Fig. 1. Csf2 regulates tissue mononuclear phagocyte frequency and effector functions required for T helper cell homeostasis.

(A) Contour plots and bar graph show percentages of CD3⁺CD4⁺Foxp3⁺ colonic T_regs among total colonic lamina propria CD45⁺ cells in C57Bl/6 and Csf2^−/− mice. (B) Bar graphs show percentages of CD3⁺CD4⁺ T cells producing IFN-γ, IL-17, IL-10, or IL-2 among total colonic CD45⁺ cells in C57Bl/6 and Csf2^−/− mice after 4 hours of stimulation with phorbol 12-myristate 13-acetate (PMA)–ionomycin in the presence of Brefeldin A. (C) Bar graphs show absolute numbers of MHCII⁺CD11c⁺ cells among total colonic CD45⁺ cells in C57Bl/6 and Csf2^−/− mice. (D) DCs were characterized as CD45⁺MHCII⁺CD11c⁺ and further subdivided into CD103⁺ DCs, CD103⁺CD11b⁺ DCs [double positive (DP) DCs], and CD11b⁺ DCs. Macrophages (MP) were characterized as MHCII⁺CD11c⁺CD11b⁺F4/80⁺CD64⁺ cells. Bar graphs show absolute numbers of CD103⁺ DCs, DP DCs, CD11b⁺ DCs, and MPs in C57Bl/6 and Csf2^−/− mice. (E) Percentages of ALDEFLUOR⁺ cells among each colonic DC subset and MPs in C57Bl/6 and Csf2^−/− mice. (F and G) Bar graphs show enzyme-linked immunosorbent assay (ELISA) measurement of TGF-β production by sorted DP DCs (F) and IL-10 production by sorted MPs (G). (H) Colonic MPs or DCs were FACS-sorted from C57BI/6 or Csf2^−/− mice and cocultured with naïve T cells alone or in the presence of exogenous Csf2. Bar graphs show absolute numbers of Foxp3⁺ T_regs. Data are representative of six independent experiments and are shown as mean ± SD. (I) Contour plots show percentages of CD45⁺CD3⁺CD4⁺Foxp3⁺ colonic T_regs in Csf2^−/− mice 12 to 14 days after injection with B16 melanoma cells or B16 cells overexpressing Csf2 (B16^Csf2). Bar graphs show absolute number of colonic T_regs in the indicated groups. All data (A to H) are shown as mean ± SD of three independent experiment with at least three mice per experiment. Student’s t test (A to F) and one-way analysis of variance (ANOVA) Bonferroni’s multiple comparison test (H and I) were performed. Statistical significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.