Supplementary Figure 4: Enhanced de novo Foxp3⁺ cell induction from _ICT_N cells is not dependent on APCs in the microenvironment.

(a) CD4⁺ T cells from ctr or pIC-treated DR mice were isolated then stimulated for 5 days with plate-bound anti-CD3 and anti-CD28 with no APC. Foxp3 expression was assessed by intracellular staining, representative FACS plot from 3 independent experiments is shown, n=3 mice per group. (b) CD4⁺ T cells from PBS-treated (ctr) or polyI:C-treated (pIC) DR mice were assessed for methylation of the Foxp3 promoter by bisulfite sequencing analysis. Control cells were obtained by FACS sorting of CD4⁺ T cells; regulatory T cells, (T_REG) were isolated by positive expression of CD25 and GITR, naïve T cells (T_naive) were CD25-negative, CD62L⁺, CD44^low. Effector T_H1 cells (T_H1) were obtained by culturing T_naive cells with plate-bound anti-CD3/CD28 with IL-12 for six days. Bar graph depicts mean plus SEM of values obtained by analysis of samples from three independent experiments, n=3 mice per group. (c) mRNA expression of Tgfbr1 from purified DR cells from control and polyI:C-treated donors. Bar graphs depict fold change in expression of indicated mRNA from polyI:C-treated cells vs controls (set to 1) after normalization to Gapdh mRNA. Mean plus SEM of fold change ratios from three independent experiments, n=3 mice per group. (d) DR cells from ctr (open histogram) or pIC-treated mice (shaded histogram) were cultured for 24 hours with OVA-peptide-pulsed APC and TGFβ, then assessed for intracellular phospho-SMAD2/3. FACS histogram representative of two independent experiments, n=2 mice per group.