SAP negative NKT cells are functionally active. (A) Tamoxifen (1 mg) was administered for 4 days in SAP^fl/fl.tgCreERT2 and SAP^+/+.tgCreERT2 mice. Ten, 24, or 60 days after the last injection mice were killed. Total number of CD1d TET⁺ TCRβ⁺ NKT cells in the spleen was determined by flow cytometry. Representative of 2 independent experiments with 3 to 4 animals per group. (B) Serum cytokine levels 3 hours after injecting taxomifen-treated WT and SAP^fl/fl.tgCreERT2 mice with αGalCer plus NP-KLH. Control group represents WT animals without αGalCer plus NP-KLH stimulation. (C) Ex vivo cytokine production of total splenocytes isolated from mice stimulated as in (B), and cultured for an additional 4 hours. (D) Splenocytes isolated freshly from mice stimulated as in panel B were subject to intracellular IL-4 and IFNγ staining. Percentages of CD1d tetramer⁺ and cytokine⁺ cells are calculated after gating on viable cells according to scatter characteristics. Percentages of cytokine-positive cells among CD1d tetramer⁺ TCRβ⁺ NKT cells are in brackets. (E) Bystander activation of B cells (B220⁺CD8⁻CD4⁻NK1.1⁻), CD8⁺ T cells (CD8⁺B220⁻CD4⁻NK1.1⁻) and NK cells (NK1.1⁺CD4⁻B220⁻) were determined by CD69 up-regulation in mice injected as in panel B.