(A) CTV dilution of CD4⁺ T cells from Ctrl and STAT1C^D4-Cre mice stimulated with plate-bound anti-CD3/CD28 for 3 days. Open histogram for WT and gray-shaded histogram for STAT1^CD4-Cre mice. (B) Percentage of live cells enumerated by flow cytometry. Data are representative of 3 experiments. Significance calculated with 2-tailed Mann-Whitney U test. (C and D) 2D2 (CD45.2⁺) and STAT1^CD4-Cre/2D2 (CD45.1⁺) CD4⁺ T cells were cotransferred at a 1:1 ratio into RAG^KO hosts, which were immunized with MOG_35–55/CFA and PT. On day 12, CNS were analyzed for the percentage of CD45.2⁺ and CD45.1⁺ cells. Representative (C) and quantified (D) percentage of CD45.1⁺ or CD45.2⁺ cells among CD4⁺Vβ11⁺ T cells (n = 5 mice/group). Significance calculated with 2-tailed Mann-Whitney U test. (E–G) Mice were immunized with MOG35-55/CFA, and 8–10 days later, CD4⁺ T cells from the dLNs were enumerated and analyzed for IFN-γ and IL-17 production. Percentage (E) and absolute number (F) of cytokine-producing cells among CD4⁺ T cells are presented. Significance was determined by a 2-tailed unpaired t test with Welch’s correction (n = 4–10 mice/group). (G) Proliferative response of dLNs from immunized mice was assessed by [3H] thymidine incorporation in response to MOG_35–55 peptide. Significance calculated with 2-way ANOVA with Sidak’s multiple comparisons. Data are representative of 2 experiments with n ≥ 3 mice in each experiment. (H–I) Naive CD4⁺ T cells from 2D2 (CD45.2⁺) and STAT1^CD4-Cre/2D2 (CD45.1⁺CD45.2⁺) mice were cotransferred into RAG^KO mice at a 1:1 ratio and immunized with MOG_35–55/CFA 1 day later. On day 6, frequencies of CD45.2⁺ and CD45.1⁺CD45.2⁺ cells in dLNs were assessed by flow cytometry. Representative (H) and quantified (I) percentage of CD45⁺ among CD4⁺Vβ11⁺ T cells (n = 8 mice/group). Significance calculated with 2-tailed Mann-Whitney U test. Data are representative of 2 experiments. (*P < 0.05, **P < 0.01, ***P < 0.001).