Figure 3: T cells require Bhlhe40 for normal cytokine production after immunization.

(a,b) ELISPOT assays for the quantitation of cells secreting (a) IL-2, IFN-γ, and IL-17A or (b) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40^−/− mice. Data for IL-2, IFN-γ, IL-17A and GM-CSF are combined from three independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). (c,e) DLN cells from immunized WT and Bhlhe40^−/− mice (n=14 per group) were cultured with or without MOG(35–55) and with or without IL-1β, IL-23 and/or IL-12 as indicated. (c) GM-CSF or (e) IL-10 was measured in the supernatant at day 4. Data are combined from five independent experiments. Cells from all mice were not used in all conditions in each of the four experiments. (d) DLN cells from immunized WT and Bhlhe40^−/− mice were cultured with MOG(35–55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4⁺ T cells. (f) DLN cells from immunized WT and Bhlhe40^−/− mice were cultured with MOG(35–55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4⁺ T cells. (g) Frequencies of GM-CSF⁺ and IL-17A⁺ γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40^−/− mice (n=3 per group) as determined by ICS. (h) DLN cells from immunized WT and Bhlhe40^−/− mice were cultured with or without MOG(35–55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 h and then analysed for IL-17A and GM-CSF by intracellular staining (that is, our normal ICS protocol). Representative plots are gated on γδ T cells.