Figure 3.
Existence of a mature Th17 population in antigen-fed and immunized CD11b−/− mice. (a) The IL-17–expressing cells represent a mature and stable Th17 population. Total CD4+ T cells, which were purified by MACS from the iLNs of OVA-fed and immunized WT or CD11b−/− mice, were cocultured with their corresponding irradiated APCs in the presence of anti-CD3 mAbs with or without 2 ng/ml TGFβ or 20 ng/ml IL-23 plus anti–IL-4 and anti-IFNγ for 3 d. They were then restimulated with PMA and ionomycin in the presence of GolgiPlug for 5 h to retain the cytokines intracellularly. The restimulated cells were analyzed by intracellular staining with anti–IL-17 and anti-IFNγ, and the percentages of CD17+IFNγ− cells within CD4+ population were determined by three-color flow cytometry and shown on the right. *, P < 0.0001, WT versus CD11b−/−. n = 4. (b) Th17 cells from CD11b−/− mice are capable of maintaining a stable Th17 phenotype under different polarizing conditions. Purified CD4+ T cells from OVA-fed and immunized WT or CD11b−/− mice were cocultured with their irradiated APCs in the presence of anti-CD3 under either Th1 (IFNγ plus anti-IL-4), Th2 (IL-4 plus anti-IFNγ), or Th17 (TGFβ plus IL-6) polarizing conditions for 3 d. The percentage of CD17+IFNγ− CD4+ T cells was determined by intracellular cytokine staining. Data shown are representative of three mice per group; these experiments were repeated three times, with similar results.