(A) Freshly-isolated purified CD4SP thymocytes from Jak3^+/− and Jak3^−/− OT-II Bcl2 mice were stimulated with or without IFN-γ (100ng/ml) for 30 min. Total cell lysates were prepared and blotted for STAT1p and total STAT1. CD4⁺ splenocytes from Jak3^+/+ OTII-transgenic mice were used as a control.
(B) Purified CD4SP thymocytes from Jak3^+/− and Jak3^−/− OT-II Bcl2 mice were cultured in Th1-skewing conditions for 4 days. Cells were rested overnight, and then stimulated with or without IL-12 (15ng/ml) for 25 min. Total cell lysates were prepared and blotted for STAT4p and total STAT4.
(C) Purified CD4SP thymocytes from Jak3^+/− and Jak3^−/− OT-II Bcl2 mice were stimulated under non-, Th1- or Th2-skewing conditions for 4 days. T-bet mRNA levels were analyzed by real-time quantitative PCR. Data are normalized to the levels of 18srRNA in each sample (left panel). Total cell lysates from freshly isolated CD4SP thymocytes (naïve) or Th1-polarized cells (Th1) were prepared and immunoblotted for T-bet and β-actin (right panel).

(A) Purified CD4SP thymocytes from Jak3^+/− and Jak3^−/− OT-II Bcl2 mice were cultured under Th1-skewing conditions for 4 days (lanes 7–12) and compared with freshly isolated cells (naïve, lanes 1–6). ChIP analysis was done with an anti-T-bet antibody (lanes 3, 6, 9, 12) or a rabbit IgG control antibody (lanes 2, 5, 8, 11).
(B) CD4⁺ splenocytes from Jak3^+/+ OTII-transgenic mice were cultured with vehicle alone or PS078507 (625nM) under Th1-skewing conditions for 4 days. ChIP analysis was done with a rabbit IgG antibody control (top) or an anti-T-bet antibody (middle). The location of PCR primers is indicated in Supplemental Figure 4 and primer sequences are shown in Supplemental Table I.

(A) CD4⁺ splenocytes were isolated from Jak3^+/+ OTII-transgenic mice and were stimulated under Th1-skewing conditions for 0h, 12h, 24h, 48h and 72h. At each timepoint, ChIP analysis was performed with a rabbit IgG control antibody (top) or an antibody to acetylated histone H3 (H3 primer set #3).
(B) CD4⁺ splenocytes from Jak3^+/+ OTII-transgenic mice were stimulated under Th1-skewing conditions, with vehicle alone or the Jak3 inhbitor (PS078507 at 625nM) added at the indicated timepoints following the initiation of the cultures. All cultures were harvested at day 4, at which time the cells were restimulated and analyzed for IFN-γ production by intracellular cytokine staining. Cell initially cultured in vehicle alone were restimulated in the presence of PS078507 at 625nM (Restim).
(C) CD4⁺ splenocytes from Jak3^+/+ OTII-transgenic mice were stimulated under Th1-skewing conditions in vehicle alone or with PS078507 (625nM) added at the indicated times following the initiation of the cultures. At day 4, ChIP analysis was performed with a rabbit IgG control antibody (top) or an antibody to acetylated histone H3 (middle) (H3 primer set #3). Freshly isolated Jak3^+/+ OTII-transgenic cells were used for comparison (naïve).

CFSE-labeled CD4SP thymocytes from Jak3^+/− and Jak3^−/− OT-II Bcl2 mice were stimulated with anti-CD3 plus anti-CD28 antibodies and cultured under non-, Th1- or Th2-skewing conditions for 4 days.
(A) Cells were restimulated with PMA plus ionomycin for 5 hours. Dot-plots show intracellular staining for IFN-γ versus CFSE fluorescence. The graphs show percentages of IFN-γ producing cells from five different experiments, with the means indicated by horizontal bars. Differences between Jak3^+/− and Jak3^−/− responses are statistically significant, with the p values indicated.
(B, C) Following 4-day culture in Th1- or Th2-polarizing conditions, cells were restimulated with PMA and ionomycin for 24h. B) Supernatants were analyzed for production of IFN-γ, IL-4, IL-5 and IL-10 by ELISA. C) Ifng mRNA levels were analyzed by real-time quantitative PCR. Data are normalized to levels of 18srRNA in each sample.

(A) Purified CD4⁺ splenocytes from Jak3^+/+ OTII-transgenic mice were labeled with CFSE and stimulated with anti-CD3 plus anti-CD28 antibodies for 4 days in the presence of vehicle alone (WT vehicle) or PS078507 at 625nM (Jak3 inhibitor). Histograms show CFSE fluorescence.
(B, C) CD4⁺ splenocytes from Jak3^+/+ OTII-transgenic mice were cultured under non-and Th1-skewing conditions for 4 days, in vehicle alone or PS078507 (625nM). Cells were restimulated with PMA plus ionomycin for 6 hours. B) Dot-plots show IFN-γ intracellular staining versus forward scatter. The graphs below show percentages of IFN-γ producing cells from four different experiments, with the means indicated by horizontal bars. Differences between cells cultured in vehicle alone or PS078507 (Jak3 inhibitor) are statistically significant, with the p values indicated. C) Total cell lysates were prepared and immunoblotted for T-bet and β-actin.

(A, B) Purified CD4SP thymocytes from Jak3^+/− and Jak3^−/− OT-II Bcl2 mice were stimulated under Th1-skewing conditions for 4 days (lanes 7–12). Freshly-isolated cells (naïve, lanes 1–6) were used for comparison. A) Nuclei were isolated, digested with HinfI, and three-fold dilutions of genomic DNA were subjected to LM-PCR with primers for the Ifng proximal promoter (LM-PCR primer set #2). B) ChIP analysis was performed with an antibody to acetylated histone H3 (lanes 3, 6, 9, 12) or a rabbit IgG control antibody (lanes 2, 5, 8, 11) (H3 primer set #3).
(C) CD4⁺ splenocytes from Jak3^+/+ OTII-transgenic mice were cultured in vehicle alone or with the Jak3 inhibitor (625nM) in Th1-skewing conditions for 4 days. ChIP analysis was performed with a rabbit IgG control antibody (top) or antibody to acetylated histone H3 (middle) (H3 primer set #3).
(D) Purified CD4SP thymocytes from Jak3^+/− and Jak3^−/− OT-II Bcl2 mice were stimulated under Th1-skewing conditions. Parallel cultures were treated with or without sodium butyrate (SB; 200µM), added at 24h post-stimulation. At day 4, cells were restimulated with PMA and Ionomycin. The graph shows the percentage of cells producing IFN-γ as assessed by intracellular staining. Data are pooled from three independent experiments. Differences between sodium butyrate treated and untreated samples for each genotype of cells are statistically significant, with p values indicated. Differences between sodium butyrate treated Jak3^+/− and Jak3^−/− cells are not significant.

(A) Purified CD4⁺ T cells from Jak3^+/+ OTII-transgenic mice were stimulated and cultured in Th1-skewing conditions for 2 days. Cells were rested overnight, and then restimulated with IL-2 (50ng/ml) for 1h. ChIP analysis was performed using an anti-STAT5a antibody. Rabbit IgG antibody was used as a negative control. Primers for each of the indicated Ifng regulatory regions were designed to detect STAT5 binding (STAT5 primer sets #5–10).
(B) Purified CD4SP thymocytes from CD2-Cre transgenic STAT5^fl/+ and STAT5^fl/fl mice were stimulated under Th1-skewing conditions. Sodium butyrate (200µM) was added at 24h post-stimulation. At day 4, cells were restimulated with PMA and Ionomycin. Bar graphs indicate the percentages of IFN-γ producing cells. Data are representative of two independent experiments with similar results.
(C) CD4⁺ splenocytes from Jak3^+/+ mice were cultured under Th1-skewing conditions for 4 days, in the indicated conditions. Jak3 inhibitor PS078507 was used at 625nM, IL-2 blocking included anti-IL-2 (10µg/ml), anti-CD25 (10µg/ml) and anti-CD122 (10µg/ml) antibodies, recombinant IL-7 (rIL-7, 10ng/ml) was added together with IL-2 blocking antibodies. Cells were restimulated with PMA plus ionomycin for 6 hours. Histograms show IFN-γ intracellular staining.
(D) Purified CD4⁺ splenocytes from Jak3^+/+ mice were stimulated with anti-CD3 plus anti-CD28 antibodies for 24h, rested for 4 hours, then stimulated with medium (first lane), IL-2 or IL-7, at 50ng/ml or 100ng/ml for 15 minutes. Cell lysates were prepared and immunoblotted for STAT5p and total STAT5.