Figure 5.

Th22 and Th17 cells are distinguished by reciprocal expression of T-bet, AhR and RORγt

(A, B) FACS-sorted naïve OTII-Tg CD4⁺ T cells were activated by Ova peptide in presence of CD4⁺ T-cell depleted splenic APCs from Il23a^−/− mice in the presence of IL-6 alone (Th22 polarization) or IL-6+TGFβ Thi7 polarization. CD4⁺ T cells were purified by magnetic sorting after 60h of culture and RNA was isolated for microarray-based gene expression analysis. Shown is a heat map of genes with more than 4-fold expression differences between the two populations (A) and transcriptome profiling of Th22- or Th17-polarized cells by scatterplot analysis (B).

(C) Naïve CD4⁺ T cells isolated from C57BL/6 mice were activated by anti-CD3 and anti-CD28 in presence of IL-6 and IL-23 (Th22 polarization), IL-6 and the indicated concentrations of TGFβ Thi7 polarization), or absence of added cytokines (Th0). On day 3 the cells were further stimulated with PMA/ionomycin for 6 hours and analyzed by RT-PCR for Rorc ,Tbx21 and Ahr transcripts. Data are means +/− SEM (*p<0.05, **p<0.01).

(D) Naïve OTII CD4⁺ T cells were simulated with Ova peptide in presence of IL-6, IL-6+IL-23 or IL-6+TGFβ and analyzed by flow cytometry for frequencies of IL-22⁺ CD4⁺ T cells on day 3 following stimulation for 4 hours with PMA/I in the presence of brefeldin A. The expression of T-bet and RORγt within the IL-22⁺ CD4⁺ gates was determined by intracellular staining (histograms).

All data are representative of three or more experiments with a minimum of 3 mice per group.