Figure 4.
GM-CSF production by CD4+ T cells promotes proliferation and differentiation of Th17, but not Th1 or Th2, cells in vitro. Naive OVA323-339 TCR transgenic CD4+ T cells purified from spleens of DO11.10/GM-CSF−/− or DO11.10/GM-CSF+/+ mice were cocultured with splenic DC purified from GM-CSF-deficient (KO) or WT mice in the presence of indicated concentrations of ovalbumin protein or OVA323-339 peptide. (A) CD4+ T cell activation measured by CD25 and CD62L surface expression after 3 d of culture. (B) CD4 T cell proliferation measured by pulsing cells with 3H-Thymidine for the last 12 h of a 60-h coculture. (C) Th1/Th2 cell differentiation driven by indicated antigen concentrations was determined by intracellular staining of IFN-γ and IL-4 after 5 d of coculture. (D and E) Cocultures were stimulated by 10 μM OVA323-339 and 1 μM CpG for 4 d. IL-17, IFN-γ, and GM-CSF production by CD4+ T cells was determined by intracellular staining in cultures containing GM-CSF+/+ CD4+ T cells and DC (D) or indicated combinations of WT and KO T cells and DC (E). (F) WT and KO CD4+ T cells and DC were cultured with anti-CD3 in Th17-polarizing conditions including 5 ng/ml TGFβ1, 20 ng/ml IL-6, 20 ng/ml IL-21, 1 μM CpG, and 100 μg/ml curdlan. Cytokine production was determined by intracellular staining at day 4.