Lack of S100A9 affects generation of MDSCs under pathological conditions. (A) Enriched HPCs were isolated from bone marrow of S100A9 KO mice (KO) and their wild-type littermates (WT). Cells were cultured with GM-CSF and IL-4 for 5 d in complete culture medium or in the presence of EL-4 TCM. The cell phenotypes were evaluated by flow cytometry. Mean ± SD of the proportions of indicated cell populations from three mice are shown. (B) EL-4 cells (3 × 105) were injected s.c. into wild-type (WT) or S100A9 knockout (KO) mice and tumor size was measured. Anti-CD8 antibody (200 μg) was injected i.p. into KO mice 3 and 1 d before injection of tumor cells. The S100A9KO group included 12 mice, WT group 15 mice, and KO mice treated with anti-CD8 antibody 5 mice. Tumor size for each mouse is shown. (C) Phenotypes of splenocytes from wild-type (S100A9+/+) and knockout (S100A9−/−) mice 15 d after EL-4 cell injection were evaluated using multicolor flow cytometry. * represents statistically significant differences between the groups (P < 0.05). (D) Wild-type and S100A9 knockout mice were injected s.c. with 200 μl of CFA. The presence of Gr-1+CD11b+ cells was monitored in peripheral blood. Each group included 4 mice. Mean ± SD are shown. (E) Gr-1+CD11b+ cells in spleens of wild-type and S100A9 knockout mice on day 12 after CFA injection. Four mice per genotype were analyzed. * represents statistically significant differences between the groups (P < 0.05). (F) S100A9−/− mice were injected s.c. with 2 × 105 EL-4 tumor cells and then split into 2 groups of 5 mice. One group was left untreated (no transfer) and the other group was treated i.v. with 3 × 106 Gr-1+CD11b+ cells isolated from tumor-bearing wild-type mice (MDSC) on days 1, 3, 5, and 7 after tumor inoculation. Tumor size for each mouse is shown.