Extended Data Fig. 6: RBPJ ablation increases iTreg stability.

a, Summarized DNA demethylation rate across FOXP3 CNS2 (n = 4 donors for (+)Ascorbate; 3 donors for (-) Ascorbate) from RBPJ-KO iTregs and non-targeting controls, related to the experiment in (Fig. 3g). For statistical assessment, the donor-level demethylation rate of NTC gRNA 1 and 2 were averaged compared to each RBPJ gRNA species. b, Statistical analysis of the mean change of FOXP3⁺ cells in control or RBPJ-ablated iTregs on Day 14 (n = 6 donors), related to the experiment in (Fig. 3h,i). Mean change was measured by averaging the change in FOXP3⁺ Day 14 versus Day 7 per donor in both gRNA species for control and RBPJ. c, Left, Gating strategy used for the experiment in (Fig. 3h,i). FOXP3^hi and FOXP3^lo cells were sorted and subjected to bisulfite sequencing at FOXP3 CNS2 to assess DNA demethylation rate. The heatmap (right) shows summarized DNA methylation status at each of the 11 CNS2 CpGs across eight independent donors. d, Foxp3 stability assessed in iTreg cells derived from RBPJ^flox/flox/Foxp3-hCD2 reporter mice. Naive CD4⁺ T cells from mice were isolated and transduced with viral particles harboring Thy1.1-T2A-Cre or mock Thy1.1 constructs to induce knockout of the RBPJ and cultured under iTreg polarizing conditions. Foxp3⁺ iTregs were sorted to purity by flow cytometry and cultured for seven days in the presence of IL-2 before analysis. Analysis of %Foxp3⁺ cells (left) and relative Foxp3 MFI (right) at the end of culture (n = 4 biological replicates). e, Assessment of the effect of Rbpj knockout in Foxp3 lineage-committed mouse iTregs. Left, Schematic of experiment for assessing in vitro Foxp3 stability. Foxp3⁺ iTregs were sorted by flow cytometry and retrovirally transduced with constructs expressing Thy1.1-T2A-Cre or mock Thy1.1. Thy1.1⁺ were rested and cultured for an additional seven days before analysis. Analysis of %Foxp3⁺ cells (middle) and relative Foxp3 MFI (right) at the end of culture (n = 4 biological replicates). f,g, Relative CTLA-4 (f) and CD25 (g) MFI analysis in control or RBPJ-ablated human FOXP3⁺ iTregs (n = 4 donors). All data are presented as mean ± s.e.m. ns, not significant. Repeated measures one-way ANOVA with Dunnett’s multiple comparison test for a and f,g; two-tailed paired and unpaired Student’s t-test in b and d,e, respectively.

Source Data