The role of iC3b on MDSC differentiation. (A) HpSCs enhances accumulation of C3 activation products on myeloid cells. HpSCs isolated from B6 mice were added at the beginning of B6 BM cell culture at a ratio of 1:80 in the presence of GM-CSF for 5 days. Culture without HpSCs served as the control (None). CD11b⁺ cells were purified with magnetic beads, stained with anti-C3 for flow analysis, and expressed as a histogram (the filled area indicates the isotype control). (B) HpSCs produce functional factor B and D. Flow analysis of C3 activation product deposition on zymosan that were incubated with sera from factor B or factor D knockout mice with compensation by control medium (Control) or HpSC culture medium. (Right panels) mRNA expression of factor B and D in HpSCs determined by semiquantitative PCR. (C) Exogenous iC3b facilitated generation of CD11b⁺CD11c⁻ cells in a dose-dependent manner. Graded doses (as indicated) of exogenous iC3b or C3d were added at the beginning into B6 BM cell culture with GM-CSF for 5 days. The floating cells were harvested and stained with anti-CD11b and -CD11c and analyzed by flow cytometry (gated on CD11b⁺ cells). The number is the percentage of positive staining in CD11b⁺ cells. The expression of CD11c was also demonstrated in a histogram (gated on CD11b⁺ cells) following incubation with various concentrations of iC3b (right panel). (D) The iC3b-conditioned myeloid cells show immature phenotype. Expression of the key molecules was analyzed by flow cytometry by gating on CD11b⁺ cells and expressed as histograms (open area refers to the isotype control). The figure shows the results of iC3b (10 μg/mL) group compared with no iC3b control. (E) The iC3b-induced myeloid cells inhibit the T-cell proliferative response. CFSE-labeled BALB/c spleen T cells were elicited with B6 DCs at a ratio of 20:1 (3 days). The tested CD11b⁺ cells were purified from the iC3b (10 μg/mL) group, and added at the beginning into the culture at a DC:CD11b⁺ cell ratio of 1:0.5 or 1:1. Addition of DCs instead of the CD11b⁺ cells at a ratio of 1:1 served as control. The proliferation of T cells was determined by CFSE dilution (gated on the CD3⁺ population). These data are representative of three separate experiments.