CD8 ⁺ Foxp3 ⁺ Tregs are revealed through B cell production of IL-2. (A) Representative gating of Foxp3⁺ CD8⁺ T cells in Cd19^creRosa^IL-2 mice and littermate controls. (B) Frequency of Foxp3 expression among CD8⁺ T cells in Rosa^IL-2 strains. n = 6–32. (C) Frequency of CD8⁺ cells among total Foxp3⁺ T cells. n = 9–32. (D) Principal component analysis of flow cytometric markers on splenic T cell populations. (E) Representative expression of indicated markers on CD4⁺ or CD8⁺ Tregs from Cd19^creRosa^IL-2 mice. (F) In vitro suppression assay comparing ability of CD4⁺ and CD8⁺ Tregs to suppress CD4⁺ Tconv (left) or CD8⁺ T conv (right) cell proliferation; pooled from three independent experiments. (G) Frequency of Foxp3⁺CD8⁺ cells in spleens of WT, MHCI^−/−, MHCII^−/−, MHCI^−/−MHCII^−/−, and CD1d^−/− mice. (H and I) UMAP of high-parameter flow cytometry data (H) from Foxp3⁻CD8⁺ Tconv cells and CD8⁺ Tregs, with frequency distribution (I) of Foxp3⁺ and CD8 Tconv cells across FlowSOM clusters (n = 3). (J) Representative flow plot showing PD-1 and CXCR5 expression. (K) Representative immunofluorescence staining of CD8α, Foxp3, and IgD of WT LN (scale bars: 50 μm; 20 μm for inset), with quantification of Foxp3⁺ cells among total CD8 T cells in the B follicle (n = 9). Data pooled from (B–D and F–I) or representative of (A, E, J, and K) at least two independent experiments. Significance was tested by one-way ANOVA (B, C, and G).