Fig3. Anti-CD47 triggers the cross-priming ability of DCs.

(a) BMDCs or BMM were cultured with MC38-OTIp in the presence of fresh GM-CSF and anti-CD47 overnight. Subsequently purified CD11c⁺ cells or F4/80⁺ cells were co-cultured with isolated CD8⁺ T cells from naive OTI mice for three days and analyzed by IFN-γ CBA. (b)–(c) MC38-OT1p bearing mice (n=5/group) were treated twice with 50µg of either anti-CD47 or rat Ig on days 11 and 14 intratumorally. Five days after the initial treatment, DLN (b) and Tumor (c) infiltrating DCs and macrophages were isolated and co-cultured with isolated CD8⁺ T cells from naive OTI mice for three days. IFN-γ production was detected by CBA. (d) A20 or (e) MC38 tumor bearing B6 mice (n=5/group) were treated with 50 µg of either anti-CD47 or rat Ig isotype control on day 10. Four days after the antibody treatment, 3×10⁴ Tumor infiltrating DCs and macrophages were isolated and co-cultured with isolated 3×10⁵ CD8⁺ T cells from A20 (d) or MC38 (e) vaccined-mice. 48 hours later, IFN-γ-producing cells were enumerated by ELISPOT assay. (f) 5 weeks after the indicated CD11c–DTR bone marrow chimera reconstitution, B6 mice (n=5/group) were injected subcutaneously with 10⁶ MC38 cells and treated with 50µg of anti-CD47 or rat Ig on days 14 and 20. Diphtheria toxin or PBS was administrated on the same day as treatment. Data are reported as the means ±s.e.m.. *p < 0.05 **p < 0.01 ***p < 0.001 (unpaired Student's t test). One representative experiment out of three independent experiments is depicted.