(A) Steady-state activation of CD4⁺ T cells from LNs and SP of 2D2xTH and 2D2xTH Il21r KO mice that were harvested ex vivo and stained for CD62L and CD44. (B) ³H proliferation assay of T cells stimulated with rMOG (left panel) or MOG_35–55 (right panel) with varying concentrations of antigen (0.01 μg/ml to 10 μg/ml). (C) Flow cytometry analysis of CD62L and CD44 gated on Vα3.2⁺CD4⁺ T cells that were cocultured in vitro with rMOG (10 μg/ml) and sorted TH B cells from either 2D2xTH or 2D2xTH Il21r KO mice. (D) Intracellular cytokine staining of IFN-γ and IL-17 from in vitro–cultured Vα3.2⁺CD4⁺ T cells that were stimulated with rMOG (0 μg/ml or 10 μg/ml) and IL-23 for 4 days. (E) Quantitative RT-PCR of Il23 mRNA from TH B cells sorted from indicated mice that were cultured in vitro for 4 days with rMOG (10 μg/ml). (F) Cytokine analysis (cytometric bead assay [CBA]) of IL-23 from TH B cells sorted from 2D2xTH and 2D2xTH Il21r KO mice that were cultured in vitro for 4 days with rMOG at indicated concentrations. (G) Cytokine analysis (CBA) of IL-23 of TH B cells and DCs sorted from 2D2xTH mice that were cultured in vitro for 4 days with no MOG, MOG_35–55 (10 μg/ml), or rMOG (10 μg/ml). (H) Cytokine analysis using CBA of IL-23 in TH B cells and DCs (CD11c⁺) sorted from 2D2xTH mice that were cultured in vitro with 2D2 T cells for 4 days with no MOG, MOG_35–55 (10 μg/ml), or rMOG (10 μg/ml). Data shown are representative of at least 3 independent experiments with at least n = 3 mice for each experiment. Data represent mean ± SEM for B and E–H. Statistics were determined by Student’s 2-tailed t test. ***P < 0.001.