CD200R blockade enhances anti-tumor immunity by unleashing NK and CD8⁺ T cells in tumor

Abstract

Immune checkpoint inhibitors have revolutionized cancer therapy, but a large proportion of patients do not respond well to current checkpoint immunotherapies. CD200R (also known as OX2R) is a transmembrane glycoprotein of the immunoglobulin superfamily that is mainly expressed on myeloid and lymphoid-derived immunocompetent cells such as myeloid cells, natural killer (NK), and CD8⁺ T cells. In this study, we investigated the therapeutic potential and cellular mechanisms of targeting CD200R in tumor immunotherapy. We established 4 subcutaneous tumor mouse models using MC38 (colon cancer), MCA205 (fibrosarcoma), LLC (lung cancer), and EO771 (mammary cancer) cell lines. We found that CD200R was highly expressed on tumor-infiltrating NK and CD8⁺ T cells with exhausted phenotypes in the four subcutaneous tumor mouse models. Either genetic ablation or antibody blockade of CD200R retarded tumor growth and prolonged the survival of tumor-bearing mice by preventing or reversing exhaustion of both NK cells and CD8⁺ T cells. The combined therapy of CD200R antibody with anti-PD-1/anti-PD-L1 synergistically inhibited tumor growth. By depletion of NK or/and CD8⁺ T cells, we demonstrated that both cell types contributed to the anti-tumor efficacy of CD200R blockade in tumor-bearing mice. Further, the blockade of human CD200R significantly enhanced human NK cell function and inhibited human tumor growth in PBMC-reconstituted xenograft mice. Our results demonstrate that CD200R is a potential immune checkpoint molecule that can suppress the tumoricidal activities of NK and CD8⁺ T cells, and could thus be exploited as a therapeutic target in the future.

Introduction

Cancer is among the most threatening diseases, and its treatment currently depends on radiotherapy or/and chemotherapy after surgery, where appropriate. Over the last 10 years, immune checkpoint blockade (ICB) has become a highly promising cancer therapy approach due to its target-specificity and relatively fewer side effects. Inhibitory immune receptors are physiologically crucial for self-tolerance and extensively expressed on immune cells to prevent their over-activation, which is referred to as an immune checkpoint. In chronic inflammation (mostly during virus infection) and the tumor microenvironment (TME), immune checkpoint receptors’ expression may be persistently upregulated, leading to functional exhaustion of immune cells and consequent viral or tumor immune escape [1–4]. Targeting immune checkpoint receptors or their ligands can substantially rejuvenate immune cells and represent a breakthrough in cancer therapy [5–9]. Notably, anti-PD-1/PD-L1 therapy has shown encouraging clinical outcomes in patients with certain types of cancer [10–15]. An increasing number of other immune checkpoint inhibitors, such as antibodies targeting TIGIT, LAG3, and TIM-3, are also being developed and entered clinical trials for translation. Despite such progress, a large proportion of patients continue to show minimal to no response to existing checkpoint immunotherapies, raising a critical unmet clinical need to explore new immune checkpoints.

CD200R (also known as OX2R) is a transmembrane glycoprotein of the immunoglobulin superfamily (IgSF) that is mainly expressed on myeloid and lymphoid-derived immunocompetent cells, such as myeloid cells, natural killer (NK), and CD8⁺ T cells [16]. Unlike most of the IgSF receptors, CD200R lacks an immunoreceptor tyrosine-based inhibitory motif (ITIM) [17]; however, its cytoplasmic domain contains three tyrosine residues, and the third of which is phosphorylated on binding of CD200R to its ligand, CD200 [18]; this results in the recruitment and phosphorylation of Dok-1 and -2, which then bind to RasGAP and SHIP [18–20]. In clinical studies, CD200 expression was associated with poor prognosis in patients with multiple myeloma, acute myeloid leukemia (AML), B cell chronic leukemia, melanoma, and lymphoid malignancies [21–23]. CD200 may directly trigger CD200R inhibitory signaling on CD200R-expressing NK cells and bone myeloid cells, and has a direct and significant inhibitory effect on NK cell activity in patients with AML [24]. In addition, the CD200:CD200R axis regulates T cell responses through indirect inhibition via myeloid cells, reversion of Th1 to a Th2 phenotype, or promoting regulatory T (Treg) cell differentiation [25, 26]. Our previous study revealed that higher CD200R expression in patients with hepatocellular carcinoma was positively associated with the levels of multiple inhibitory receptors and negatively associated with patient survival [27]. NK and CD8⁺ T cells function as effector lymphocytes to mediate tumor killing, but whether and how CD200R regulates NK and CD8⁺ T cell-mediated anti-tumor responses remains to be elucidated.

In this study, we found that CD200R expression on tumor-infiltrating NK and CD8⁺ T cells was associated with tumor progression and functional exhaustion of NK and CD8⁺ T cells. Cd200r deficiency in mice prevented tumor-infiltrating NK and CD8⁺ T cell exhaustion and slowed tumor progression in various mouse tumor models. Further, we generated a rat anti-mouse CD200R monoclonal antibody (mAb), 6G₃, that specifically blocks the interaction between CD200R and its ligand, CD200. Treatment of tumor-bearing mice with anti-CD200R mAb phenocopied the observations in CD200R-deficient mice, and the therapeutic effects of CD200R blockade were dependent on both NK and CD8⁺ T cells. Furthermore, combined blockade of CD200R and PD-1/PD-L1 had synergistic effects in tumor-bearing mice. Besides, we generated and purified anti-human CD200R mAb and found that anti-human CD200R (anti-hCD200R) significantly slowed tumor growth in peripheral blood mononuclear cell (PBMC)-reconstituted xenograft mouse models. These findings indicate that CD200R is involved in NK and CD8⁺ T cell exhaustion, and represents a promising potential target for tumor immune checkpoint immunotherapy.

Materials and methods

Mice

All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China. C57BL/6 wild-type (WT) mice were purchased from the Shanghai SLAC Laboratory (Shanghai, China). C57BL/6 CD200R-knockout (KO) mice were generated by the Laboratory Animal Center of the University of Science and Technology of China (Hefei, China), and bred in-house. NCG (NOD/ShiLtJGpt-Prkdc^em26Cd52Il2rg^em26Cd22/Gpt) mice were purchased from GemPharmatech Corporation Limited (Nanjing, China). All mice were housed in a specific pathogen-free facility with unrestricted access to food and water, in compliance with the guidelines for the use of experimental animals at the University of Science and Technology of China. Mice were used between 6 and 8 weeks of age.

Cell lines

Cell lines (LLC, B16F10, SW620, A375, K562, and HEK293T) were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MCA205 fibrosarcoma cell line was kindly provided by Prof Yu-ting Ma (Suzhou Institute of Systems Medicine, Suzhou, China). RMA-s cell line was gifted by Prof Zhong-jun Dong (Tsinghua University, Beijing, China). MC38 colon adenocarcinoma cell lines were kindly provided by Prof Yang-xin Fu (University of Texas Southwestern Medical Center, Dallas, TX, USA). PK136 (anti-NK1.1; in vivo depletion) hybridoma cells, the NK92 cell line, and the EO771 mammary gland carcinoma cell line were purchased from ATCC (Manassas, VA, USA). SW620, A375, HEK293T, MCA205, LLC, B16-F10, and MC38 cell lines were maintained in DMEM (Viva Cell, Shanghai, China) medium containing 10% fetal bovine serum (FBS; Thermo, Waltham, MA, USA) and 1× penicillin-streptomycin solution (Beyotime, Shanghai, China). EO771 cell line was cultured in DMEM medium containing 10% FBS, 1× penicillin-streptomycin solution, and 20 mM HEPES (Thermo, Waltham, MA, USA). K562 and RMA-s cell line was maintained in RPMI-1640 (Viva Cell, Shanghai, China) medium containing 10% FBS and 1× penicillin-streptomycin solution at 37 °C in 5% CO₂. NK92 cells were cultured in MEM alpha (1×)-Minimum Essential Medium (Thermo, Waltham, MA, USA) containing 12.5% FBS, 12.5% donor equine serum (Thermo, Waltham, MA, USA), recombinant human interleukin-2 (100 U/mL) (Kingsley, Jiangsu, China), 2-mercaptoethanol (0.1 mM), folic acid (0.02 mM) and 1× penicillin-streptomycin solution at 37 °C in 5% CO₂. 6G₃ (rat IgG1, κ), a mAb blocking mouse CD200R, was a novel clone generated in-house by our laboratory with no prominent antibody-dependent cellular cytotoxicity. All cell lines were confirmed to be mycoplasma-free by PCR.

Identification of anti-CD200R antibodies

For the rat anti-mouse CD200R mAb (6G₃) binding assay, 2 × 10⁵ 293T-mouse CD200R cells were incubated with different concentrations of 6G₃ antibody, and the binding frequency detected using APC-conjugated goat anti-rat IgG antibody (BioLegend, San Diego, CA, USA). To assess CD200R-CD200 antagonistic activity, 2 × 10⁵ 293T mouse CD200R cells were incubated with different concentrations of 6G₃ antibody and 1 μg/mL mouse CD200-hFc fusion protein. APC-conjugated mouse anti-human IgG Fc antibody (BioLegend, San Diego, CA, USA) was used to detect the binding frequency of mCD200-hFc fusion protein.

To assess binding between anti-CD200R (6G₃) and mouse CD200R, 96-well flat bottom plates were coated overnight at 4 °C with 100 μL/well of protein (10 μg/mL; mouse CD200R-Fc) in coating buffer. After removing the coating solution, non-specific binding was blocked by the addition of 300 μL blocking solution containing 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and incubated at 37 °C for 2 h. Plates were then washed six times in PBS supplemented with 0.05% Tween-20 (wash buffer), and serial dilutions of 6G₃ antibody were added at 100 μL/well and incubated for 2 h at room temperature. Plates were then washed with wash buffer and 100 μL/well HRP-conjugated affinipure rabbit anti-rat IgG (H + L) (BOSTER, Wuhan, China) diluted 1:2000 in ELISA buffer (PBS containing 1% BSA and 0.05% Tween 20) was added and incubated at room temperature for 1 h. Plates were washed with wash buffer and developed for 3 min by addition of 80 μL/well of tetramethylbenzidine (TMB; Beyotime, Shanghai, China). Enzymatic color development was stopped with 80 μL/well of TMB stop solution (Beyotime, Shanghai, China). Plates were analyzed at 450 nm using an ELISA Microplate Reader (BioTek ELx800). To assay anti-CD200R (6G₃) blocking by ELISA, 96-well flat bottom plates were coated overnight at 4 °C with 10 μg/mL mouse CD200R-Fc (100 μL/well) in PBS. After removing the coating solution, non-specific binding was blocked with PBS containing 5% BSA (300 μL/well) and incubated at 37 °C for 2 h. Then, plates were washed six times in PBS supplemented with 0.05% Tween-20 (wash buffer), 50 μL/well of 1 μg/mL biotinylated mouse CD200 protein (ACROBiosystems, Newark, DE, USA) and 100 μL/well serial dilutions of 6G₃ antibody added for 1 h at room temperature, followed by washing with wash buffer, and adding 100 μL/well streptavidin-HRP (Thermo, Waltham, MA, USA) diluted 1:5000 in ELISA buffer (PBS containing 1% BSA and 0.05% Tween 20) at room temperature for 1 h. Plates were then washed with wash buffer and developed for 5 min by addition of 80 μL/well of TMB. Enzymatic color development was stopped with 80 μL/well of TMB stop solution, and plates were analyzed at 450 nm using an ELISA microplate reader (BioTek ELx800).

Syngeneic tumor studies

The MC38, MCA205, LLC, EO771, RMA-S, and B16F10 cells were cultured as described above. To evaluate tumor growth in mice, 2 × 10⁵ MC38, 2 × 10⁵ MCA205, 1 × 10⁶ LLC, or 2 × 10⁵ EO771 cells in endotoxin-free PBS were injected subcutaneously (s.c.) into the flanks of 6-week-old WT (C57BL/6) or CD200R KO mice. For the anti-CD200R (6G₃) treatment experiment, WT (C57BL/6) mice were inoculated s.c. with 5 × 10⁴ MC38 or 2 × 10⁵ MCA205 cells. Mice were randomized into treatment groups 3 days later, then treated with control antibody (200 μg; purified in-house from rat serum) or anti-CD200R (6G₃, 200 μg; purified in-house from 6G₃ Hybridoma ascites) at different doses, administered every 3 days for five times by intraperitoneal (i.p.) injection. To evaluate the effect of combined therapy, WT (C57BL/6) mice were injected s.c. with 2 × 10⁵ MCA205 cells on day 0, then randomized into treatment groups 3 days later and treated with rat IgG (200 μg, purified in-house), anti-CD200R (6G₃) (200 μg), anti-PD-L1 (200 μg; clone 10 F.9G2) or anti-PD-L1 (200 μg) combined with CD200R (6G₃) (200 μg) five times (once every 3 days) through i.p. injection. WT (C57BL/6) mice were injected s.c. with 2 × 10⁵ MC38 cells on day 0, then grouped randomly on day 5. When tumors reached an average volume of 150–200 mm³, typically 11 days after implantation, mice were injected with control mAb (rat IgG) (200 μg, purified in-house), anti-CD200R mAb (6G₃, 200 μg), anti-PD-1 (200 μg; clone 29F.1A12) or anti-CD200R (6G₃, 200 μg) + anti-PD-1 (200 μg) i.p. starting on day 11 for five times. For the human PBMC cell-reconstituted xenograft model, NCG mice were injected intravenously (i.v.) with human PBMCs on day −5. Mice were inoculated subcutaneously with SW620-hCD200 or A375-hCD200 tumor cells on day 0. Reconstruction efficiency was detected on day 5. Mice were then treated with anti-hCD200R mAb (250 μg) or control mAb (human IgG) (250 μg) intraperitoneally (i.p.) every 3 days starting on day 5 for five times. Tumors were measured every 2 days by caliper, and tumor volume was calculated as 0.5 × length × width × width. When tumor size reached 1000 mm³, mice were euthanized. Mice whose tumors were unmeasurable or below 20 mm³ were considered to be tumor-free.

In vivo cell depletion

For NK or CD8⁺ T cell depletion, 200 μg mAb to NK1.1 (PK136) or 200 μg mAb to CD8β (53-5.8) was injected i.p. into WT (C57BL/6) mice 24 h before the challenge and then the antibodies were injected once every week.

Isolation of mouse tumor-infiltrating lymphocytes (TILs)

To obtain tumor-infiltrating cells, mice were euthanized and their tumor burdens carefully removed, tumor tissue mechanically cut into small pieces using surgical scissors and digested in RPMI 1640 with collagenase IV (1 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) and DNase I (15 U/mL, Sigma-Aldrich, St. Louis, MO, USA) with shaking at 37 °C for 1 h, followed by collection and purification of supernatants by Percoll gradient centrifugation (GE Healthcare, Chicago, IL, USA). Isolated cells were then used in various assays to evaluate the phenotype and function of NK and T cells. Data were collected using LSR Fortessa or Celesta (BD, Franklin Lakes, NJ, USA) flow cytometers and analyzed with FlowJo software (BD, Franklin Lakes, NJ, USA).

Isolation of human PBMCs and NK cells

Peripheral blood samples from healthy controls were collected from The First Affiliated Hospital of Anhui Medical University (Hefei, China). PBMCs were isolated by centrifugation through a density gradient according to the manufacturer’s instructions (GE Healthcare, Chicago, IL, USA). NK cells were purified by negative selection using a human NK Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The purity of NK cells was >90% as determined by flow cytometry. Human PBMCs and purified NK cells were cultured in RPMI-1640 medium with 10% FBS and 100 IU/mL recombinant human IL-2. All experiments were approved by the Ethics Committee of the University of Science and Technology of China. All subjects provided written informed consent.

Cytotoxicity assay

RMA-s cell lines were labeled with CellTrace™ Violet (CTV) (Thermo, Waltham, MA, USA), at 37 °C for 15 min according to the manufacturer’s instructions. Labeled target cells (1 × 10⁴) were added to 96-well round-bottom plates along with effector cells (tumor-infiltrating NK cells) for 4 h in complete RPMI at an effector:target ratio of 10:1. Human tumor cell lines were labeled with CTV at 37 °C for 15 min according to the manufacturer’s instructions. Labeled target cells (1 × 10⁴) were added to 96-well round-bottom plates along with effector cells (NK92 cells, PBMCs, or purified human NK cells) for 4 h in the presence of anti-human CD200R antibody or control antibody (human IgG) in complete RPMI at different effector/target ratios. As a spontaneous cell death control, CTV-labeled target cells were cultured alone under identical conditions. After 4 h, cells were harvested, and 7-aminoactinomycin D (7-AAD) (BD, San Diego, CA, USA) was added before analyses. Samples were mixed thoroughly and analyzed by flow cytometry.

In vitro co-culturing system

1 × 10⁵ NK92 cells or 1 × 10⁶ human PBMCs were co-cultured with 4 × 10⁴ SW620-hCD200 or A375-hCD200 for 24 h either in the presence of anti-human CD200R antibody or control antibody (human IgG). CD107a antibody and 2 µg/mL monensin (eBioscience, San Diego, CA, USA) were added to the culture 4 h before harvest.

Antibodies

Anti-hCD200R, a mAb targeting CD200R and blocking the interaction between human CD200R and CD200 was generated and purified in-house. mAb to mouse NK1.1 (PK136) was purified in-house from the hybridoma cell supernatant. Mouse CD200R (6G₃) was purified in-house from hybridoma ascites. Anti-PD-L1 (10F.9G2), anti-PD-1 (29F.1A12), and anti-CD8β (53-5.8) antibodies were purchased from Bio X Cell (Lebanon, NH, USA). The control antibody (rat IgG) was purified in-house from rat serum.

Other antibodies were: FITC-conjugated antibodies to mouse CD200R (OX-110; BioLegend, San Diego, CA, USA), NKG2A/C/E (20d5; BD, San Diego, CA, USA), CD69 (H1.2F3; BD, San Diego, CA, USA), and CD107a (1D4B; BD, San Diego, CA, USA); PE-conjugated antibodies to mouse NKp46 (29A1.4; BioLegend, San Diego, CA, USA), CD200R (OX-110; BioLegend, San Diego, CA, USA), CD96 (3.3; BioLegend, San Diego, CA, USA), NKG2D (CX5; BD, San Diego, CA, USA), and GzmB (16G9; eBioscience, San Diego, CA, USA); APC-conjugated antibodies to mouse CD200R (OX-110; BioLegend, San Diego, CA, USA), CD200 (OX-90; BioLegend, San Diego, CA, USA), NKG2D (CX5; BD, San Diego, CA, USA), LAG3 (C9B7W; BioLegend, San Diego, CA, USA), CD27 (LG.3A10; BioLegend, San Diego, CA, USA), and Perforin (S16009A; BioLegend, San Diego, CA, USA); BV605-conjugated antibody to mouse NK1.1 (PK136; BioLegend, San Diego, CA, USA); BUV395-conjugated antibodies to mouse CD3ε (145-2C11; BD, San Diego, CA, USA) and TCRβ (H57-597; BD, San Diego, CA, USA); BUV563-conjugated antibody to mouse CD4 (GK1.5; BD, San Diego, CA, USA); BUV737-conjugated antibody to mouse CD8α (53-6.7; BD, San Diego, CA, USA); BV510-conjugated antibody to mouse CD45 (30-F11; BD, San Diego, CA, USA); BV785-conjugated antibody to mouse PD-1 (29F.1A12; BioLegend, San Diego, CA, USA); BV786-conjugated antibodies to mouse NKG2A/C/E (20d5; BD, San Diego, CA, USA) and IFN-γ (XMG1.2; BD, San Diego, CA, USA); BV421-conjugated antibodies to mouse Tim-3 (RMT3-23; BioLegend, San Diego, CA, USA), NKG2D (CX5; BD, San Diego, CA, USA), and TNF-α (MP6-XT22; BioLegend, San Diego, CA, USA); PE-Cy7-conjugated antibodies to mouse CD226 (10E5; BioLegend, San Diego, CA, USA) and Ki67 (SolA15; eBioscience, San Diego, CA, USA); PerCP-Cy5-5-conjugated antibodies to mouse TRAIL (N2B2; BioLegend, San Diego, CA, USA), IFN-γ (XMG1.2; BioLegend, San Diego, CA, USA), and CD11b (M1/70; BioLegend, San Diego, CA, USA); BB700-conjugated antibody to mouse LAG3 (C9B7W; BD, San Diego, CA, USA); and PerCP-eFluor 710-conjugated antibody to mouse TIGIT (GIGD7; Thermo, Waltham, MA, USA). BD Horizon™ Fixable Viability Stain 780 (APC-Cy7) was from BD (San Diego, CA, USA). BV510-conjugated antibody to human CD45 (2D1; BioLegend, San Diego, CA, USA) and CD107a (H4A3; BioLegend, San Diego, CA, USA); BV605-conjugated antibody to human CD56 (5.1H11; BioLegend, San Diego, CA, USA); PerCP-Cy5.5-conjugated antibody to human CD3 (UCHT1; BD, San Diego, CA, USA); PE-Cy7-conjugated antibody to human CD8α (RPA-T8; BD, San Diego, CA, USA); BV786-conjugated antibody to human CD4 (L200; BD, San Diego, CA, USA); Alexa-488-conjugated antibody to human Ki67 (Ki-67; BioLegend, San Diego, CA, USA); PE-conjugated antibody to human TNF-α (Mab11; BioLegend, San Diego, CA, USA); BV421-conjugated antibody to human GzmB (GB11; BD, San Diego, CA, USA), CD200R (OX-108; BioLegend, San Diego, CA, USA) and CD200 (OX-104; BioLegend, San Diego, CA, USA); APC-conjugated antibody to human IFN-γ (4S.B3; BD, San Diego, CA, USA).

Flow cytometry analysis

Isolated cells were incubated with rat serum for 30 min at 4 °C to block Fc receptors, followed by staining with fluorescently labeled mAbs against surface molecules. For CD107a and intracellular cytokine staining, splenocytes and TILs were stimulated for 4 h with 30 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO, USA) and 1 µg/mL ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of 2.5 μg/mL monensin (eBioscience, San Diego, CA, USA). After stimulation, cells were stained for surface markers. Then, cells were fixed and permeabilized with FoxP3 fixation buffer (eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions and stained with mAbs against intracellular molecules. All samples were acquired on an LSRFortessa cytometer and analyzed using FlowJo software.

Statistical analysis

Statistical analyses were performed in GraphPad Prism v9.0 (La Jolla, USA) using appropriate tests (unpaired two-tailed t-test, paired two-tailed t-test, one-way ANOVA followed by Tukey’s multiple comparisons test, two-way ANOVA, or Mantel–Cox test) as indicated in the figure legends. Significance was defined as follows: ns, P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. In animal experiments, “n” refers to the number of animals per experimental group.

Results

Higher CD200R expression is associated with the exhaustion of tumor-infiltrating lymphocytes (TILs)

To investigate the role of CD200R in anti-tumor immunity, we established four subcutaneous tumor mouse models using MC38 (colon cancer), MCA205 (fibrosarcoma), LLC (lung cancer), and EO771 (mammary cancer) cell lines. We found that tumor-infiltrating NK and CD8⁺ T cells expressed significantly higher levels of CD200R in different tumor models, compared with their counterparts in the spleen of normal mice or tumor-bearing mice (Fig. 1a, b and Supplementary Fig. S1a). To determine the effect of CD200R on tumor-infiltrating lymphocytes (TILs), we compared the phenotype of CD200R⁺ NK cells with that of CD200R⁻ NK cells in the four subcutaneous tumor models. We found that tumor-infiltrating CD200R⁺ NK cells expressed higher levels of the inhibitory receptors, including CD96, TIGIT, NKG2A, and LAG3, compared with tumor-infiltrating CD200R⁻ NK cells (Fig. 1c–f). Also, tumor-infiltrating CD200R⁺ CD8⁺ T cells in different tumor models showed higher surface expression of CD96, PD-1, TIM-3, and LAG3 (Fig. 1g–j). Together, these data indicate that CD200R expression is associated with exhaustion of tumor-infiltrating NK and CD8⁺ T cells in tumor-bearing mice.

Fig. 1. Upregulation of CD200R and functional exhaustion of tumor-infiltrating NK and CD8⁺ T cells in various subcutaneous tumor models.

Frequency of CD200R⁺ NK cells (a) and CD200R⁺ CD8⁺ T cells (b) in splenocytes (Spl) and TILs from WT (B6) mice (n = 3) or tumor-bearing mice injected s.c. with either 2 × 10⁵ MC38 (n = 5), 2 × 10⁵ MCA205 (n = 5), 1 × 10⁶ LLC (n = 5), or 2 × 10⁵ EO771 (n = 4) cells. Geometric MFI of CD96, TIGIT, NKG2A, or LAG3 on tumor-infiltrating CD200R⁻ NK vs CD200R⁺ NK cells from MC38-bearing mice (s.c., 2 × 10⁵ MC38 cells, n = 5) (c), MCA205-bearing mice (s.c., 2 × 10⁵ MCA205 cells, n = 5) (d), LLC-bearing mice (s.c., 1 × 10⁶ LLC cells, n = 5) (e), or EO771-bearing mice (s.c., 2 × 10⁵ EO771 cells, n = 4) (f). Geometric MFI of CD96, PD-1, TIM-3, or LAG3 on tumor-infiltrating CD200R⁻ CD8⁺ T cells vs CD200R⁺ CD8⁺ T cells from MC38-bearing mice (s.c., 2 × 10⁵ MC38 cells, n = 5) (g), MCA205-bearing mice (s.c., 2 × 10⁵ MCA205 cells, n = 5) (h), LLC-bearing mice (s.c., 1 × 10⁶ LLC cells, n = 5) (i), or EO771-bearing mice (s.c., 2 × 10⁵ EO771 cells, n = 4) (j). Tumor tissues were harvested when tumor volume was ~300 mm³ (a–j). Data are representative of at least two independent experiments. Error bars represent mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple-comparisons test; ns not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

CD200R deficiency delays tumor growth and prevents TIL exhaustion

To further explore CD200R function, we generated CD200R-KO mice, and confirmed their lack of CD200R expression by flow cytometry (Supplementary Fig. S2a). No significant differences were observed in overall appearance, weight, or organ size between CD200R-KO and WT mice. The percentages of NK cells in CD200R-KO mice were similar to those in WT mice, indicating that Cd200r knockout did not affect NK cell development and homeostatic maintenance (Supplementary Fig. S2b). By analyzing NK cell maturation stages, based on cell surface expression of CD27 and CD11b, we found that the maturation state of splenic NK cells was also unaltered by Cd200r KO (Supplementary Fig. S2c, d). In addition, we compared the expression of activating receptors, inhibitory receptors, and effector molecules in splenic NK cells between WT and CD200R-KO mice, and confirmed that there were no significant differences between the two groups at steady state (Supplementary Fig. S2e–g). Further, effector molecule expression in CD8⁺ T cells from CD200R-KO mice was also similar to that in WT mice (Supplementary Fig. S2h). Thus, Cd200r ablation did not affect the development, phenotype, or function of NK or CD8⁺ T cells.

To determine the role of CD200R in tumor progression, we inoculated (s.c.) MC38 colon cells into WT and CD200R-KO mice. Tumor growth was markedly decreased and survival was significantly prolonged in CD200R-KO compared with WT mice (Fig. 2a, b). Similar results were observed in other subcutaneous tumor models, including MCA205, LLC, and EO771 tumors (Fig. 2c–h). Together, these results indicate that Cd200r knockout inhibits tumor progression in multiple tumor models.

Fig. 2. CD200R deficiency delays tumor growth in various subcutaneous tumor models.

a, b WT (n = 8) and CD200R KO (n = 8) mice were injected s.c. with 2 × 10⁵ MC38 cells on day 0, and tumor volume was measured every 2 days. Median tumor volume (a) and mouse survival (b) over time were shown. c, d WT (n = 7) and CD200R KO (n = 7) mice were injected s.c. with 2 × 10⁵ MCA205 cells on day 0, and tumor volume was measured every 2 days. Median tumor volume (c) and mouse survival (d) over time were shown. e, f WT (n = 7) and CD200R KO (n = 7) mice were injected s.c. with 1 × 10⁶ LLC cells on day 0, and tumor volume was measured every 2 days. Median tumor volume (e) and mouse survival (f) over time were shown. g, h WT (n = 8) and CD200R KO (n = 7) mice were injected s.c. with 2 × 10⁵ EO771 cells on day 0, and tumor volume was measured every 2 days. Median tumor volume (g) and mouse survival (h) over time were shown. Data are representative of at least two independent experiments. Error bars represent mean ± SEM. Statistical significance was determined using two-way ANOVA (a, c, e, g) or Mantel–Cox test (b, d, f, h); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Further, in both the MC38 and MCA205 models, tumor size and weight were reduced in CD200R-deficient mice on day 15 after the challenge (Fig. 3a, b and Supplementary Fig. S3a, b), and there was a significant increase in the absolute numbers of tumor-infiltrating NK cells and CD8⁺ T cells (Fig. 3c, d and Supplementary Fig. S3c, d). Tumor-infiltrating NK cells in CD200R-KO mice exhibited upregulated expression of the activation marker, CD226, and downregulated expression of the inhibitory receptors, TIGIT and NKG2A (Fig. 3e, Supplementary Fig. S3e). Further, tumor-infiltrating NK cell proliferation and survival were enhanced in CD200R-KO mice, as evidenced by increased Ki67 and Bcl2 expression (Fig. 3f, Supplementary Fig. S3f). Furthermore, higher frequencies of tumor-infiltrating NK cells expressing CD107a, IFN-γ, TNF-α, or GzmB were observed in CD200R-KO mice (Fig. 3g, Supplementary Fig. S3g).

Fig. 3. CD200R KO prevents the exhaustion of tumor-infiltrating NK and CD8⁺ T cells in the MC38 subcutaneous tumor model.

a, b WT (n = 6) and CD200R KO (n = 6) mice were subcutaneously injected with 2 × 10⁵ MC38 cells on day 0, and the tumor volume was measured every 2 days. Representative photograph (a) and weight of tumor (b) on day 15 after challenge. Absolute number of tumor-infiltrating NK cells (c) and CD8⁺ T cells (d) in WT and CD200R KO mice. The geometric MFI of cell surface molecules (e) and intracellular molecules (f) among tumor-infiltrating NK cells in WT and CD200R KO mice, which were analyzed on day 15 after tumor challenge. g Frequency of cells expressing CD107a, IFN-γ, GzmB, and TNF-α among tumor-infiltrating NK cells in WT and CD200R KO mice. The geometric MFI of cell surface molecules (h) and intracellular molecules (i) among tumor-infiltrating CD8⁺ T cells in WT and CD200R KO mice analyzed on day 15 after tumor challenge. j Frequency of cells expressing CD107a, IFN-γ, GzmB, and TNF-α among tumor-infiltrating CD8⁺ T cells in WT and CD200R KO mice. Data were representative of at least two independent experiments. Each symbol represents an individual mouse (b–j); Error bars represent mean ± SEM. Statistical significance was determined using unpaired two-tailed t-test (b–j) *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

In both the MC38 and MCA205 subcutaneous tumor models, similar to NK cells, tumor-infiltrating CD8⁺ T cells in CD200R-KO mice exhibited upregulated expression of the activation marker, CD226, and downregulated levels of the inhibitory receptors, TIM-3 and NKG2A (Fig. 3h, Supplementary Fig. S3h). Tumor-infiltrating CD8⁺ T cell proliferation and survival were also enhanced in CD200R-KO mice (Fig. 3i, Supplementary Fig. S3i). Furthermore, higher percentage of CD107a⁺, IFN-γ⁺, GzmB⁺, or TNF-α⁺ tumor-infiltrating CD8⁺ T cells was observed in CD200R-KO mice (Fig. 3j, Supplementary Fig. S3j). Taken together, these data indicate that CD200R deficiency slows tumor growth and prolongs survival of tumor-bearing mice, possibly due to enhanced function and proliferation of tumor-infiltrating NK and CD8⁺ T cells, by preventing the exhaustion of these TILs.

CD200R blockade inhibits tumor growth and reverses TIL exhaustion

To evaluate the anti-tumor effect of CD200R blockade in vivo, we generated a rat anti-mouse CD200R mAb (6G₃) and verified its ability to block CD200-CD200R interaction (Supplementary Fig. S4a–d). WT mice were inoculated s.c. with MC38 tumor cells to establish mouse colon tumor models, followed by treatment with PBS, rat IgG, or anti-CD200R mAb on day 3 post-inoculation (Fig. 4a). Early treatment with anti-CD200R mAb significantly reduced tumor growth (Fig. 4b) and extended the overall survival (Fig. 4c) of tumor-bearing mice. Smaller tumor volume and lower tumor weight were also observed on day 21 after tumor challenge in mice treated with anti-CD200R (Fig. 4d, e). Consistent with the MC38 model, anti-CD200R mAb treatment resulted in significantly slower tumor growth and longer survival in mice injected s.c. with MCA205 tumor cells (Supplementary Fig. S5a–c), accompanied by smaller tumor mass 16 days after challenge (Supplementary Fig. 5d, e). These data indicate that CD200R blockade alone exhibits therapeutic efficacy against syngeneic tumors in mice.

Fig. 4. Blockade of CD200R inhibits tumor growth in the MC38 subcutaneous tumor model and synergizes with anti-PD-1.

a–e Experimental protocol (a) for the MC38 subcutaneous tumor model: WT mice were injected s.c. with 5 × 10⁴ MC38 cells on day 0, followed by treatment with control (black) or anti-CD200R (6G₃) mAb (red) (n = 8 per group) on days 3, 6, 9, 12, and 15. Median tumor volume (b) and overall survival (c) were shown. Representative photographs (d) and weights (e) of tumors on day 21 after challenge. f–h Experimental protocol (f) for the MC38 subcutaneous tumor model: WT mice were injected s.c. with 2 × 10⁵ MC38 cells on day 0 and randomly grouped on day 5. When tumor volume has reached ~150–200 mm³, mice were intraperitoneally (i.p.) injected with control mAb (rat IgG), anti-CD200R (6G₃), anti-PD-1, or anti-CD200R (6G₃) + anti-PD-1 mAbs, five times starting on day 11 (n = 10 per group). Median tumor volume (g) and overall survival (h) were shown. i–n Experimental protocol (i, l) for subcutaneous tumor models: WT mice were treated five times with control (rat IgG), anti-CD200R (6G₃), anti-PD-1, or anti-CD200R (6G₃) + anti-PD-1 mAbs after s.c. injection of 2 × 10⁵ MC38 tumor cells, then tumor-free (survivor, n = 5) and age-matched control (n = 6) mice were re-challenged s.c. with 1 × 10⁶ MC38 tumor cells (i–k) or 1 × 10⁵ B16F10 tumor cells (l–n). Median tumor volume (j, m) and overall survival (k, n) were shown. Data are representative of at least two independent experiments. Error bars represent mean ± SEM. Statistical significance was determined using two-way ANOVA (b, g, j, m), Mantel–Cox test (c, h, k, n), or unpaired two-tailed t-test (e); ns not significant (P > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001, and ****P < 0.0001.

We also evaluated the combined therapeutic value of anti-CD200R plus anti-PD-1 mAbs. WT mice bearing subcutaneous MC38 tumors were treated with anti-PD-1, anti-CD200R, or anti-CD200R plus anti-PD-1 mAbs when tumors reached ~150–200 mm³ (Fig. 4f). Anti-PD-1 mAb treatment alone effectively controlled tumor growth, and the combination with of anti-PD-1 and anti-CD200R mAbs resulted in synergistic therapeutic effects, markedly more effective than either mAb alone (Fig. 4g, h). We observed the same synergistic effect of combination treatment in the MCA205 tumor model (Supplementary Fig. S5f–h). These data suggest that mAb targeting CD200R could serve as an immunotherapeutic alone or in combination with PD-1 blockade.

Next, we investigated whether mice that survived tumor challenge following combination treatment with anti-CD200R and anti-PD-1 mAbs had anamnestic responses to tumor re-challenge. For this purpose, MC38 tumor-bearing mice were initially treated with anti-PD-1 plus anti-CD200R mAbs and subsequently re-challenged with a five-fold amount of MC38 cells on day 134 after the first tumor inoculation, without any further treatment (Fig. 4i). We observed that tumor growth was inhibited and mouse survival prolonged, relative to those observed in age-matched tumor-naïve mice (Fig. 4j–k). To verify tumor-specificity, we also challenged survivors with un-related B16-F10 cells on day 113 after the first MC38 tumor challenge (Fig. 4l). Mice did not eliminate the un-related B16-F10 tumors, and there were no significant differences in tumor size or overall survival between survivors and age-matched controls (Fig. 4m, n). Together, these results indicate that dual blockade of PD-1 and CD200R immunotherapy can elicit anti-tumor immune memory.

We then compared the functional status of TILs in mice treated with anti-CD200R and those treated with control mAb. In both the MC38 and MCA205 tumor models, we found significant increases in the absolute numbers of tumor-infiltrating NK cells after CD200R blockade (Fig. 5a, Supplementary Fig. 6a). Tumor-infiltrating NK cells from tumor-bearing mice treated with anti-CD200R mAb showed increased expression of the activating receptor, CD226, and decreased expression of the inhibitory receptors, TIGIT and NKG2A (Fig. 5b, Supplementary Fig. 6b). Further, tumor-infiltrating NK cell proliferation and survival were also enhanced after anti-CD200R mAb treatment (Fig. 5c, Supplementary Fig. 6c). Furthermore, CD200R blockade led to increased expression of CD107a and the cytotoxic molecules, perforin and TRAIL, as well as the cytokine, IFN-γ, on tumor-infiltrating NK cells (Fig. 5d, Supplementary Fig. 6d). Besides, tumor-infiltrating NK cells exhibited enhanced cytotoxic activity against RMA-s target cells in vitro after anti-CD200R mAb treatment (Fig. 5e). These data indicate that CD200R blockade can reverse exhaustion of tumor-infiltrating NK cells.

Fig. 5. Blockade of CD200R reverses exhaustion of tumor-infiltrating NK and CD8⁺ T cells in the MC38 subcutaneous tumor model.

a–i WT mice were subcutaneously injected with 5 × 10⁴ MC38 cells, followed by treatment with anti-CD200R or control mAbs (Rat IgG) from day 3, at 3-day intervals for five times injection in total. Tumor tissues were harvested when tumor volume was around 500–800 mm³ (n = 8 per group). a Absolute number of tumor-infiltrating NK cells. b, c GMFI of the indicated molecules among tumor-infiltrating NK cells. d The percentages of CD107a, IFN-γ, Perforin, and TRAIL expression among tumor-infiltrating NK cells. e Killing assays using tumor-infiltrating NK cells as effector cells and CTV-labeled RMA-s tumor cells as target cells, with an effector-target (E:T) ratio of 10:1. Percentages of 7-AAD⁺ cells among CTV-labeled RMA-s cells (n = 3/group). f Absolute number of tumor-infiltrating CD8⁺ T cells. g, h GMFI of the indicated molecules among tumor-infiltrating CD8⁺ T cells. i Frequency of cells expressing CD107a, IFN-γ, GzmB and TNF-α among tumor-infiltrating CD8⁺ T cells. Data were representative of at least two independent experiments. Error bars represent mean ± SEM. Statistical significance was determined using unpaired two-tailed t-test (a–i) not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Anti-CD200R treatment also increased the number of tumor-infiltrating CD8⁺ T cells (Fig. 5f, Supplementary Fig. 6e). The frequency of tumor-infiltrating CD8⁺ T cells expressing the activating receptor, CD226, was higher, while that of CD8⁺ T cells expressing the inhibitory receptors, TIM-3 and NKG2A, was lower, in tumor-bearing mice treated with anti-CD200R mAb (Fig. 5g, Supplementary Fig. 6f). Tumor-infiltrating CD8⁺ T cell proliferation and survival were also enhanced after anti-CD200R mAb treatment (Fig. 5h, Supplementary Fig. 6g). Further, these therapeutic effects were accompanied by a higher frequency of tumor-infiltrating CD8⁺ T cells with surface expression of CD107a, IFN-γ, GzmB, and TNF-α (Fig. 5i, Supplementary Fig. 6h), indicating that treatment with anti-CD200R mAb could also reverse CD8⁺ T cell exhaustion.

The anti-tumor efficacy of CD200R blockade depends on NK and CD8⁺ T cells

As both NK and CD8⁺ T cells have increased CD200R expression in the TME and exhibited enhanced tumoricidal activities after CD200R blockade, we next attempted to investigate the roles of these cells in CD200R therapeutic blockade. We depleted NK and/or CD8⁺ T cells in tumor-bearing mice using mAbs against the NK cell receptor NK1.1 (PK136) and CD8β (53-5.8), respectively (Fig. 6a); successful depletion of NK or CD8⁺ T cells was confirmed by flow cytometric analysis (Fig. 6b). Consistent with the aforementioned results, anti-CD200R mAb significantly inhibited tumor growth and prolonged overall survival of MC38 colon tumor-bearing mice (Fig. 6c–f); however, NK cell depletion abrogated the therapeutic effects of CD200R blockade, as evidenced by significantly accelerated tumor growth and shortened survival of tumor-bearing mice treated with anti-CD200R and NK cell-depleting mAbs (Fig. 6c, d). As expected, CD8⁺ T cell depletion had a similar effect (Fig. 6c, f), suggesting that both NK and CD8⁺ T cells contribute to anti-CD200R mAb therapeutic efficacy. Also as expected, combined depletion of NK and CD8⁺ T cells resulted in the worst tumor growth and survival in tumor-bearing mice (Fig. 6c, f). Overall, these results indicate that both NK and CD8⁺ T cells contribute to the therapeutic outcome of CD200R blockade.

Fig. 6. Both NK and CD8⁺ T cells contribute to the anti-tumor efficacy of CD200R blockade.

a Experimental protocol for the MC38 subcutaneous tumor model used in (c–f). Effects of NK or CD8 cell depletion using anti-NK1.1 (PK136) or anti-CD8β (53-5.8) in MC38 syngeneic model mice treated with either anti-CD200R (6G₃) or control (rat IgG) mAbs (n = 10 per group). b Representative flow plots of splenic NK or CD8⁺ T cells from mice treated with anti-NK1.1 (PK136), anti-CD8β (53-5.8), or control mAbs. Median tumor volume (c, e) and overall survival (d, f) of mice receiving different treatments. Data are representative of two independent experiments. Error bars represent mean ± SEM. Statistical significance was determined using two-way ANOVA (c, e) or Mantel–Cox test (d, f); *P < 0.05, ***P < 0.001, ****P < 0.0001.

Blockade of human CD200R inhibits human tumor growth in PBMC-reconstituted xenograft mice

To further evaluate the therapeutic potential of CD200R blockade in human tumor immunotherapy, we generated and purified a mAb targeting human CD200R (hCD200R). We established a humanized mouse model by reconstituting NCG mice (lack T, B, and NK cells) with human PBMCs before inoculation with human CD200-overexpressing SW620 (SW620-hCD200) colon cancer cells or A375 (A375-hCD200) malignant melanoma cells. Mice were grouped randomly on day 5 post-tumor cell inoculation and treated with anti-hCD200R mAb or human IgG mAb on days 5, 8, 11, 13, and 17 (Fig. 7a, e). We observed significantly slower tumor growth in humanized tumor-bearing mice treated with anti-hCD200R mAb (Fig. 7b, f). On day 19 or 21 afterthe tumor cell challenge, we observed that treatment with anti-hCD200R mAb significantly reduced tumor mass in the PBMC-reconstituted xenograft mice (Fig. 7c, d, g, h).

Fig. 7. Blockade of CD200R delays human tumor growth in PBMC-reconstituted xenograft mice.

a NCG mice were transferred with human PBMCs on day −5. Mice were inoculated subcutaneously with SW620-hCD200 tumor cells on day 0. Mice were then treated with anti-hCD200R mAb or control (human IgG) mAb intraperitoneally (i.p.) every 3 days starting on day 5 five times (n = 10 per group). b Median tumor volume was measured at various time points. Representative photograph (c) and weight of tumor (d) on day 21 after challenge (n = 10 per group). e NCG mice were transferred with human PBMCs on day −5. Mice were inoculated subcutaneously with A375-hCD200 tumor cells on day 0. Mice were then treated with anti-hCD200R mAb or control (human IgG) mAb intraperitoneally (i.p.) every three days starting on day 5 five times (n = 10 per group). f Median tumor volume was measured at various time points. Representative photograph (g) and weight of tumor (h) on day 19 after challenge (n = 10 per group). Data are representative of at least two independent experiments. Error bars represent mean ± SEM. Statistical significance was determined using unpaired two-tailed t-test (d, h) or two-way ANOVA (b, f). **P < 0.01; ****P < 0.0001.

To explore the effects of CD200R on human NK cells, we first examined the surface expression of CD200R on NK92 cell line by flow cytometry, and found that almost all NK92 cells expressed CD200R (Supplementary Fig. 7a). Anti-hCD200R mAb significantly enhanced the expression of effector molecules (NKG2D, TNF-α and IFN-γ) in NK92 cells co-cultured with SW620-hCD200 cells (Supplementary Fig. 7b) or A375-hCD200 colon cancer cells (Supplementary Fig. 7c). Consistently, anti-hCD200R mAb improved NK92 cell cytolytic activities against SW620-hCD200 cells (Supplementary Fig. 7d) or K562-hCD200 cells (Supplementary Fig. 7e) at different effector/target ratios in vitro. We also found that human primary NK cells derived from peripheral blood expressed CD200R (Supplementary Fig. 7f). We co-cultured human PBMCs with SW620-hCD200 cells (Supplementary Fig. 7g) or A375-hCD200 cells (Supplementary Fig. 7h) in the presence or absence of anti-hCD200R mAb, and found that anti-hCD200R mAb significantly boosted the cytokine secretion (IFN-γ and TNF-α) and degranulation (CD107a) of human NK cells. Furthermore, anti-hCD200R mAb significantly improved the cytotoxicity of PBMCs against SW620-hCD200 (Supplementary Fig. 7i) or A375-hCD200 (Supplementary Fig. 7j) tumor cells in vitro. In addition, enhanced lysis of SW620-hCD200 cells by purified human NK cells in vitro was observed upon treatment with anti-hCD200R mAb (Supplementary Fig. 7k). Taken together, our results demonstrate that anti-hCD200R mAb can enhance the cytotoxicity of human NK cells, and more importantly, inhibit human tumor growth in PBMC-reconstituted xenograft mice, suggesting the potential value of CD200R blockade in translational applications.

Discussion

Radiotherapy and chemotherapy are currently effective against the majority of tumors but are associated with a number of unpleasant side effects and high relapse rates. In contrast to traditional anti-tumor therapies, immunotherapies based on checkpoint blockade have been praised for their specific targeting and reduced side effects and have revolutionized the cancer treatment [28]. ICB has been used to treat various tumors, including melanoma, lung cancer, head and neck squamous cell carcinoma, gastric cancer, and ovarian cancer [29, 30]. Immune checkpoint inhibitors, such as anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) and anti-programmed cell death protein 1 (anti-PD-1), are promising targets for cancer treatment [31]; however, there remain a large proportion of patients who do not respond to, and therefore do not benefit from, these therapies [32], emphasizing the importance of identifying novel immune checkpoint targets or combination strategies. In this study, we propose CD200R as a potential novel target for tumor immunotherapy.

Unlike most IgSF receptors, CD200R lacks ITIM domains [17]; however, its cytoplasmic tail contains three tyrosine residues, the third of which is located within an NPXY motif that is phosphorylated on binding of its ligand [18]. Currently, the only known ligand for CD200R is CD200, and their ligation can trigger inhibitory signaling in CD200R-expressing cells. Excessive CD200R signaling results in functional exhaustion of immune cells, manifesting as progressive loss of proliferation, cytokine secretion, and tumor cell lysis abilities [33, 34]. Our group has reported that CD200R is associated with hepatocellular carcinoma progression and negatively correlated with the survival of patients, suggesting that CD200R has the potential to serve as a novel immunotherapeutic target [27]. In the present study, we used CD200R-KO mice and anti-CD200R mAb to demonstrate that targeting CD200R can inhibit tumor progression and have therapeutic effects in various tumor models. Consistent with these findings, a previous study found reduced growth and metastasis of CD200-positive EMT6 tumors in CD200R-KO mice [35]. In contrast, another group observed that CD200R deficiency led to accelerated tumor progression in the 4THM model [36]. This discrepancy may be explained by the use of different tumor models with distinct immune cell composition in the TME, as CD200R is expressed by both anti-tumor immune cells, such as NK and CD8⁺ T cells, as well as tumor-suppressing cells, including tumor-associated macrophages and myeloid-derived suppressor cells, in the TME [22, 24, 25, 33, 37]. The relative abundance of CD200R-expressing cell types may differ among distinct tumors, resulting in the variation in the effects of CD200R deficiency on tumor growth and progression.

NK and CD8⁺ T cells are cytotoxic lymphocytes with critical roles in tumor immunosurveillance. On chronic exposure to signals from the TME, NK, and CD8⁺ T cells upregulate the expression of immune checkpoint receptors and exhibit impaired effector functions, which are typical features of an exhausted state. Previous studies on CD200R-mediated regulation of immune cells during tumor development have focused primarily on myeloid cells. In this study, we discovered that CD200R expression was increased on tumor-infiltrating NK and CD8⁺ T cells during tumor progression and that these CD200R⁺ TILs were more exhausted than their CD200R⁻ counterparts. CD200R deficiency or blockade led to enhanced effector functions of tumor-infiltrating NK and CD8⁺ T cells, accompanied by increased expression of activating receptors and decreased expression of inhibitory receptors, respectively. Depletion of NK and/or CD8⁺ T cells significantly impaired the therapeutic effects of CD200R blockade. These findings suggest that targeting CD200R enhances anti-tumor activity by unleashing the anti-tumor effects of both NK and CD8⁺ T cells.

Although our findings highlight the functional significance of CD200R signaling in cytotoxic lymphocytes, we do not rule out the possibility that the intrinsic effects of CD200R deficiency or blockade in TAMs and MDSCs may impact their phenotypes [38]. TAMs and MDSCs express CD200R, and the CD200-CD200R axis contributes to the expansion of MDSCs within the TME, thereby increasing tumor-associated immunosuppression. Blockade of CD200R significantly inhibits tumor growth and reduces both the percentage and number of MDSCs infiltrating tumor tissue [38, 39]. Interestingly, CD200R signaling appears to maintain M2 macrophage polarization by suppressing classical M1 activation, thereby favoring a Th2-skewed immune milieu [40]. Current evidence suggests that the functional outcomes of CD200-CD200R pathway modulation exhibit context-dependent variability across tumor models, likely reflecting differences in the baseline inflammatory states and immune cell composition of distinct TMEs [33]. Furthermore, CD200R signaling in Tregs might modulate effector T cell activity through cell-cell interactions. Given the complex cellular crosstalk mediated by the CD200-CD200R axis in the TME, future studies employing conditional knockout models will be required to dissect the relative contributions of distinct cellular compartments across different tumor types. A comprehensive understanding of these dynamic interactions is essential for developing effective CD200R-targeted therapies.

Tumor-associated endothelial cells and activated T lymphocytes within TME exhibit substantial CD200 expression [41]. While the functional role of endothelial CD200 in tumorigenesis remains incompletely characterized, existing evidence suggests its involvement in mediating immune cell-endothelial crosstalk and suppressing anti-tumor immunity [42]. Our analysis of the MC38 subcutaneous tumor model corroborates previous findings, demonstrating elevated CD200 expression across multiple immune subsets including TAMs, MDSCs, and T cells [43]. Notably, the TME also contains soluble CD200 (sCD200) capable of engaging CD200R to propagate immunosuppressive signals [44]. A critical unresolved question arising from our study is the precise cellular origin of the CD200 that interacts with CD200R⁺ NK/T cells. Specifically, it remains unclear whether CD200 is expressed on tumor cells or adjacent immune cell populations, such as TAMs and MDSCs. This mechanistic ambiguity necessitates further investigation using conditional knockout models and spatial transcriptomic approaches to clarify the compartment-specific interactions between CD200 and CD200R.

Data from a recent phase I clinical trial has suggested that the anti-CD200 antibody, samalizumab, could not fully saturate CD200 on CD200-expressing B-cell malignancies from patients, potentially limiting its efficacy [45]. Since CD200, the only known CD200R ligand, is also widely expressed in normal tissues, anti-CD200 mAb may not mainly bind with CD200-expressing cells in the TME, leading to a partial loss of therapeutic efficacy. Anti-CD200 mAb may also induce extensive side effects because of their binding with CD200 on normal tissues. Targeting CD200R may be a more feasible alternative for tumor treatment, due to its limited expression in normal tissues while its abundant presence in most types of solid tumors. To determine the therapeutic value of CD200R blockade, we developed a rat anti-mouse CD200R mAb and an anti-human CD200R mAb and found that treatment using anti-mCD200R mAb significantly slowed tumor growth and extended survival of tumor-bearing mice, and treatment using anti-hCD200R mAb significantly slowed tumor growth in PBMC-reconstituted xenograft tumor models. Furthermore, the blockade of CD200R using anti-hCD200R can enhance the cytokine secretion and cytotoxicity of NK92 cells and primary NK cells against various tumor cell lines in vitro. Thus, targeting CD200R should be a feasible approach for tumor therapy.

Anti-PD-1/PD-L1 immunotherapy is clinically approved, but the response rates remain unsatisfactory in certain tumor types [46]. The synergistic effect of newly discovered immunotherapeutic targets with existing immunotherapies in the treatment of cancer has drawn increasing interest, highlighting the importance of combination immunotherapy [5, 6]. Clinical trials have also demonstrated that a combination of immunotherapeutic targets can effectively prolong the survival of patients with cancer [47]. Our study revealed that combined blockade of CD200R and PD-1/PD-L1 significantly inhibited tumor growth and prolonged survival of tumor-bearing mice, relative to the blockade of either CD200R or PD-1/PD-L1 alone. Therefore, CD200R can be considered as a novel target to further enhance anti-tumor immunity in combination with anti-PD-1/PD-L1, with broader clinical application prospects.

For future CD200-CD200R targeted cancer therapy, it is essential to conduct thorough studies to evaluate the tumor types that benefit the most. Assessing the expression levels of CD200 and CD200R across different cancers is critical. Tumors exhibiting high levels of CD200 expression, as well as those with tumor-infiltrating NK cells and CD8⁺ T cells highly expressing CD200R, could emerge as primary candidates for blockade therapy. We anticipate that tumors with a predominance of M2 macrophages may particularly benefit from CD200R blockade. Furthermore, tumors that demonstrate a poor response to conventional therapies may especially benefit from a combination strategy involving CD200R blockade, aimed at restoring effective anti-tumor immunity. In summary, it is vital to conduct expression profile analyses, characterize TME features, and correlate these with clinical outcomes. Such comprehensive evaluations will be crucial in identifying the cancer types most likely responding to CD200R blockade therapy.

In conclusion, our study reveals that increased CD200R expression is correlated with tumor-infiltrating NK and CD8⁺ T cell exhaustion and that CD200R deficiency or blockade can prevent or reverse the exhaustion of NK and CD8⁺ T cells respectively, thereby inhibiting tumor progression. CD200R blockade improves the anti-tumor therapeutic efficacy of PD-1/PD-L1 blockade, with combined therapy exhibiting synergistic anti-tumor effects. Overall, these findings suggest that CD200R is a potential checkpoint molecule for tumor-infiltrating NK and CD8⁺ T cells and provides an attractive prospect for tumor immunotherapy.

Author contributions

HYS, ZGT, and HP initiated, designed, and supervised the study. ZFZ performed experiments, analyzed data, and interpreted the results. ZFZ, HYS, ZGT, and HP wrote the manuscript. ZFZ, YZ, YWC, GSC, XDZ, and RS contributed to the discussion of the results. All of the authors reviewed the manuscript before submission.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-025-01556-0.