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Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy

Abstract

Overexpression of the B7-H1 (PD-L1) molecule in the tumor microenvironment (TME) is a major immune evasion mechanism in some patients with cancer, and antibody blockade of the B7-H1/PD-1 interaction can normalize compromised immunity without excessive side-effects. Using a genome-scale T cell activity array, we identified Siglec-15 as a critical immune suppressor. While only expressed on some myeloid cells normally, Siglec-15 is broadly upregulated on human cancer cells and tumor-infiltrating myeloid cells, and its expression is mutually exclusive to B7-H1, partially due to its induction by macrophage colony-stimulating factor and downregulation by IFN-γ. We demonstrate that Siglec-15 suppresses antigen-specific T cell responses in vitro and in vivo. Genetic ablation or antibody blockade of Siglec-15 amplifies anti-tumor immunity in the TME and inhibits tumor growth in some mouse models. Taken together, our results support Siglec-15 as a potential target for normalization cancer immunotherapy.

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Acknowledgements

We thank B. Cadugan for editing the manuscript; other members in the laboratories of Yale and NextCure for helpful discussions and technical assistance; and D. Pardoll and J. Fu at Johns Hopkins University for the B16-GMCSF cell line. This research was supported partially by the National Institutes of Health grants nos. P30 CA16359 and P50 CA196530, sponsored research funding from NextCure Inc. and the United Technologies Corporation Endowed Chair.

Author information

L.C., J.W. and J.S. designed the study, interpreted data and wrote the manuscript. J.S. and J.W. conducted the majority of the experiments. L.N.L., S.L. and D.B.F. helped with the design of experiments, coordinated the study and contributed key reagents. M.T., D.L.R., A.N.B. and J.Z. conducted immunohistochemistry and pathological analysis. R.S.H. leads lung cancer clinical team/core that provides samples for analysis. X.N., M.F.S., X.H. and C.S. assisted mouse model studies and immune phenotyping. M.Z., K.A.A., T.O. and X.Z. assisted technically on molecular biology, antibody production and functional analysis in vitro.

Competing interests

In the past 12 months, L.C. has been a consultant/advisory board member for NextCure, AstraZenca, Pfizer, Junshi, Vcanbio and GenomiCare. L.C. is a scientific founder of NextCure and Tayu Biotech Group and has sponsored research grants from NextCure, Pfizer and Boehringer Ingelheim. D.R. is a consultant/advisory board member for Amgen, AstraZeneca, Agendia, Biocept, Bristol-Myers Squibb, Cell Signaling Technology, Cepheid, Daiichi Sankyo, Merck, NanoString, Perkin Elmer, PAIGE and Ultivue and has sponsored research grants from AstraZeneca, Cepheid, Navigate/Novartis, NextCure, Lilly, Ultivue and Perkin Elmer. R.H. is a consultant/advisory board member for Abbvie Pharmaceuticals, AstraZeneca, Biodesix, Bristol-Myers Squibb, Eli Lilly and Company, EMD Serrano, Genentech/Roche, Heat Biologics, Infinity Pharmaceuticals, Junshi Pharmaceuticals, Loxo Oncology, Merck and Company, Nektar, Neon Therapeutics, NextCure, Novartis, Pfizer, Sanofi, Seattle Genetics, Shire PLC, Spectrum Pharmaceuticals, Symphogen, TESARO and has sponsored research grants from AstraZeneca, Eli Lilly and Company, Merck and Company, and is a board member (non-executive/independent) for Junshi Pharmaceuticals. There are patent applications pending related to this work.

Correspondence to Lieping Chen.

Extended data

  1. Extended Data Fig. 1 Schematic representation of the outcome from the TCAA screening.

    The 293T cells stably expressing membrane-associated anti-human CD3 antibody (OKT3) scFv were used to stimulate the activation of Jurkat-NF-κB reporter cells to generate GFP signals. Expression of an individual plasmid encoding a human transmembrane gene would engage a potential receptor on Jurkat T cells to co-stimulate or co-inhibit OKT3-induced T cell activation. Unchanged GFP signal on the transfection of 293T cells indicates the lack of co-stimulatory or co-inhibitory activity.

  2. Extended Data Fig. 2 Siglec-15 mRNA expression in normal tissues of human and mouse origin.

    a, Siglec-15 mRNA relative levels in human tissues and immune cell subsets from BioGPS database. A dash line was added to indicate the flow cytometry detection threshold (verified by Siglec-15 negative staining on CD8+ T cells). Data are mean ± s.e.m. (tissues, n = 2; monocyte, n = 6; macrophage, n = 10; other immune subsets, n = 4 samples). b, Siglec-15 mRNA expression in mouse tissues was tested by RT–PCR, quantified by ImageJ software (NIH) and normalized to the levels of reference gene GAPDH. The 293T cells overexpressing Siglec-15 were used as a positive control. E7, day 7 embryos.

  3. Extended Data Fig. 3 Siglec-15 protein expression on human and mouse immune cells.

    a, Flow cytometry analysis of 293T cells transfected with empty vector (pcDNA) or plasmid with full-length Siglec-15 gene and stained by m03 (anti-Siglec-15 mAb). b, Siglec-15 expression on lymphocytes and neutrophils from S15KO and WT mice by flow cytometry analysis with m03. In a and b, data are representative of three independent experiments. c, Flow cytometry analysis of Siglec-15 expression on the RAW264.7 macrophage line treated with or without 20 ng ml–1 recombinant murine IFN-γ for 48 h. d, Human CD14+ monocytes from peripheral blood were incubated for 7 days in the presence of 100 ng ml−1 M-CSF (M-CSF group) or M-CSF for 4 days followed by M-CSF plus 50 ng ml–1 IFN-γ for 3 more days (M-CSF + IFN-γ group) or medium only as control (Medium). Siglec-15 mRNA levels was determined by RT–PCR. Data are presented as mean ± s.e.m. after intrasample normalization to the reference gene GAPDH (n = 4 cell cultures). P values by an two-tailed unpaired t-test. In c and d, data are representative of two independent experiments.

  4. Extended Data Fig. 4 Effect of Siglec-15 as recombinant protein or cell surface protein on human and mouse T cell functions.

    a, The effect of plate-coated hSiglec-15-hIg or control hIg (5 μg ml−1) on human T cell proliferation in the presence of plate-coated anti-human CD3 mAb at the indicated concentrations. Proliferation of T cells was indicated by 3H-thymidine incorporation at 72 h. b, The effect of plate-coated mSiglec-15-mIg or control mIg (5 μg ml−1) on mouse splenic T cell proliferation in the presence of plate-coated anti-mouse CD3 mAb (1 μg ml−1). Proliferation of T cells was indicated by 3H-thymidine incorporation at 72 h. c,d, The effect of soluble mSiglec-15-mIg or control mIg (5 μg ml−1) on mouse splenic CD8+ T cells in the presence of coated anti-mouse CD3 mAb (1 μg ml−1). The cell proliferation as indicated by CFSE dilution (c) and IFN-γ in the culture medium (d) at 72 h are shown. e, The 293T-KbOVA-S15+ or S15 negative control cells were placed in a 384-well plate at 1 × 104 per well for 24 h, followed by the addition of OT-I (1 × 104 per well) pre-activated with OVA257–264. Real-time survival of target cells was monitored by the xCELLigence cellular impedance assay (left panel) and normalized by the value right before adding OT-I cells (normalized cell index). Data at 72 h are shown as a bar in the right panel. All data above are mean ± s.e.m. (n = 3 or 4 cell cultures) and representative of two independent experiments. P values by an two-tailed unpaired t-test.

  5. Extended Data Fig. 5 Normal phenotype of Siglec-15 deficient mice.

    a. Tissue histological analysis was performed on 18-month-old Siglec-15 KO mice and WT littermate and is shown as the pathological score (see Materials and Methods). The indicated tissues were fixed in formalin, embedded with paraffin, and stained with hematoxylin and eosin. The inflammatory status of tissues was evaluated on the basis of a semi-quantitative method that describes the level of immune infiltration. Data are presented as mean. bd, The body (b), spleen (c) and liver (d) weight of Siglec-15 KO and WT mice. Data are presented as mean ± s.d. e,f, The levels of anti-dsDNA IgG antibodies (e) and anti-nuclear antibodies (ANA) (f) in sera of 18-month-old Siglec-15 KO and WT mice were quantified by specific sandwich ELISA. Data are presented as mean ± s.e.m. In a–f, data are analyzed by two-tailed unpaired t-test (WT n = 27 mice; KO n = 34 mice; n.s., not significant). g, Gating strategy for OT-I T cell EdU incorporation and apoptosis analysis by flow cytometry. h, Siglec-15 KO or WT BMDCs pulsed with OVA257–264 peptide were injected intraperitoneally into WT mice at 5 × 105 per mouse followed by intraperitoneal injection of OT-I T cells at 2 × 106 per mouse 6 h later. The OT-I in the blood at the indicated time points were analyzed by flow cytometry. The results are shown as the percentage of OT-I among total CD8+ T cells. Data are mean ± s.e.m. (n = 5 mice per group). Data are analyzed by two-way ANOVA.

  6. Extended Data Fig. 6 Analysis of Siglec-15 mRNA expression in human cancers.

    a, The inverse correlation of mRNA expression levels between Siglec-15 and T cell signature genes (CD3E, IFNG, GZMA and GZMB) in bladder cancer by meta-analysis of TCGA databases. Pearson r score and P value are shown (n = 407 human samples). bd, Validation of anti-Siglec-15 antibody clone PA5-48221. b, Representative quantitative immunofluorescence images of positive staining on 293T cells overexpressing Siglec-15 (293T-S15+, left panel) compared to mock transfected 293T cells (293T-S15-, right panel) (DAPI, blue and S15, red).Scale bars, 100 um. Data are representative of four independent experiments. c, QIF scores of 293T-S15+ and 293T-S15 cell lines. Data are mean ± s.e.m. (n = 4 independent experiments). P values by two-tailed unpaired t-test. d, Comparison of Siglec-15 protein and RNA expression using RNAscope in situ detection by QIF. Pearson r score and P value are shown (n = 27 human samples). e, The relative levels of Siglec-15 mRNA in human cancer cell lines from the BioGPS database. f, Cell surface expression of Siglec-15 on LOX IMVI and U87 human cancer lines by staining with anti-Siglec-15 and control mAb and analyzed with flow cytometry. Data are representative of three independent experiments.

  7. Extended Data Fig. 7 Expression and function of Siglec-15 in mouse tumors.

    a, Siglec-15 mRNA expression in tumors from indicated mouse models by comparison to B7-H1, analyzed from the CrownBio MuBase database. b, Siglec-15 mRNA levels in tumors of B16-GMCSF and GL261 model was determined by RT–PCR on day 14 after inoculation. Spleen from a S15KO mouse was used as negative control. Data are relative levels to reference gene RPL13a. c, Flow cytometric analysis of Siglec-15 expression on infiltrating immune cell subsets of B16-GMCSF tumors from Siglec-15 WT and KO mice on day 14 after inoculation. Data are representative of two independent experiments. d,e, B16-GMCSF tumor cells at 1.5 × 106 per mouse or wild type B16 tumor cells at 1 × 106 per mouse were injected subcutaneously into Siglec-15 WT, KO or LysM-Cre KO as indicated. Tumor growth was measured regularly and is shown as the mean tumor diameter ± s.e.m. (n = 6 mice per group). P values by two-way ANOVA (n.s., not significant; P = 0.3180).

  8. Extended Data Fig. 8 Immunophenotyping of B16-GMCSF tumors.

    a–c, Mass cytometry analysis of tumor-infiltrating leukocytes isolated at day 14 after B16-GMCSF tumor cell inoculation as described in Fig. 5 (n = 3 mice per group). t-SNE plot of tumor-infiltrating leukocytes overlaid with the expression of indicated markers (a). Density t-SNE plots of an equal number of CD45+ tumor-infiltrating leukocytes in Siglec-15 KO and WT mice (b). The normalized expression value (mean mass intensity) of checkpoint receptors on tumor-infiltrating CD8+ T cells (c). d,e, On day 14 after B16-GMCSF tumor cell inoculation, spleens and lymph nodes (LN) from WT and KO mice were dissected (d). The percentage of CD4+ and CD8+ T cells in the draining and non-draining lymph nodes (LN) was analyzed by flow cytometry (e). Data are mean ± s.e.m. (n = 4 mice per group). P values by two-tailed unpaired t-test. f, B16-GMCSF tumor cells were injected subcutaneously into Siglec-15 WT and KO at 1.5 × 106 per mouse. Mice were treated with 200 μg anti-CD8 antibody every 3–4 days since 3 days before tumor inoculation. Tumor growth was measured regularly and is shown as the mean tumor diameter ± s.e.m. (n = 5 mice per group). P values by two-way ANOVA (n.s., not significant; P = 0.9372).

  9. Extended Data Fig. 9 Growth of GL261 Glioblastoma in Siglec-15 deficient mice and analysis of immune infiltration.

    a,b, GL261-luc cells were injected intracranially into Siglec-15 WT, KO or LysM-Cre KO mice at 4×105 per mouse. Mice were subsequently treated with a 4Gy whole brain radiation on day 4. Tumor volume in mice was measured by the IVIS imaging system every 4–5 days. Tumor growth in individual Siglec-15 WT or KO mice (left) and imaging at days 13 and 18 after tumor inoculation (right) are shown in (a) (n = 10 mice per group). Data are representative of two independent experiments. The GL261-luc tumor growth in Siglec-15 WT, KO and LysM-Cre KO mice is shown as mean bioluminescence in radiance ± s.e.m. (p/sec/cm2/sr) over time (b) (WT, n = 10 mice; KO, n = 10 mice; LysM-Cre KO, n = 8 mice). P values by two-tailed Mann-Whitney test. c–e, Flow cytometry analysis of tumor-infiltrating immune cells at day 14 after GL261 tumor inoculation (n = 4 per group). CD8+ T cells, CD4+ T cells, CD11b+ CD45high macrophages (MØ), CD11b+ CD45low microglia, and CD11c+ dendritic cells (DC) in brain (c) or spleen (d) were quantified by flow cytometry. Brain mononuclear cells were further re-stimulated with irradiated GL261-luc cells for 5 days. Total number of IFN-γ-producing CD8+ T cells and CD4+ T cells was determined by live cell counting and intracellular staining (e). Data are mean ± s.e.m. (n = 4 mice per group) and representative of two independent experiments. P values by two-tailed unpaired t-test (n.s., not significant; c, P = 0.1937; d, P = 0.0916 and 0.0624; e, P = 0.4820).

  10. Extended Data Fig. 10 Effect of α-S15 on established mouse tumors with tumor-associated macrophages.

    a, Binding of PE-labeled α-S15 (5G12) to 293T cells overexpressing human or mouse Siglec-15. 293T parental cells served as controls. Data are representative of three independent experiments. b,c, Human PBMCs were stimulated by coated OKT3 (0.1 μg ml−1) in 96-well plates for 3 days in the presence of 5 μg ml−1 hS15-hIg or control hIg with or without α-S15 at 12 μg ml−1. The proliferation of CD4+ T cell (b) and CD8+ T cell (c) was indicated by CFSE dilution. Data are mean ± s.e.m. (n = 6 cell cultures) and representative of three independent experiments. P values by two-tailed unpaired t-test. d, B16-GMCSF tumor cells were subcutaneously injected into WT C57BL/6 mice at 1.5 × 106 per mouse and subsequently treated with 200 μg α-S15 or isotype control mAb at day 5, 9, 13 and 17 (n = 7 mice per group). P values by two-tailed unpaired t-test. e, MC38 tumor cells (3 × 105) mixed with or without WT or KO BMDMs (2 × 105) were subcutaneously injected into C57BL/6 mice (n = 5 mice per group). P values by two-way ANOVA (n.s., not significant; P = 0.4920). f, MC38 tumor cells (3 × 105) mixed with Siglec-15 KO BMDMs (2 × 105) were subcutaneously injected into C57BL/6 mice and subsequently treated with 200 μg α-S15 or isotype control mAb at day 5, 9, 13 and 17 (n = 7 mice per group). P values by two-way ANOVA (n.s.; P = 0.9727). g, CT26 tumor cells (1.5 × 105) mixed with Balb/c BMDMs (1.5 × 105) were subcutaneously injected into Balb/c mice and subsequently treated with 200 μg α-S15 or isotype control mAb as described in the methods (n = 10 mice per group). Data are representative of two independent experiments. P values by two-way ANOVA. h,i, On day 15 after CT26 tumor inoculation as described in (g), tumor-infiltrating CD8+ T cells (h) and CT26 tumor-specific CD8+ T cells (i) were stained with anti-CD8 mAb and AH1 MHC-I dextramer and analyzed by flow cytometry (control, n = 5 mice; α-S15, n = 3 mice). P values by two-tailed unpaired t-test. j, CT26 tumor cells (1.5 × 105) mixed with Balb/c BMDMs (1.5 × 105) were subcutaneously injected into Balb/c mice. Mice were treated with 200 μg α-S15 or isotype control mAb and/or 100 μg anti-PD-1 mAb as described in the methods (n = 10 mice per group). P values by two-way ANOVA. In dj, data are presented as mean ± s.e.m. k, Expression of Siglec-15 on transduced MC38 cells (MC38-S15+) or parental cells (MC38-WT) as determined by staining with m03 mAb or control antibody and flow cytometry analysis. Data are representative of three independent experiments. l, OT-I T cells from OT-I/Rag-1 KO mice were injected intravenously into C57BL/6 mice that are subsequently immunized with OVA257–264 peptide and adjuvant as described in Fig. 3. Spleen cells were isolated on day 5 and stained by mouse Siglec-15 recombinant fusion protein or by control Ig for flow cytometry analysis. Data are shown in a histogram as specific binding to OT-I T cells gated by anti-CD8 mAb and OT-I tetramer positive staining. Data are representative of two independent experiments.

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Fig. 1: Identification of Siglec-15 as a T cell suppressive molecule in the TCAA.
Fig. 2: Expression of Siglec-15 by macrophages and its inhibitory activity for T cells.
Fig. 3: Inhibitory effect of Siglec-15 on antigen-specific T cell responses in vivo.
Fig. 4: Siglec-15 is abundant in human cancers.
Fig. 5: Effect of Siglec-15 on tumor growth in syngeneic mice.
Fig. 6: Effect of Siglec-15 mAb on established tumors in syngeneic mice.
Extended Data Fig. 1: Schematic representation of the outcome from the TCAA screening.
Extended Data Fig. 2: Siglec-15 mRNA expression in normal tissues of human and mouse origin.
Extended Data Fig. 3: Siglec-15 protein expression on human and mouse immune cells.
Extended Data Fig. 4: Effect of Siglec-15 as recombinant protein or cell surface protein on human and mouse T cell functions.
Extended Data Fig. 5: Normal phenotype of Siglec-15 deficient mice.
Extended Data Fig. 6: Analysis of Siglec-15 mRNA expression in human cancers.
Extended Data Fig. 7: Expression and function of Siglec-15 in mouse tumors.
Extended Data Fig. 8: Immunophenotyping of B16-GMCSF tumors.
Extended Data Fig. 9: Growth of GL261 Glioblastoma in Siglec-15 deficient mice and analysis of immune infiltration.
Extended Data Fig. 10: Effect of α-S15 on established mouse tumors with tumor-associated macrophages.