Figure 2.

Shp1-deficient iNKT cells produce more T_H2 cytokines. (A) Shp1^fl/fl and Shp1^fl/fl CD4-cre mice were injected i.v. with αGC (0.5 μg). Intracellular FACS analysis of IFN-γ and IL-4 in spleen iNKT cells 90 min post-injection. Data shows representative flow cytometry plots (left), as well as the corresponding frequency (individual mice and mean values +/− s.e.m.) of total, single and double producers of IFN-γ and/or IL-4 (right). (B, C) Thymocytes (B) and splenocytes (C) from Shp1^fl/fl and Shp1^fl/fl CD4-cre mice were stimulated with plate-bound CD1d-αGC, or PMA/ionomycin (P/I) for 6 hours. Intracellular FACS analysis of IFN-γ and IL-4 in iNKT cells. Data shows representative flow cytometry plots (left), as well as individual and mean values +/− s.e.m. of IL-4/IFN-γ ratios. (D) Shp1^fl/fl and Shp1^fl/fl CD4-creER^T2 mice were treated with tamoxifen and subsequently injected i.v. with αGC (0.2 μg). Data shows individual and mean values +/− s.e.m. of IFN-γ⁺ or IL-4⁺ iNKT cells, as well as IL-4/IFN-γ ratios. (E) iNKT cells from Shp1^fl/fl or Shp1^fl/fl CD4-cre mice were sorted and cultured with BMDCs alone, BMDCs pre-loaded with αGC, or BMDCs in the presence of the indicated cytokine for 48 h. in the presence of indicated cytokines. IFN-γ, IL-4, IL-13, IL-17A and IL-22 production was assessed using a multiplex assay. (F) iNKT cells from Shp1^fl/fl or Shp1^fl/fl CD4-cre mice were sorted and cultured with BMDCs with isotype control, anti-CD25 or anti-CD127 antibodies (40 μg/ml) for 48 h. Data shows the mean +/− s.e.m. of triplicate values and is representative of 2 individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student t test (A-D) or two-way ANOVA (E).