Supplementary Figure 1: T_reg cell surface phenotype and proliferative capacity in the absence of a functional TCR.

(a) CD4⁺eGFP⁺ TCRβ⁺ and TCRβ^- cells were sorted from Trac^FL/WTFoxp3^Cre-ERT2 mice 9 days after tamoxifen treatment on days 0 and 1 (a), labeled with CellTrace Violet and plated in 96-well plates coated with anti-CD3ɛ (clone 2C11) (b), anti-TCRβ (clone H57-597) (c) or PMA and Ionomycin (d) in the presence of 25U/mL IL-2 for 84 hours before analysis. All conditions were performed in triplicate wells with similar results. (e) Expression of T_reg cell markers on TCRβ⁺ and TCRβ^- CD4⁺Foxp3⁺ cells in lymph nodes of Trac^WT/WTFoxp3^Cre-ERT2 (WT/WT), Trac^FL/WTFoxp3^Cre-ERT2 (FL/WT) and Trac^FL/FLFoxp3^Cre-ERT2 (FL/FL) mice. Gray histograms are gated on CD4⁺Foxp3^- cells in Trac^WT/WTFoxp3^Cre-ERT2 mice. (f) Percentages and absolute numbers of total Foxp3⁺ among CD4⁺ cells in spleens and lymph nodes of the indicated mice. (g) Gating strategy for analysis of TCRβ⁺ and TCRβ^- cells among CD4⁺Foxp3⁺ lymph node cells. For (e-g), mice were analyzed on day 9 following tamoxifen treatment on days 0 and 1. Mice in (f-h) are indicated as in (e). (h) BrdU incorporation vs. TCRβ expression on CD4⁺eGFP⁺ cells sorted to >99% purity from pooled spleens and lymph nodes of the indicated mice on day 9 following tamoxifen treatment on days 0 and 1 and i.p. injection of BrdU on day 8. Data is representative of two experiments with four or more mice per group each (e-g) or three experiments with two or more mice per group each (h). P-value in (f) was calculated using a two-tailed unpaired t-test.