (n = 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.

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 IFNγ. Cell lines were treated with or without IFNγ (1,000 IU/mL) for 24 hours and were subsequently subjected to flow cytometry. Gray, isotype control; blue, untreated; red, IFNγ 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 = 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 = 5 per group). C, Tumor antigen (MuLV p15E)-specific CD8+ T cells in TILs (top). TILs were prepared from MC38 tumors on day 14, and tumor antigen–specific CD8+ T cells were detected by MuLV p15E/H-2Kb tetramers. β-galactosidase/H-2Kb tetramer staining served as the control. Summary of the MuLV p15E/H-2Kb tetramer+CD8+ T-cell frequencies (n = 6 per group; bottom). D, Effector CD8+ T cells detected by CD44+CD62LCD8+ T cells, PD-1+CD8+ T cells, and cytokine-producing CD8+ T cells in TILs. TILs were prepared from MC38 tumors as in C, and CD44+CD62LCD8+ TILs, PD-1+CD8+ TILs, and TNFα+IFNγ+CD8+ TILs were examined (n = 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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PD-L2 expression is a mechanism for evading antitumor immunity

Because PD-L2 was minimally expressed by the murine tumor cell lines that are frequently used in preclinical studies for PD-1 signal blockade although some human tumor cells highly express PD-L2 (20, 25), we hypothesized that the lesser appreciation of PD-L2 in PD-1 signal blockade treatment may solely reflect the lack of appropriate animal models using PD-L2–expressing tumor cell lines. The PD-L2–overexpressing cell lines MC-38 (MC-38-L2) and CT26-NY-ESO-1 (CT26-NY-ESO-1-L2) were thus developed to evaluate influence of PD-L2 on PD-1 signal blockade efficacies using anti-PD-1 mAb, anti-PD-L1 mAb, and anti-PD-L2 mAb. PD-L2 expression was confirmed 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 of endogenous PD-L1 expression after IFNγ treatment (Fig. 2A and B). PD-L2–expressing tumors grew rapidly compared with those in control mice injected with parental tumors or GFP-expressing tumors (Fig. 2C) although these tumors exhibited comparable tumor growth in immunocompromised mice (Supplementary Fig. S5A). Furthermore, to elucidate the role of CD4+ T cells and CD8+ T cells, the tumor growth of MC-38/-GFP/-L2 and CT-26-NY-ESO-1/-GFP/-L2 was evaluated in CD4+ T-cell– or CD8+ T-cell–depleted mice. In CD8+ T-cell–depleted mice, the rapid tumor growth in PD-L2–expressing tumors was totally abrogated although PD-L2–expressing tumors grew rapidly compared with the parental tumors or GFP-expressing tumors in CD4+ T-cell–depleted mice (Supplementary Fig. S5B and 5C), indicating the importance of CD8+ T-cell suppression by PD-L2. Accordingly, in the MC-38-L2 tumors, MuLV p15E tetramer+ CD8+ T cells were reduced compared with those of the control mice (Fig. 2D). In addition, activated CD8+ T cells (CD44+CD62L effector/memory CD8+ T cells and PD-1+CD8+ T cells) and TNFα+IFNγ+CD8+ T cells in TILs were significantly lower in MC-38-L2 tumors than in the control parental tumors or GFP-expressing tumors (Fig. 2E; Supplementary Fig. S6). Taken together, PD-L2 expressed by tumor cells functions as an immunosuppressive molecule against antitumor immunity, particularly CD8+ T cells.

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 IFNγ (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, IFNγ 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 = 6 per group). D, Tumor antigen (MuLV p15E)-specific CD8+ 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 CD8+ T cells were detected by MuLV p15E/H-2Kb tetramers. Summary of the MuLV p15E/H-2Kb tetramer+CD8+ T-cell frequencies (n = 6 per group; right). E, Effector CD8+ T cells detected by CD44+CD62LCD8+ T cells, PD-1+CD8+ T cells, and cytokine-producing CD8+ T cells in TILs. TILs were prepared from each tumor as in C, and CD44+CD62LCD8+ TILs, PD-1+CD8+ TILs, and TNFα+IFNγ+CD8+ TILs were examined (n = 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.

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 IFNγ (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, IFNγ 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 = 6 per group). D, Tumor antigen (MuLV p15E)-specific CD8+ 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 CD8+ T cells were detected by MuLV p15E/H-2Kb tetramers. Summary of the MuLV p15E/H-2Kb tetramer+CD8+ T-cell frequencies (n = 6 per group; right). E, Effector CD8+ T cells detected by CD44+CD62LCD8+ T cells, PD-1+CD8+ T cells, and cytokine-producing CD8+ T cells in TILs. TILs were prepared from each tumor as in C, and CD44+CD62LCD8+ TILs, PD-1+CD8+ TILs, and TNFα+IFNγ+CD8+ TILs were examined (n = 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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PD-L2 blockade is necessary for controlling PD-L2–expressing tumors

We next addressed how PD-L2 influenced the antitumor effects of anti-PD-1 mAb, anti-PD-L1 mAb, and anti-PD-L2 mAb. Anti-PD-1 mAb significantly inhibited PD-L2–expressing tumor growth compared with anti-PD-L1 mAb or anti-PD-L2 mAb alone. The combination of anti-PD-L1 mAb and anti-PD-L2 mAb showed 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 additional antitumor effects against PD-L2–expressing tumors when combined with anti-PD-L2 mAb, indicating that anti-PD-1 mAb alone can inhibit PD-1/PD-L2 interaction sufficiently to reinvigorate an effective antitumor immunity. (Supplementary Fig. S7). The tumor growth inhibition by these antibodies including anti-PD-L2 mAb was totally abrogated in immunocompromised mice or CD8+ T-cell–depleted mice although they exhibited antitumor effects against PD-L2–expressing tumors in CD4+ T-cell–depleted mice as well as in control mice (Supplementary Fig. S8A), indicating that the antitumor efficacy against PD-L2–expressing tumors was attributed for unleashing CD8+ T cells. In addition, anti-PD-L2 mAb did not exhibit antitumor immunity against parental tumors or GFP-expressing tumors in immunocompromised mice (Supplementary Fig. S8B). Thus, the antitumor efficacy of anti-PD-L2 mAb against PD-L2–expressing tumors was dependent on the reactivation of CD8+ T cells, but not caused by a direct effect of PD-L2 blockade against PD-L2–expressing tumors. Accordingly, MuLV p15E tetramer+CD8+ T cells were significantly primed/augmented by anti-PD-1 mAb or the combination of anti-PD-L1 mAb and anti-PD-L2 mAb compared with anti-PD-L1 mAb or anti-PD-L2 mAb alone in MC-38-L2 tumor–bearing mice (Fig. 3B). Activated CD8+ T cells (CD44+CD62L effector/memory CD8+ T cells and PD-1+CD8+ T cells) and TNFα+IFNγ+CD8+ T cells in TILs were also significantly higher in mice treated with the anti-PD-1 mAb or the combination of anti-PD-L1 mAb and anti-PD-L2 mAb compared with untreated mice or mice treated either by anti-PD-L1 mAb or by anti-PD-L2 mAb alone (Supplementary Fig. S9). Therefore, PD-L2 endows resistance to anti-PD-L1 mAb monotherapy, which can be overcome by combined treatment with anti-PD-L1 mAb and anti-PD-L2 mAb.

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 = 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 = 5 per group). B, Tumor antigen (MuLV p15E)-specific CD8+ 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 CD8+ T cells were detected by MuLV p15E/H-2Kb tetramers. Summary of the MuLV p15E/H-2Kb tetramer+CD8+ T-cell frequencies (n = 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 = 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 = 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 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 = 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 = 5 per group). B, Tumor antigen (MuLV p15E)-specific CD8+ 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 CD8+ T cells were detected by MuLV p15E/H-2Kb tetramers. Summary of the MuLV p15E/H-2Kb tetramer+CD8+ T-cell frequencies (n = 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 = 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 = 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.

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To further confirm the role of PD-L2, a PD-L1–knockout MC-38 cell line (MC-38-L1KO) was generated using a CRISPR/Cas9 system, and the lack of PD-L1 protein expression was confirmed with flow cytometry (Supplementary Fig. S10). Either PD-L2 or GFP was then expressed in MC-38-L1KO cells (MC-38-L1KO-L2 and MC-38-L1KO-GFP, respectively; Supplementary Fig. S10). MC-38-L1KO tumors grew more slowly than the parental MC-38 tumors, whereas PD-L2 expression allowed MC-38-L1KO-L2 tumors to grow comparably with the parental MC-38 tumors (Fig. 3C). In contrast, these tumors grew similarly in immunocompromised mice (Supplementary Fig. S10). Anti-PD-1 mAb and anti-PD-L2 mAb significantly inhibited tumor growth, but anti-PD-L1 mAb did not (Fig. 3D). Thus, PD-L2 alone sufficiently hampers antitumor immunity, allowing rapid tumor growth.

PD-L2 expression is associated with antitumor immune responses in various cancers

Because PD-L2 significantly suppressed antitumor immunity in animal models, we investigated the correlation between immune-related gene expression and CD274 (encoding PD-L1) or PDCD1LG2 (encoding PD-L2) in humans using TCGA datasets. The expression levels of 43 genes were defined for the immune cell types, CD8+ 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 the efficacy of anti-PD-1 mAb or anti-PD-L1 mAb has been demonstrated in these cancers, and the published datasets were available (4, 12, 14–16, 27–30). The correlation coefficient between the expression level of each gene and that of CD274 or PDCD1LG2 was calculated (Fig. 4A; Supplementary Fig. S11). Especially in RCC, PDCD1LG2 expression rather than CD274 expression was significantly positively correlated with immune-related gene expression (Fig. 4A). We further evaluated the correlation between CD274, PDCD1LG2, or PDCD1 expression and the INTERFERON_GAMMA_RESPONSE gene signature (Fig. 4B; ref. 31). The heatmap highlights the quantitative differences in association between PD-1–related molecules and immune responses in the TME across tumor types. In RCC and LUSC, PDCD1LG2 expression was strongly correlated with IFNγ-related gene expression, although CD274 expression was less significantly correlated. These data suggest that PD-L2 expression in RCC and LUSC strongly controls antitumor immune responses in the TME.

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, and PDCD1, are shown in red. B, Heatmaps of correlation coefficients between 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.

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, and PDCD1, are shown in red. B, Heatmaps of correlation coefficients between 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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We next examined the relationship between CD274 or PDCD1LG2 expression and survival from RCC and LUSC TCGA datasets. In RCC, no correlation between CD274 or PDCD1LG2 expressions and survival was observed. On the other hand, high expression of CD274 or PDCD1LG2 equally correlated with a better survival in LUSC, indicating the possible correlation between antitumor immune responses and a favorable prognosis (Supplementary Fig. S12).

CD8+ T-cell infiltration is accompanied by PD-L2 expression in RCC and LUSC

Because the correlation between immune-related genes and CD274 expression was relatively weak in RCC and LUSC, the expression of PD-L1 or PD-L2 in tumor cells and the number of infiltrated CD8+ T cells were assessed by IHC in RCC (n = 29) and LUSC (n = 27) cohorts to address a role of PD-L2 expression for the suppression against antitumor immunity, particularly CD8+ T cells. 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. Correlations between PD-L1 or PD-L2 expression and clinicopathologic features of RCC and LUSC are summarized in Supplementary Tables S2 and S3, respectively. In RCC, age, sex, Fuhrman nuclear grade, pathological stage (pStage), and histology did not correlate with PD-L1 or PD-L2 expression (Supplementary Table S2). In LUSC, age, pStage, smoking status, and driver gene status did not correlate with PD-L1 or -L2 expression (Supplementary Table S3). In RCC, CD8+ 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, CD8+ T cells showed higher infiltration into PD-L2–expressing tumors, such as LUSC_21, than PD-L2–negative tumors, such as LUSC_27. In contrast, CD8+ T-cell infiltration was unrelated to PD-L1 expression in both tumors (Fig. 5; Supplementary Tables S2 and S3). In accordance with our animal models, PD-L2 expression in immune cells was very low compared with tumor cells. Together with TCGA dataset analyses, PD-L2 in tumor cells appears to play an important role in evading antitumor immunity in several cancers, including RCC and LUSC.

Figure 5.

PD-L2 expression is accompanied by CD8+ T-cell infiltration in the TMEs of RCC and LUSC. PD-L1, PD-L2 expression, and CD8+ 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 CD8+ T cells 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 μm; N.S., not significant; ***, P < 0.001.

Figure 5.

PD-L2 expression is accompanied by CD8+ T-cell infiltration in the TMEs of RCC and LUSC. PD-L1, PD-L2 expression, and CD8+ 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 CD8+ T cells 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 μm; N.S., not significant; ***, P < 0.001.

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Accumulating evidence shows that the important immunosuppressive role of PD-L1 in the TME, while that of PD-L2 has not been fully elucidated. Using animal models, we showed that PD-L2 played an important role in evading antitumor immunity. In addition, we revealed that PD-L2 was dominantly correlated with antitumor immune responses in the TME rather than PD-L1 in RCC and LUSC using TCGA datasets and clinical samples. In preclinical animal models, anti-PD-1 mAb was effective against the PD-L2–expressing tumors, whereas PD-L2–expressing tumors were resistant to anti-PD-L1 mAb alone. This limitation of anti-PD-L1 mAb was overcome by combined treatment with anti-PD-L2 mAb. To our knowledge, this is the first report to directly show the importance of PD-L2 as a mechanism for evading antitumor immunity using preclinical animal models and clinical samples.

CD8+ T cells play major roles in antitumor immunity in the TME by directly killing tumor cells (32). PD-L1 expressed by tumor cells inhibits antitumor effects of CD8+ T cells (7). PD-L1 expression in tumor cells is induced by two mechanisms: intrinsic induction by genetic alterations (innate expression) and stimulation by IFNγ released from effector T cells, including CD8+ T cells (acquired expression; ref. 33). In many cancers, tumor cells express PD-L1 through the latter mechanism (acquired expression; ref. 34, 35); that is, PD-L1 expression seems to be a surrogate marker for the presence of antitumor immune responses, such as CD8+ T cell responses in the TME. PD-1 signal blockade unleashes suppressed antitumor T-cell immunity, resulting in tumor regression; thereby, PD-L1 expression becomes a predictive biomarker (12, 36). However, in some cancers, including RCC and LUSC, PD-L1 expression was not accompanied by clinical effectivity of PD-1 signal blockade treatment (4, 13, 14). Consistent with this finding, we found that PDCD1LG2 expression was more strongly correlated with immune-related gene expression than CD274 expression, especially in RCC and LUSC, using TCGA datasets. Our cohort also revealed that PD-L2 expression in tumor cells was correlated with CD8+ T-cell infiltration rather than PD-L1 expression in RCC and LUSC, suggesting that PD-L2 expression can be a more dominant immunosuppressive mechanism than PD-L1 in these cancer types. In addition, our in vivo animal models showed that PD-L2 can be related to resistance to anti-PD-L1 mAb alone, which can be overcome by combined treatment with anti-PD-L2 mAb. Thus, the PD-1/PD-L2 interaction plays a more important role in evading antitumor immunity than the PD-1/PD-L1 interaction in these cancers.

Approximately 30% of patients with RCC possess locally advanced or metastatic RCC at diagnosis, and 40% of patients with localized RCC develop metastasis after primary surgical treatment (37). Therefore, developing effective systemic therapies is critical in RCC treatment. Immunotherapeutic agents, such as IL2 and IFNα, have been used to treat advanced RCC with limited success (a ∼10%–22% response rate; refs. 38, 39). Along with understanding RCC biology, several molecularly targeted agents, including VEGF and mTOR, have been clinically introduced (40, 41). While VEGF and mTOR inhibitors have provided marked clinical benefits in advanced RCC, some patients are inherently resistant to these therapies, and most patients acquire resistance to them (42). Lung cancers, in which approximately 80% are classified as NSCLC, are leading causes of cancer-related mortality worldwide (43). Among NSCLCs, several molecular-targeted therapies have provided significant clinical benefits in lung adenocarcinoma (44). However, systemic therapeutic options against LUSC remain limited, resulting in poor prognoses compared with lung adenocarcinoma (44). Thus, more effective systemic therapies against these cancers are necessary, and PD-1 signal blockade treatment, which has been approved to treat these cancer types (4, 14), is thought to play a major role as a key therapeutic strategy. Yet, as the clinical efficacy is unsatisfactory, predictive biomarkers are urgently needed. PD-L1 expression evaluated by IHC, which is used as a biomarker of PD-1 signal blockade, is not a predictive biomarker of PD-1 signal blockade in RCC or LUSC (4, 14). Furthermore, in recent clinical trials, anti-PD-L1 mAb monotherapy was ineffective against RCC (45) and seemed less valuable against LUSC compared with lung adenocarcinoma (28). These findings are consistent with our current findings showing that not only PD-1/PD-L1 but also PD-1/PD-L2 interaction could be important in evading antitumor immunity in RCC and LUSC.

While PD-L2 expression was initially thought to be restricted in APCs, such as dendritic cells (6), that was negligible in our in vivo mouse models in contrast to PD-L1. In addition, we showed that PD-L2 expression in several murine cancer cell lines was kept in low 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 models. Thus, little attention has been paid to PD-L2 expression. It has been shown that PD-L2 is expressed by tumor cells similar to our current human clinical sample study (20, 25), suggesting a discrepancy in PD-L2 expression between mouse models and humans. We therefore generate genetically engineered PD-L2–expressing murine cancer cell lines harboring the comparable level of PD-L2 expression with endogenous PD-L1. The PD-L2–overexpressing tumors grew faster than the controls regardless of PD-L1 expression, although this was not observed in immunocompromised mice, clearly indicating that PD-L2 expressed by tumor cells can be involved in evading antitumor immunity. Accordingly, PD-L2–expressing tumors were resistant to anti-PD-L1 mAb alone, which was overcome by combined treatment with anti-PD-L2 mAb, thus highlighting that more attention should be paid to PD-L2 expression in clinical settings. However, why some cancers, including RCC and LUSC, exhibit PD-L2 expression rather than PD-L1 expression is unclear. A recent study revealed a difference between the regulations of PD-L1 and PD-L2 expression (46). It was reported that the IFNγ–JAK1/JAK2–STAT1/STAT2/STAT3–IRF1 axis primarily regulated PD-L1 expression with IRF1 binding to its promoter. On the other hand, PD-L2 responded equally to IFNβ and γ and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter (46). In addition, genetic and/or epigenetic abnormalities may inhibit PD-L1 expression, and PD-L2 compensates the immunosuppressive role of PD-L1, or genetic and/or epigenetic abnormalities in the PD-L2 gene induce a more dominant expression than those of PD-L1 (47, 48).

In conclusion, we demonstrated that PD-L2 expression in tumor cells appears to be strongly correlated with antitumor immune responses in the TME especially in RCC and LUSC. Although little attention has been paid to PD-L2 expression in animal models and clinical settings, mainly due to the lack of appropriate cell lines and examination methods for PD-L2 expression, respectively. PD-L2 clearly contributes to evading antitumor immunity, suggesting that the PD-1/PD-L2 interaction should be blocked when treating PD-L2–expressing tumors. Currently, both anti-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 types of cancer expressing either PD-L1 or PD-L2. Yet, as it is still an open question that PD-L1 may have other receptors, it is difficult to conclude that anti-PD-1 mAb is more useful in any types of cancer (49). Furthermore, because anti-PD-L1 mAb does not inhibit PD-L2, the frequencies of immune-related adverse events (irAE), such as endocrine system disorders and pneumonitis, are reported to be lower in patients treated with anti-PD-L1 mAb than in those treated with anti-PD-1 mAb (50). Thus, considering the proper use of PD-1 signal blockade treatment with reflective PD-L2 and PD-L1 expression in addition to minimizing irAEs is an important issue for optimal cancer immunotherapy and our findings help clinicians to select optimal immunotherapy.

Y. Togashi reports receiving other commercial research support from Ono Pharmaceutical, Bristol-Myers Squibb, and AstraZeneca, and reports receiving speakers bureau honoraria from Ono Pharmaceutical and Chugai. K. Azuma reports receiving speakers bureau honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, AstraZeneca, and Chugai. M. Eto reports receiving commercial research grants from Ono Pharmaceutical, Takeda, Pfizer, Astellas, and Kissei, and reports receiving speakers bureau honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, Pfizer, Novartis, Bayer, and Takeda. H. Nishikawa reports receiving speakers bureau honoraria and other commercial research support from Ono Pharmaceutical, Bristol-Myers Squibb, and Chugai. No potential conflicts of interest were disclosed by the other authors.

Conception and design: Y. Togashi, M. Eto, H. Nishikawa

Development of methodology: T. Tanegashima, Y. Togashi

Acquisition 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. Eto

Analysis 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. Nishikawa

Writing, review, and/or revision of the manuscript: T. Tanegashima, Y. Togashi, H. Nishikawa

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E. Kashiwagi

Study supervision: H. Nishikawa

The authors thank Ms. Tomoka Takaku, Miyuki Nakai, Konomi Onagawa, Megumi Takemura, Chie Haijima, Megumi Hoshino, Kumiko Yoshida, Eriko Gunshima, and Noriko Hakoda for their technical assistance. This study was supported by Grants-in-Aid for Scientific Research (S grant number 17H06162, to H. Nishikawa; Challenging Exploratory Research grant number 16K15551, to H. Nishikawa; Young Scientists number 17J09900, to Y. Togashi; and JSPS Research Fellowship 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. Nishikawa) from the Japan Agency for Medical Research and Development; the National Cancer Center Research and Development Fund (number 28-A-7, to H. Nishikawa); the Naito Foundation (to Y. Togashi and H. Nishiskawa); the Takeda Foundation (to Y. Togashi); the Kobayashi Foundation for Cancer Research (to Y. Togashi); the Novartis Research Grant (to Y. Togashi); the Bristol-Myers Squibb Research Grant (to Y. Togashi); the SGH Foundation (to Y. Togashi); and Mitsui Life Social Welfare Foundation (to M. Eto).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Hodi
FS
,
O'Day
SJ
,
McDermott
DF
,
Weber
RW
,
Sosman
JA
,
Haanen
JB
, et al
Improved survival with ipilimumab in patients with metastatic melanoma
.
N Engl J Med
2010
;
363
:
711
23
.
2.
Topalian
SL
,
Hodi
FS
,
Brahmer
JR
,
Gettinger
SN
,
Smith
DC
,
McDermott
DF
, et al
Safety, activity, and immune correlates of anti-PD-1 antibody in cancer
.
N Engl J Med
2012
;
366
:
2443
54
.
3.
Brahmer
JR
,
Tykodi
SS
,
Chow
LQ
,
Hwu
WJ
,
Topalian
SL
,
Hwu
P
, et al
Safety and 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
.
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 family member leads to negative regulation of lymphocyte activation
.
J Exp Med
2000
;
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 mechanism 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 of B7-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 costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2
.
J Exp Med
2012
;
209
:
1201
17
.
10.
Hui
E
,
Cheung
J
,
Zhu
J
,
Su
X
,
Taylor
MJ
,
Wallweber
HA
, et al
T cell costimulatory receptor CD28 is a primary target for PD-1–mediated inhibition
.
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-cell lung 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 J Med
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
Pembrolizumab 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 in patients with advanced gastric or gastro-oesophageal junction cancer refractory 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 clinical application
.
Int Immunol
2007
;
19
:
813
24
.
18.
Rozali
EN
,
Hato
SV
,
Robinson
BW
,
Lake
RA
,
Lesterhuis
WJ
. 
Programmed death ligand 2 in cancer-induced immune suppression
.
Clin Dev Immunol
2012
;
656340
.
19.
Zhong
X
,
Tumang
JR
,
Gao
W
,
Bai
C
,
Rothstein
TL
. 
PD-L2 expression extends beyond dendritic cells:macrophages to B1 cells enriched for VH11:VH12 and 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-L2 expression 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
Peptide vaccine induces enhanced tumor growth associated with apoptosis induction in CD8+ 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 of mouse sarcoma
.
Proc Natl Acad Sci U S A
1997
;
94
:
6375
9
.
23.
Spranger
S
,
Dai
D
,
Horton
B
,
Gajewski
TF
. 
Tumor-residing Batf3 dendritic cells 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
Clinicopathologic 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 genetic properties of tumors associated with local immune cytolytic activity
.
Cell
2015
;
160
:
48
61
.
27.
Ferris
RL
,
Blumenschein
G
 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 randomised controlled trial
.
Lancet
2017
;
389
:
255
65
.
29.
Cerami
E
,
Gao
J
,
Dogrusoz
U
,
Gross
BE
,
Sumer
SO
,
Aksoy
BA
, et al
The cBio cancer genomics portal: an open platform for exploring multidimensional cancer 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 using the cBioPortal
.
Sci Signal
2013
;
6
:
pl1
.
31.
Liberzon
A
,
Birger
C
,
Thorvaldsdottir
H
,
Ghandi
M
,
Mesirov
JP
,
Tamayo
P
. 
The Molecular Signatures Database (MSigDB) hallmark gene set collection
.
Cell Syst
2015
;
1
:
417
25
.
32.
Chen
DS
,
Mellman
I
. 
Oncology meets immunology: the cancer-immunity cycle
.
Immunity
2013
;
39
:
1
10
.
33.
Topalian
SL
,
Drake
CG
,
Pardoll
DM
. 
Immune checkpoint blockade: a common 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 bladder cancer
.
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 MPDL3280A in 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-cell lung cancer
.
N Engl J Med
2016
;
375
:
1823
33
.
37.
Fisher
R
,
Gore
M
,
Larkin
J
. 
Current and future systemic treatments for renal cell carcinoma
.
Semin Cancer Biol
2013
;
23
:
38
45
.
38.
Unverzagt
S
,
Moldenhauer
I
,
Coppin
C
,
Greco
F
,
Seliger
B
. 
Immunotherapy for metastatic renal cell carcinoma [Protocol]
.
Cochrane Database Syst Rev
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'Immunotherapie
.
N Engl J Med
1998
;
338
:
1272
8
.
40.
Dancey
J
. 
mTOR signaling and drug development in cancer
.
Nat Rev Clin Oncol
2010
;
7
:
209
19
.
41.
Vachhani
P
,
George
S
. 
VEGF inhibitors in renal cell carcinoma
.
Clin Adv Hematol 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 Clin
2018
;
68
:
7
30
.
44.
Herbst
RS
,
Morgensztern
D
,
Boshoff
C
. 
The biology and management of non-small cell lung cancer
.
Nature
2018
;
553
:
446
54
.
45.
McDermott
DF
,
Huseni
MA
,
Atkins
MB
,
Motzer
RJ
,
Rini
BI
,
Escudier
B
, et al
Clinical activity and molecular correlates of response to atezolizumab alone or in combination with bevacizumab versus sunitinib in renal cell carcinoma
.
Nat Med
2018
;
24
:
749
57
.
46.
Garcia-Diaz
A
,
Shin
DS
,
Moreno
BH
,
Saco
J
,
Escuin-Ordinas
H
,
Rodriguez
GA
, 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 3′-UTR disruption in multiple 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-L1 and 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
. 
Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to 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: systematic review and meta-analysis
.
BMJ
2018
;
360
:
k793
.

Supplementary data