Figure 4. Satb1 is required for the generation of induced regulatory T cells.

(A) Natural CD3⁺CD4⁺FoxP3⁺ Treg from the spleens of adult (week 10) Satb1^−/− mice effectively suppressed CD3/CD28-induced T cell splenocyte activation in a dose-dependent manner. Representative of two independent experiments. (B) CD3⁺CD4⁺Foxp3⁻CD62L⁺CD44⁻ naïve T cells sorted from CD4^CreSatb1^f/fFoxP3^GFP mice or control Satb1^f/fFoxP3^GFP littermates were in vitro differentiated to Treg cells for 3 days. Conversion into FoxP3⁺ Treg cells was compromised in CD4^CreSatb1^f/fFoxP3^GFP cells. (C) (Left) Satb1-dependent differences in Treg conversion (n=15); (Right) Similar proliferation rates were observed under differentiation conditions for Satb1⁺/Satb1⁻ populations. (D) ChIP-PCR for the CNS1 (left panel) and CNS2 (right panel) enhancers in Foxp3. Chromatin was immunoprecipitated with anti-Satb1 or control IgG from FoxP3⁺ Treg cells, in vitro differentiated from naïve Satb1-competent CD4⁺ T cells. Percent of input before immunoprecipitation (2.5% input values) was quantified by Real-Time Q-PCR (n=5). Mean and SEM are shown. (E) Representative dot plots showing the gating strategy for Tfh and Tfr cells in OVA-vaccinated mice carrying Satb1^−/− T cells, compared to Satb1-competent littermates. (F) Reduced generation of PD-1^hiCXCR5⁺Foxp3⁺ Tfr cells with corresponding increases in FoxP3⁻ Tfh cells (n≥20). (G) Higher ratios Tfh to Tfr cells in Satb1^−/− T cells compared to control littermates (n≥20). Data are presented as median and pooled from ≥2 independent experiments. ***p≤0.001.