Figure4. Type I IFNs are induced during anti-CD47 mediated tumor Inhibition and required.
(a) Four days after anti-CD47 or Rat Ig treatment (n=5/group), the single cell suspensions from tumors were sorted into CD45+CD11c CD11b+Ly6chi(Monocytes), CD45+CD11c−/+ CD11b+Ly6c F4/80+(Macrophage) and CD45+CD11c+CD11b−/+Ly6C F4/80 (DC)populations. mRNA level of ifna and ifnb in different cell subsets were quantified by real-time PCR assay. Representative data are reported as mean copy numbers ±s.e.m. after intrasample normalization to the levels of reference gene hprt in three independent experiments. *p < 0.05, **p<0.01, ***p < 0.001 (unpaired Student's t test). (b) C57BL/6 mice (n = 6) were injected s.c. with 106 MC38 cells and treated intratumorally with 50µg of anti-CD47 or isotype control rat Ig on days 11 and 14. 50µg anti-IFNAR1 antibody was administered intratumorally on days 0 and 2 after anti-CD47 treatment. (c)Ifnar1flox/flox and CD11c–CreIfnar1flox/flox mice (n = 6) were injected s.c. with 106 MC38 cells and treated with 50µg of anti-CD47 or rat Ig on days 10 and 15. (d) BMDCs from WT mice or ifnar1−/− mice were cultured with MC38-OTIp in the presence of anti-CD47 or rat Ig for 16h. Subsequently purified CD11c+ cells were cocultured with isolated CD8+ T cells from naive OT-I mice for 3 days. IFN-γ production in supernatant was determined by CBA. Data are reported as means ±s.e.m. Tumor growth is reported as the mean tumor size ±s.e.m. over time. **p < 0.01; One representative experiment out of three independent experiments is depicted.