Loss of TCF-1 results in the emergence of IL-17–producing CD8⁺ T (Tc17) cells. (A) Expression of RORγt (upper panel) and TCF-1 (lower panel) in various T cell populations from thymus and spleen of WT mice. Populations include DP (TCRβ⁻CD4⁺CD8⁺) thymocytes and CD4⁺ (TCRβ⁺CD4⁺CD8⁻) or CD8⁺ (TCRβ⁺CD4⁻CD8⁺) T cells isolated from spleen or thymus. Control for RORγt expression represents fluorescence minus one stain of CD8⁺ T cells from WT mice and for TCF-1 expression shows spleen CD8⁺ T cells from Tcf7^−/− mice. Data show representative plots of one of two independent experiments (n = 4 mice/experiment). (B) tSNE analysis of TCRβ⁺ cells analyzed by flow cytometry for CD4, CD8, CD62L, CD44, and RORγt from spleen of Tcf7^+/+ and Tcf7^−/− mice. Dot plots are displayed as pseudocolor plots denoting areas of high and low population density. Orange shading indicates CD8⁺ T cells. Histogram shows RORγt expression. Representative of three independent experiments (n = 6 mice). (C) Contour plots gated on TCRβ⁺CD8⁺CD4⁻ T cells show the frequency (upper panels) and number (lower panel) of RORγt⁺CD8⁺ T cells in various tissues in Tcf7^+/+ and Tcf7^−/− mice. Data show representative plots (upper panels) and the mean ± SEM of individuals pooled from three independent experiments (lower panels; n = 6, P values calculated using Student’s t test). (D) IL-17 production and RORγt⁺ expression by Tcf7^−/− Tc17 cells (red, TCRβ⁺CD8⁺CD4⁻CD44⁺CD62L⁻CD25⁺) and Tcf7^+/+ or Tcf7^−/− effector cells (blue and black, TCRβ⁺CD8⁺CD4⁻CD44⁺CD62L⁻CD25⁻) FACS-purified from the spleen and lymph nodes of Tcf7^+/+ and Tcf7^−/− mice, followed by stimulation with PMA and ionomycin for 4 h in vitro. Histograms are representative of one of two independent experiments (n = 3 mice/genotype/experiment). (E) Flow-cytometric analyses of MAIT cells in spleen of WT and Tcf7^−/− mice. Data show representative staining from an individual mouse for each genotype and data from one of two similar experiments (n = 3–5 mice/genotype). (F) Single-cell PCR analysis of TCR Vα (upper panel) and TCR Vβ (lower panel) usage of effector CD8⁺ T cells (TCRβ⁺CD4⁻CD8⁺CD44⁺CD62L⁻Rorc(gt)^+/+) and MAIT cells from Tcf7^+/+Rorc(gt)^+/GFP mice (TCRβ⁺CD4⁻CD8⁺CD44⁺CD62L⁻Rorc(gt)^+/GFP) and Tc17 cells from Tcf7^−/− mice (TCRβ⁺CD4⁻CD8⁺CD44⁺CD62L⁻CD25⁺). Data show the mean expression ± SEM of 50 individual cells analyzed for TCR Vα and TCR Vβ usage from each of three mice.