Figure 5. GM-CSF production by T_H1 and T_H17 cells is required for their encephalitogenicity.

Splenocytes from wild-type- or Csf2^−/−-MBP_Ac1-11 transgenic mice were activated with MBP_Ac1-11 peptide in the presence of IL-12 (T_H1 conditions) or TGF-β plus IL-6, anti-IFN-γ and anti-IL-4 during 72 h (T_H17 conditions). Cells were rested 2 days in the presence of IL-2 and then reactivated with MBP_Ac1-11 peptide in the presence of IL-12 (T_H1 conditions) or IL-23 (T_H17 conditions). After 72 h, cells were stimulated with PMA and ionomycin in the presence of GolgiPlug. (a) Flow cytometric analysis of IL-17A and IFN-γ expression in wild-type and Csf2^−/− T_H1 and T_H17 cells after the second stimulation. CD4⁺ cells are shown. (b) IL-17A, IFN-γ and GM-CSF concentrations were measured by ELISA in the supernatants after the second stimulation. (c) RORγt and T-bet expression on gated CD4⁺IFN-γ⁺ (T_H1) or CD4⁺IL-17A⁺ cells (T_H17) analyzed by flow cytometry after the second stimulation. Filled histograms represent isotype controls. (d) Real time PCR analysis of Ahr, CCL20, CCR6, IL-23R and RORα expression in CD4⁺ T cells enriched by magnetic beads after the second stimulation from wild-type or Csf2^−/− MBP_Ac1-11-splenocytes cultured in T_H17 conditions. (e) Clinical scores of recipient mice that received 5×10⁶ of either MBP_Ac1-11-specific wild-type or Csf2^−/− T_H1 or T_H17 cells enriched by magnetic beads after the second stimulation. Pertussis toxin was injected i.p. on days 0 and 2 post transfer. *p< 0.01; **p < 0.001. Data are representative of two independent experiments. (error bars, s.e.m).