Supplementary Figure 4: Maintenance of TCR-deficient T_reg cell responsiveness to IL-2 and proliferation in the presence of activated DCs in vitro.

(a) p-STAT5 was assessed in eGFP⁺ (T_reg) cells among bulk CD4⁺ cells purified on day 9 from Trac^FL/WTFoxp3^Cre-ERT2 mice treated with tamoxifen on days 0 and 1, stained for CD4 and TCRβ, and cultured with increasing concentrations of IL-2 for 20 minutes before fixation. Data is representative of two experiments with a total of two mice. (b) In vitro IL-2 capture assay. Trac^FL/WTFoxp3^Cre-ERT2 mice were administered tamoxifen on days 0 and 1, and on day 9 CD4⁺eGFP⁺TCRβ⁺, CD4⁺eGFP⁺TCRβ^-, and CD4⁺TCRβ⁺eGFP^- cells were sorted and cultured in media with increasing concentrations of IL-2. PE median fluorescence intensity (MFI) directly correlates with IL-2 remaining in the media at the time of analysis. (c) CD80 expression on DC populations in lymph nodes of Trac^WT/WTFoxp3^Cre-ERT2 (black lines, left, and white circles, right) and Trac^FL/FLFoxp3^Cre-ERT2 (red lines, left, and black circles, right) mice on day 13 following tamoxifen administration on days 0, 3, 7 and 10. (d) In vitro proliferation of T_reg cells was assessed as follows: CD4⁺eGFP⁺ cells were sorted on day 9 from Trac^FL/WTFoxp3^Cre-ERT2 mice treated with tamoxifen on days 0 and 1, labeled with CellTrace Violet and cultured for 84 hrs with IL-2 in the presence or absence of DCs with or without LPS. Cells were stained for CD4, TCRβ and Foxp3 for analysis. All conditions were performed in triplicate with similar results. **, P < 0.001; *, P = 0.002. P-values were calculated using a two-tailed unpaired t-test.