Impact of C3 produced by HpSCs on induction of MDSCs in vitro. (A) mRNA expression of C3 in hepatocytes and HpSCs from normal B6 mice were determined by qPCR assay. (B) Impact of C3 produced by HpSCs on myeloid cell development. HpSCs prepared from C3^−/− or WT B6 mice were stained with anti-C3 Ab (red) and examined under a microscope (left panels). HpSCs from C3^−/− or WT 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 a control. The floating cells were harvested, stained for CD11b, CD11c, and analyzed by flow cytometry (middle panels). The number represents the percentage of the positive cells in the whole-cell population. The absolute number of CD11c⁺ cells per well were calculated based on flow analysis (right panel). (C) Expression of key surface molecules. The cells were analyzed by gating on CD11b⁺ cells and expressed as histograms. The clear areas are isotype controls. The number is a percentage of the positive cells in the CD11b⁺ cells. (D) Expression of iNOS and arginase 1. CD11b⁺ cells were purified (magnetic beads), examined for mRNA expression of iNOS and arginase 1 by qPCR, and expressed as a mean ± 1 SD (n = 3). (E) Allostimulatory activity. BALB/c spleen T cells (2 × 10⁵ per well) were cultured with a graded number of irradiated CD11b⁺ cells in triplicate for 3 days. The proliferative response was determined by ³H-thymidine incorporation and expressed as mean cpm ± 1 SD. (F) CD11b⁺ cells from the C3^−/− HpSC group largely lose the ability to inhibit the T-cell proliferative response. In an MLR culture, CFSE-labeled B6 spleen T cells were cultured with irradiated BALB/c DCs at a ratio of 20:1 for 3 days. CD11b⁺ cells purified from the DC culture in the presence of WT or C3^−/− HpSCs (referred as MDSCs) were added at the beginning into the MLR culture at a DC:MDSC ratio of 1:1. Addition of DCs, instead of MDSCs, served as control. The proliferative response was determined by CFSE dilution gated in the CD3⁺ population. The data are representative of 3 separate experiments.