Foxp3 expression in the CD8 lineage. (A) Hierarchical gating of CD8α⁺Foxp3⁺ cells in Cd19^creRosa^IL-2 and littermate controls, pregated on viable lymphocytes. FSC, forward scatter. (B–D) Expression of the Foxp3^Thy1.1 reporter (B), Foxp3^YFP-cre reporter (C), and Foxp3^BAC reporter (D) in CD8α⁺CD4⁻ T cells. (E) tSNE representation of high-parameter flow cytometry data from splenic T cell populations from Cd19^creRosa^IL-2 mice and littermate controls. FlowSOM clusters annotated based on differential expression of key markers. (F) Expression of CD8β in CD8α⁺Foxp3⁺ cells from the spleen of WT mice. (G) Usage of E8I enhancer (RFP⁺) in CD8⁺ T cells from CD8^creRosa^RFP mice. n = 4, unpaired t test. (H) Representative gating of CD8⁺Foxp3⁺ cells in spleens of WT, MHCI^−/−, MHCII^−/−, MHCI^−/−MHCII^−/−, and CD1d^−/− mice. (I) Number of CD8⁺Foxp3⁺ cells in spleen. (J) Frequency and number of CD8⁺Foxp3⁺ cells in thymus. For H–J, n = 3–5, data pooled from two independent experiments. Significance tested by one-way ANOVA.