NKT-cell activation by IL-12 and IL-18. (A) Addition of IL-12 and IL-18 does not induce detectable calcium flux by autoreactively activated NKT cells. Similar results were obtained in 4 independent experiments using 2 different NKT-cell clones. (B) IL-12 and IL-18 costimulate IFN-γ secretion by autoreactively activated NKT cells in a JAK-STAT–dependent manner. NKT cells were coincubated with untransfected (UT) or CD1d-transfected APCs (Autoreactive) in the presence of IL-12 and IL-18 (■) or without these cytokines (□), and IFN-γ secretion was quantitated by ELISA. Where indicated, the coincubation was performed in the presence of JAK inhibitor 1. (C) Autoreactive activation potentiates subsequent NKT-cell IFN-γ secretion in response to IL-12 and IL-18. NKT cells were preexposed for 4 hours to the APCs shown on the x-axis; then these APCs were removed and the NKT cells were coincubated with untransfected APCs (ie, CD1d-negative) in the presence of IL-12 and IL-18 (■) or in the absence of these cytokines (□). Where indicated, the CD1d-transfected APC preexposure was performed in the presence of the MEK inhibitor U0126. (D) NKT cells were preexposed to the indicated APCs; these were then removed and the NKT cells were coincubated with α-GalCer–pulsed CD1d-transfected APCs and IFN-γ secretion was quantitated by ELISA. The plots in panels B to D show 1 representative experiment of 5. In each case, similar results were obtained from 3 different NKT-cell clones.