Fig. 1. Three assays are developed to dissect RORγt function.

(A) Representative flow cytometric analysis (left) of CD4⁺ and CD8⁺ thymocytes ex vivo developed from indicated genotypes of CD4⁻CD8⁻ thymocytes placed on the OP9-DL4 stroma cells for 3 days (n = 3 per genotype). The number indicates the percentage of cells in the gated area throughout. Right: Percentage of CD4⁺CD8⁺ and CD4⁺ cells. (B) Representative flow cytometric analysis (left) of CD4⁺ and CD8⁺ thymocytes ex vivo developed from sorted RORγt^−/−CD4⁻CD8⁻ thymocytes transduced with retrovirus expressing GFP alone (EV) or with RORγt, and cultured on OP9-DL4 stroma cells for 3 days (n = 3 per genotype). Right: Percentage of CD4⁺CD8⁺ + CD4⁺ cells. (C) Representative flow cytometric analysis of IL-17A (left) and percentage of IL-17A⁺ cells (right) among indicated genotypes of CD4⁺ T cells transduced with retrovirus expressing GFP alone (EV) or with RORγt and polarized under T_H17 conditions for 3 days (n = 3 per genotype). (D) Mean clinical EAE score of Rag1^−/− mice adoptively transferred with same number of Tg^Tcr2D2 or RORγt^−/−/Tg^Tcr2D2 CD4⁺ T cells transduced with retrovirus expressing GFP alone (EV) or with RORγt and polarized under T_H17 conditions for 3 days. Bars are means ± SE. ***P < 0.001 (two-tailed Student’s t test).