Chemokines produced during DC–T-cell interactions induce DC chemotaxis. (A,B) DCs were seeded on coverslips and exposed to supernatants from 24-hour cocultures of DC plus T cells plus superantigens. Videomicroscopy was performed as in Figure 1. (A) Individual frames from Video S4 and corresponding DC mobility tracks are shown; the results are representative of 4 experiments. Bar, 20 μm. (B-D) Dunn chamber assay. (B) Migration tracks of DCs in a gradient of supernatant from DC plus T cells plus superantigens (one representative experiment). DC individual trajectories in the chamber are represented in colors and direction of the gradient is figured with black lines. (C) Mean horizontal and radial velocity of DCs. (D) DC trajectories are plotted in a scatter diagram with the starting point for each cell at the intersection between the x- and y-axes, and the direction of the gradient vertical upwards. Left panel: DCs exposed to “active” supernatants as in panel B. Percentage of cells that ended up within a 120° arc facing the supernatant source is indicated (mean ± SD of 3 independent experiments with ≥ 400 cells tracked). Middle panel: DCs exposed to supernatant from DC plus T cells. Right panel: PTX-treated DCs exposed to “active” supernatant. (E) Transwell chemotaxis assays. Untreated immature DCs or DCs treated with PTX (DC_PTX) or LPS-treated DCs (DCm) were placed in the upper chamber of a transwell, and supernatants from the indicated 24-hour cocultures in the lower chamber. Percentages of DCs attracted to the lower chambers after 2 hours are shown. Data are means plus or minus SD of 3 independent experiments.