Dynamics of autoreactive NKT cell interactions with APCs. (A) Time-lapse microscopy of NKT cells interacting with human myeloid DCs. Top rows show fluorescence images taken at the indicated times with NKT cells labeled green and the DCs labeled red, and the bottom rows show phase-contrast views. Data are from 1 representative experiment of 3. Videos S1 through S7 provide kinetic views of NKT cell-APC interactions. (B) Analysis of the velocity of NKT cells in the presence of the indicated APCs. Single-cell tracking analysis was performed using Image J software (NIH), and the average velocity of each T cell was calculated by dividing the distance traveled by the time (20 minutes). Horizontal bars within the datasets show the means with the P values calculated by ANOVA shown at the top. The results are from a representative experiment using clone JC2.4, and similar results were obtained with 1 additional clone. (C) Flow cytometric analysis of tight adherence of NKT cells to APCs. Fluorescently labeled NKT cells and APCs were coincubated for the indicated times, and tightly adhered conjugates were analyzed by flow cytometry. Data are mean plus or minus SD of 3 replicate samples and are from 1 representative experiment of 3 using clone J3N.5. Similar results were observed using 2 other NKT cell clones. (D) Microscopic analysis of carboxyfluorescein diacetate succinimidyl ester-labeled NKT cells (green) conjugated to APCs.