Fig. 5: Foxp3 is preferentially required for regulation of gene expression during early T_reg cell differentiation.

a, Experimental design for transcriptional profiling of developing thymic T_reg cells. Each genotype consisted of three independent biological replicates. b, Gating strategy used to sort CD73⁻ nascent thymic T_reg cells. c, Bar graph comparing the number of Foxp3 degradation-induced DEGs in thymic, resting and activated T_reg cells from Foxp3^AID/WT mice. d, Pearson correlation between Foxp3 degradation-induced and Foxp3-dependent DEGs in thymic, resting and activated T_reg cells. e, Scatter-plot and cumulative distribution function (CDF) plots comparing Foxp3 degradation-induced and Foxp3-dependent DEGs across the three T_reg populations. Data were analyzed using a Mann–Whitney U-test. f, Metacell analysis of thymocyte scRNA-seq data³⁰ correlating UMI-normalized Foxp3 expression levels in each metacell with the expression of TIR1-up and TIR1-down gene signatures identified in a–c. UMAP plots are colored by scaled expression levels of TIR1-up, TIR1-down and UMI-normalized counts of Foxp3. Dashed red line depicts line of best fit. Correlations and corresponding P values were calculated with Pearson correlation over all genes. g, Experimental design for in vivo Foxp3 degradation in 1-day-old neonatal Foxp3^AID mice and adult Foxp3^AID mice. h, CD4⁺ and CD8⁺ T cell activation in adult and neonatal Foxp3^AID mice following Foxp3 degradation. i, Expansion of eosinophils and neutrophils in adult and neonatal Foxp3^AID mice after Foxp3 degradation. j,k, Representative H&E staining (j) and histology scores of liver inflammation (k) in neonatal Foxp3^AID mice following Foxp3 degradation. l, In vitro suppression assay of T_reg cells sorted from Foxp3^AIDR26^WT and Foxp3^AIDR26^TIR1(F74G) neonatal mice after 7 days of in vivo 5-ph-IAA administration. 5-ph-IAA was also included in culture to maintain Foxp3 degradation. Each point represents a unique mouse (h–l). Data are pooled from two independent experiments. Scatter-plots represent mean ± s.e.m. Data were analyzed using a one-way ANOVA. m, Bar graphs summarizing the number of Foxp3 degradation-induced DEGs in T_reg cells from neonatal and adult Foxp3^AID/y mice after 14 days of Foxp3 degradation. n, Scatter-plot correlating gene expression changes induced by Foxp3 degradation and Foxp3 gene deficiency⁷ in neonatal mice. o, Scatter-plots comparing gene expression changes induced by 7 days of Foxp3 degradation in neonatal T_reg cells to those in adult thymic, resting and activated T_reg cells from Foxp3^AID/WT mice.

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