Extended Data Fig. 10: Effect of α-S15 on established mouse tumors with tumor-associated macrophages.
From: Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy
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 d–j, 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.
