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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 2. Expression of Siglec-15 by macrophages and its inhibitory activity for T-cells.

Figure 2.

(a, b) Quantitative PCR (Q-PCR) estimation on Siglec-15 mRNA levels in human macrophages derived from CD14+ peripheral blood monocytes with 100ng/ml M-CSF (a), or LPS-treated mouse BMDMs or BMDCs (b) at indicated time points. Data are mean ± s.e.m. after normalization to reference gene GAPDH (a), or RPL13a (b), and representative of two independent experiments.

(c) Flow cytometry analysis of Siglec-15 expression by anti-Siglec-15 mAb (clone m03) staining on mouse myeloid cell subsets from blood, spleen, bone marrow (BM) or peritoneal cavity of S15KO and WT mice. MΦ, macrophage. Data are representative of three independent experiments.

(d) Human macrophages were generated from CD14+ peripheral blood monocytes from two healthy donors (D#1 and D#2) with M-CSF for 7 days. Alternatively, monocytes were cultured with M-CSF for 4 days followed by M-CSF + IFN-γ for 3 more days. Siglec-15 expression was analyzed by flow cytometry with anti-Siglec-15 (clone 1H3) or an isotype control mAb staining.Data are representative of two independent experiments.

(e-g) The % of divided human peripheral CD8+ (e) or CD4+ T-cells (f) as indicated by CFSE dilution, as well as IFN-γ in the culture medium (g) after stimulation with 0.1 μg/ml of anti-CD3 in the presence of 5 μg/ml human Siglec-15 fusion protein (hS15-hIg) or control (hIg) for 3 days. (e and f, n = 6 cell cultures; g, n = 3 cell cultures from the same donor)

(h, i) OT-I T-cells pre-activated with OVA257-264 (1×105/well) were co-cultured with irradiated 293T-KbOVA cells stably expressing Siglec-15 (293T-KbOVA-S15+) or mock (293T-KbOVA-Control) (2×104/well) in a 96-well plate. OT-I T-cell proliferation was determined by 3H-thymidine incorporation at 72 hrs (h). The cytokine levels in the culture medium were analyzed at 48 hrs (i). (n = 4 cell cultures from the same mouse)

(j) The % of divided OT-I T-cells as indicated by CFSE dilution after co-cultured for 3 days with S15KO or WT peritoneal macrophages pulsed with OVA257-264 at the indicated concentrations. (n = 3 cell cultures)

In e-j, data are presented as mean ± s.e.m. and representative of two or three independent experiments. P values by two-tailed unpaired t-test.

See also Extended Data Figs. 2-4.