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. Author manuscript; available in PMC: 2020 Apr 22.
Published in final edited form as: Nat Med. 2019 Mar 4;25(4):656–666. doi: 10.1038/s41591-019-0374-x

Figure 6. Effect of Siglec-15 mAb on established tumors in syngeneic mice.

Figure 6.

(a) The % of divided OT-I T-cells (1×105/well) was analyzed by CFSE dilution after co-culture with irradiated 293T-KbOVA-S15+ or control cells (2×104/well) in a 96-well plate for 5 days with 10 μg/ml Siglec-15 antibody (α-S15) or isotype control mAb (Control).

(b) The % of divided OT-I T-cells (2×105/well) was analyzed by CFSE dilution after co-culture for 3 days with S15KO or WT BMDMs (2×104/well) pulsed with 0.1ng/ml OVA257-264 in the presence of 10 μg/ml α-S15 or isotype control antibody. In a and b, data are mean ± s.e.m. (n = 3 cell cultures). P values by two-tailed unpaired t-test.

(c) MC38 tumor cells (3×105) mixed with WT BMDMs (2×105) were s.c. injected into WT C57BL/6 mice. Mice were treated with 200μg α-S15 or control antibody from day 5, every 4 days for 4 doses in total. Data are mean ± s.e.m. (n = 5 mice per group). P values by two-way ANOVA.

(d, e) Naïve splenic CD8+ T-cells isolated from PD-1 KO mice were labeled with CFSE and co-cultured with WT BMDMs in the presence of anti-CD3 and α-S15 or control antibody. T-cell proliferation (d) and cytokine production (e) was analyzed at 72 hrs. Data are mean ± s.e.m. (n = 3 or 4 cell cultures). P values by two-tailed unpaired t-test.

(f, g) IL-2 (f) and TNF-α (g) production from human PBMCs after stimulation with anti-CD3 and SEB in the presence of α-S15, α-PD-1 (Nivolumab) or their control antibodies for 72hrs. Data are mean ± s.e.m. (n = 5 cell cultures). P values by two-tailed unpaired t-test (n.s., not significant; P = 0.1759).

(h) MC38-S15+ cells were s.c. injected into WT C57BL/6 mice (4×105/mouse). Mice were treated with 200μg α-S15 or control antibody from day 6, every 4 days for 4 doses in total. Data are mean ± s.e.m. (n = 5 mice per group). P values by two-way ANOVA.

(i) MC38-S15+ cells were i.v. injected into WT C57BL/6 mice (1×105/mouse). Mice were treated with 400 μg α-S15 or control antibody from day 2, every 4 days for 6 doses in total. Lungs were harvested on day 28 and tumor nodules were counted. Data are mean ± s.e.m. (n = 8 mice per group). P values by two-tailed unpaired t-test.

All data are representative of two or three independent experiments. See also Extended Data Fig. 10.