Figure S4.
ICOS–mTORC2 signaling in iNKT cell development.(A) Flow cytometry analysis of pMob1 (Thr35) MFI in thymic S1–S2 and S3 iNKT cells from WT mice (n = 4 samples/group). (B) Flow cytometry analysis of pMob1 (Thr35) MFI in thymic WT and Cd4CreMst1fl/fl iNKT cells. (C) Immunoblot analysis of total Mst1 and β-actin protein in thymic S1–S2 and S3 iNKT cells from WT mice after stimulation by cross-linked CD3 and CD28. (D) Flow cytometry analysis of relative MFI of indicated molecule expression on thymic iNKT cell effector subsets from WT and Cd4CreMst1fl/fl mice (n = 3 or 4 mice/group). Data are relative to the average MFI of each WT iNKT effector subset. (E) Flow cytometry analysis (upper) and statistics of MFI (lower) of ICOS expression on total iNKT cells in the thymus of WT and Cd4CreMst1fl/fl mice (n = 4 mice/group). (F) Flow cytometry analysis (upper) and relative MFI (lower) of 4-1BB, OX40 and GITR expression on total iNKT cells in the thymus of WT and Cd4CreMst1fl/fl mice (n = 3–6 mice/group). Data are relative to the average MFI of WT iNKT cells. (G) Flow cytometry analysis (left) and percentages (right) of iNKT developmental stages (S1, S2, and S3) from the thymus of WT, ICOS−/− (Cd4CreMst1fl/+ or +/+), Cd4CreMst1fl/fl, and Cd4CreMst1fl/flICOS−/− mice (n = 4 or 5 mice/group). (H) Induction of Akt phosphorylation (Ser473) in WT S1–S2 and S3 iNKT cells after cross-linking ICOS, CD3, or ICOS and CD3. (I) Flow cytometry analysis of ICOS expression on WT S1, S2, and S3 iNKT cells. (J) Flow cytometry analysis (left) and percentages (right) of iNKT developmental stages from the thymus of WT, Cd4CreRictorfl/fl (Mst1fl/+), Cd4CreMst1fl/fl (Rictorfl/+ or +/+), and Cd4CreMst1fl/flRictorfl/fl mice (n = 4 or 5 mice/group). Numbers indicate percentage of cells in gates or quadrants or MFI. Data are mean ± SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Two-way ANOVA with Bonferroni’s test (A), two-tailed unpaired Student’s t test (D and E), one-way ANOVA with Tukey’s test (F, G, and J). Data are representative of two (A), three (B), or four (H and I) or combined from two (E and F) or three (D, G, and J) independent experiments.

ICOS–mTORC2 signaling in iNKT cell development. (A) Flow cytometry analysis of pMob1 (Thr35) MFI in thymic S1–S2 and S3 iNKT cells from WT mice (n = 4 samples/group). (B) Flow cytometry analysis of pMob1 (Thr35) MFI in thymic WT and Cd4CreMst1fl/fl iNKT cells. (C) Immunoblot analysis of total Mst1 and β-actin protein in thymic S1–S2 and S3 iNKT cells from WT mice after stimulation by cross-linked CD3 and CD28. (D) Flow cytometry analysis of relative MFI of indicated molecule expression on thymic iNKT cell effector subsets from WT and Cd4CreMst1fl/fl mice (n = 3 or 4 mice/group). Data are relative to the average MFI of each WT iNKT effector subset. (E) Flow cytometry analysis (upper) and statistics of MFI (lower) of ICOS expression on total iNKT cells in the thymus of WT and Cd4CreMst1fl/fl mice (n = 4 mice/group). (F) Flow cytometry analysis (upper) and relative MFI (lower) of 4-1BB, OX40 and GITR expression on total iNKT cells in the thymus of WT and Cd4CreMst1fl/fl mice (n = 3–6 mice/group). Data are relative to the average MFI of WT iNKT cells. (G) Flow cytometry analysis (left) and percentages (right) of iNKT developmental stages (S1, S2, and S3) from the thymus of WT, ICOS−/− (Cd4CreMst1fl/+ or +/+), Cd4CreMst1fl/fl, and Cd4CreMst1fl/flICOS−/− mice (n = 4 or 5 mice/group). (H) Induction of Akt phosphorylation (Ser473) in WT S1–S2 and S3 iNKT cells after cross-linking ICOS, CD3, or ICOS and CD3. (I) Flow cytometry analysis of ICOS expression on WT S1, S2, and S3 iNKT cells. (J) Flow cytometry analysis (left) and percentages (right) of iNKT developmental stages from the thymus of WT, Cd4CreRictorfl/fl (Mst1fl/+), Cd4CreMst1fl/fl (Rictorfl/+ or +/+), and Cd4CreMst1fl/flRictorfl/fl mice (n = 4 or 5 mice/group). Numbers indicate percentage of cells in gates or quadrants or MFI. Data are mean ± SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Two-way ANOVA with Bonferroni’s test (A), two-tailed unpaired Student’s t test (D and E), one-way ANOVA with Tukey’s test (F, G, and J). Data are representative of two (A), three (B), or four (H and I) or combined from two (E and F) or three (D, G, and J) independent experiments.

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