(A) Expression of RasGRP1 and RasGRP2 in WT iNKT cells. mRNA levels in FACS sorted iNKT cells and cαβT cells were determined by real-time PCR. (B-C) Thymocytes, splenocytes, and liver mononuclear cells from RasGRP1^-/- (KO) and RasGRP1^+/- (Ctrl) littermates were subjected to flow cytometry analysis. Data shown are representative of five mice per group. (B) Flow cytometry of cells stained with α-Galcer-loaded CD1d-tetramer (CD1d-Tet) and anti-TCRβ. (C) Percentage (left) and number (right) of live CD1d-Tet⁺TCRβ⁺ cells (mean ± SEM). (D) iNKT cells staining after enrichment for CD1d-Tet⁺ cells with magnetic beads. Thymocytes from WT and RasGRP1^-/- mice were stained with PE-CD1d-Tet. iNKT cells were enriched with a PE-enrichment kit. Top panels, TCRβ and CD1d-Tet staining of enriched cells. Bottom panels, CD24 expression on gated TCRβ⁺CD1d-Tet⁺NK1.1^- cells. (E). Total CD24⁺CD44^-NK1.1^- iNKT cell numbers in WT and RasGRP1^-/- thymi (n=6). (F) Assessment of iNKT cell development within CD24^- iNKT cells. Dot plots show expression of CD44 and NK1.1 on CD1d-Tet⁺CD24^- gated live thymocytes. Bar figure shows percentages (mean ± SEM) of stages 1, 2, and 3 iNKT cells in RasGRP1^-/- and control mice. (G) CD4 and CD8 staining of WT and RasGRP1^-/- thymi. (H) Ratios of iNKT cells to cαβT cells in the thymus (Thy), spleen (Spl), and liver (Li) from WT and RasGRP1^-/- mice. *p<0.05; **, p<0.01; ***, p<0.001 (Student t-test).

(A-C) Generation and analysis of sublethally irradiated TCRβ^-/-δ^-/- recipient mice reconstituted WT (CD45.1⁺CD45.2⁺) and RasGRP1^-/- (CD45.1^-CD45.2⁺) bone marrow (BM) at 1:1 ratio. Chimeric mice were analyzed 7-8 weeks after reconstitution. (A) Expression of CD45.1 and CD45.2 on mixed WT and RasGRP1^-/- BM cells before adoptive transfer. (B) Analysis of cαβT cells and non-T cells in recipient mice. Left panels show CD4 and CD8 staining of thymocytes and splenocytes from recipient mice. Middle and right panels show CD45.1 and CD45.2 staining in the DN, DP, and SP populations based on CD4 and CD8 expression. (C) Analysis of iNKT cells in recipient mice. Left panels show CD45.1 and CD45.2 staining in the indicated organs from recipient mice. Middle and right panels show expression of CD1d-Tet and CD24 on gated CD45.1⁺CD45.2⁺ WT and CD45.1^-CD45.2⁺ RasGRP1^-/- cells. (D) Analysis of lethally irradiated WT C57B6 recipient mice reconstituted with WT (Thy1.1) and RasGRP1^-/- (Thy1.2) BM cells at 1:10 ratio. Left panel, Thy1.1 and Thy1.2 staining of recipient thymocytes; Middle and right panels, CD24 and CD1d-Tet staining as well as CD4 and CD8 staining gated on Thy1.1⁺ and Thy1.2⁺ thymocytes. (E) CD1d expression on RasGRP1^-/- and control CD4⁺CD8⁺ DP thymocytes. Data are representative of three (A-C) or two (D, E) experiments.

(A) Semi-quantitative PCR analysis of sorted CD4⁺CD8⁺ thymocytes from RasGRP1^+/- and RasGRP1^-/- mice with primers for Vα14-Jα2, Vα14-Jα18, Vα14-Jα56, and CD14 (loading control). (B) Expression of CD1d, SLAM (CD150), and SLAMF6 (Ly108) on CD4⁺CD8⁺ thymocytes from RasGRP1^+/- and RasGRP1^-/- mice. Data shown are representative of three mice per group. (C) Percentages of cell death of CD1d-Tet⁺TCRβ⁺ iNKT cells and CD1d-Tet^-TCRβ⁺ cαβT cells from thymus (mean ± SEM, n=4). (D, E) Increased Ki67 expression in RasGRP1^-/- iNKT cells. Ki67 expression in iNKT cells gated from WT and RasGRP1^-/- thymocytes were determined by intracellular staining. (D) Overlay of histogram for Ki67 expression of gated iNKT cells; (E) Mean ± SEM of Ki67⁺ iNKT cells from WT and RasGRP1^-/- thymus (n=5). (F) Impaired proliferation of RasGRP1^-/- iNKT cells in response to α-Galcer stimulation in vitro. CFSE-labeled WT and RasGRP1^-/- thymocytes were left unstimulated or stimulated with 125 ng/ml α-Galcer at 37°C for 72 hours. Cells were then stained for APC-CD1d-Tet and TCRβ. Overlaid histograms show CFSE levels in gated WT and RasGRP1^-/- iNKT cells. (G) Real-time PCR analysis of mRNA expression of various proteins in sorted CD4⁺CD8⁺ thymocytes from RasGRP1^+/- and RasGRP1^-/- mice. *p<0.05; **, p<0.01 (Student t-test).

(A) Expression of CD4 on CD1d-Tet⁺CD24^- gated cells from thymus, spleen, and liver. (B) Percentage of CD4⁺ in the CD1d-Tet⁺CD24^- iNKT cells the indicated organs (n=4). (C) Expression of CD4 and NK1.1 on CD1d-Tet⁺CD24^- gated thymocytes with various cell surface phenotypes. (D) Percentage of CD4⁺ iNKT cells in different stages (n=4). (E) Schematic illustration of RasGRP1 for cαβT and iNKT cell development. RasGRP1 plays a critical role for positive selection of both cαβT and iNKT cells. RasGRP1 is crucial for the generation/maintenance of CD4⁺ iNKT cells. In addition, RasGRP1 may promote late stage iNKT cell maturation. ***, p<0.001 (Student t-test).