Figure 2: Ets1-SE is dispensable for thymic T cell generation but is required for CD4⁺ Th1 differentiation.

A, Plots demonstrate percentage of cells defined by flow cytometry analysis in the thymus from age matched wildtype and Ets1-SE^−/− female mice. Data are representative of three independent experiments. Each dot represents an individual mouse (wildtype; n=7 - Ets1-SE^−/−; n=7). Error bars = SEM; and P: ns = not significant, (DN, DP, nTregs, CD4⁺/CD8⁺ SP: Mann-Whitney U test; DN1-DN4, TCRβ^low/high, Semi-Mature/Mature CD4⁺/CD8⁺: Two-way ANOVA with multiple comparisons and Bonferroni correction).

B, Plots demonstrate percentages of cells defined by flow cytometry analysis in the lung Frequencies at steady state of lungs parenchyma CD4⁺ T cells (TCRβ⁺, CD4⁺), activated CD4⁺ T cells (TCRβ⁺, CD4⁺, CD44⁺), CD8⁺ T cells (TCRβ⁺, CD8⁺), activated CD8⁺ T cells (TCRβ⁺, CD8⁺, CD44⁺), CD4⁺ Th2 cells (TCRβ⁺, CD4⁺, GATA-3⁺) from age-matched wildtype and Ets1-SE^−/− male mice. Data are representative of two independent experiments. Each dot represents an individual mouse (wildtype; n=4 - Ets1-SE^−/−; n=4). Error bars = SEM; and P: ns = not significant, * =P≤0.05 (Mann-Whitney U test).

C, (left) Representative flow cytometry contour plot of naive CD4⁺ T cells from wildtype or Ets1-SE^−/− mice cultured under Th1, Th2 or Th17 polarizing conditions for 6 days. Unstained wildtype cells are shown for each polarizing condition as a negative control (wildtype FMO Control). (right) Frequencies of Th1 (IFNγ⁺), Th2 (IL-13⁺) or Th17 (IL-17⁺) cytokines producing CD4⁺ T cells cultured under Th1, Th2 or Th17 polarizing conditions for 6 days. All populations were pre-gated on SSC-A/FSC-A, Singlets and Viability⁻ (Live), TCRβ⁺ and CD4⁺ cells. Two independent experiments were pooled and were repeated five times. Each dot represents an individual mouse (wildtype; n=8 - Ets1-SE^−/−; n=8). Error bars = SEM; and P: ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Two-way ANOVA with multiple comparisons and Bonferroni correction).

D, (left) Frequencies of induced Tregs (iTregs, FoxP3⁺ CD4⁺ cells) and mean fluorescence intensity (MFI) of FoxP3, CD25 and T-bet from naïve CD4⁺ T cells cultured under iTreg polarizing conditions for 3 days. (right) Representative histogram of the proportion of FoxP3+ CD4⁺ T cells 3 days after culturing in iTregs polarizing conditions. Data are representatives of two independent experiments. Each dot represents an individual mouse (wildtype; n=3 - Ets1-SE^−/−; n=3). Error bars = SEM; and P: ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Mann and Whitney).