RasGRP1 is required for efficient TCRαβ⁺ CD8αα IEL development through control of thymic IELp generation. IELs were isolated from small intestines of WT and 1KO animals and analyzed by flow cytometry. (A) CD45⁺CD4⁻ cells were gated and analyzed for expression of TCRβ and TCRγδ (left). TCRβ⁺ (middle) and γδTCR⁺ (right) IELs were analyzed for expression of CD8α and CD8β. (B) Numbers of TCRαβ⁺ CD8αα IELs (top) and γδTCR⁺CD8αα (bottom) isolated from the indicated mice. Data were obtained from the indicated number of mice over 14 individual experiments; WT (n = 16), 1KO (n = 10). (C) Thymi were harvested from the indicated mice and analyzed for the presence of IELp cells. Total thymocytes were analyzed for expression of CD4 and CD8 and CD4^−/dullCD8^−/dull were gated (left). NK1.1⁻ cells were subsequently analyzed for expression of TCRβ and CD5 (right). (D) Number of IELp cells (CD4^−/dullCD8^−/dullNK1.1⁻TCRβ⁺CD5⁺) from the indicated mice. Data were obtained from the indicated number of mice over 17 individual experiments; WT (n = 17), 1KO (n = 11). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test). Error bars indicate SEM.