Figure 2

Cytokine production and transcription factor expression in antigen reactive CD4⁺ T cells. At day 10 days post first type II collagen (CII) immunization, draining lymph node (LN) cells from C57BL/6 mice (n = 3 or 4) and RORγt Tg mice (n = 3 or 4) were cultured with or without 100 μg/mL of denatured CII for 72 h. (A) IL-17 and IFNγ levels in supernatants measured by ELISA. (B) Cells were analyzed by staining for intracellular IL-17 and IFNγ. Data represent gated CD3⁺ and CD4⁺. Bar graphs show percentages of cytokine-positive cells. (C) The expression levels of RORγt and T-bet were evaluated by intracellular staining. Data represent gated CD3⁺ and CD4⁺. In the histograms, the gray shaded area represents data for the isotype control. Bar graphs show mean fluorescence intensity (MFI) of T-bet and RORγt Tg. (D) IL-10 levels in supernatants measured by ELISA. (E , F) Cells were analyzed by staining for intracellular IL-17 and IL-10 (E), and propium iodide (PI) and Annexin V (F). Data represent gated CD3⁺ and CD4⁺. Data are representative of two independent experiments with similar results, and are mean ± standard error of the mean. *P < 0.05, **P < 0.01, versus C57BL/6 mice, Student’s t-test.