Defective calcium signaling during NKT-cell autoreactive activation. (A) NKT cells were labeled with the intracellular calcium indicator dyes Fluo-4 and Fura-Red and stimulated by contact with APCs. UT indicates the untransfected parent cell (CD1d-negative); Autoreactive, unpulsed CD1d-transfected APCs; α-GalCer, antigen-pulsed CD1d transfectants. Intracellular calcium levels were assessed by flow cytometry. The cells in flux were defined as those cells with a ratio of Fluo-4 to Fura-Red that exceeded the 95% confidence interval of the baseline. The data shown are from 1 representative experiment of 3 using clone J24N.22, and similar results were obtained from 3 other clones. (B) NKT cells were coincubated for 5 minutes (left panels) or 20 minutes (right panels) with APCs, and NFAT1 activation was assessed by immunofluorescence analysis of nuclear translocation. NFAT1 is shown in green and nuclear staining (6-diamidino-2-phenylindole dihydrochloride) is shown in blue. (C) The percentage of NKT cells showing nuclear colocalization of NFAT1 after the indicated APC coincubation times. At least 80 events in 10 independent fields were quantitated. The data in panels B and C are from 1 representative experiment of 2 using clone J24N.22, and similar results were observed from analysis of 1 other NKT-cell clone.