Figure 4.

C5a drives Th17 cell differentiation in synergy with TLR or GM-CSF. (A) C57BL/6 CD4⁺ T cells were cultured with TLR4^−/−, MyD88^−/−, or wild-type C57BL/6 macrophages in the presence of TGF-β with or without C5a. (B) Macrophages alone from these mice or co-cultured with anti-CD3–stimulated CD4⁺ T cells from IL-6^−/− mice were stimulated with C5a overnight. IL-6 in the supernatant was determined by ELISA. (C) MyD88^−/− macrophages were stimulated by C5a overnight in the presence or absence of cytokines at the indicated doses. IL-6 in the supernatant was determined by ELISA (triplicates). Error bars are means ± SD. (D) Freshly isolated BABL/c or SKG splenic CD4⁺ T cells were stained for intracellular IL-17 and GM-CSF. (E) BALB/c CD4⁺ T cells were cultured with macrophages in the presence or absence of C5a and/or TGF-β, stimulated with anti-CD3, and stained for intracellular cytokines on day 3. Results in A–E represent three independent experiments. Numbers in A, D, and E indicate percentages.