Prolonged interactions between DCs and T reg cells. (A) Intracellular IL-2 staining of OT-II T cells and polyclonal T cells from draining LNs after brief stimulation with PMA and ionomycin, ∼20 h after s.c. injection of OVA-pulsed DCs, coinciding with the time point when T reg cell–DC interactions were visualized. (B) The protocol for imaging T cell–DC interactions in vivo. (C and D) Snapshot for T conv cells (C) or T reg cells (D) and DCs interaction analysis. UT, untreated; IL-2, pre-treated with IL-2. (E) Duration of contacts quantitated for T cell–DC conjugates under different conditions. For CD25⁻ T conv cells 63 untreated and 67 IL-2 pretreated T cells are analyzed; for CD25⁺ T reg cells, 72 untreated and 70 treated cells are analyzed. Data are pooled from four independent experiments (see Videos 3 and 4). **, P < 0.01; ***, P < 0.001; N.S., not significant, Student’s t test.