Rate of wound closure in organ culture is increased after activation of skin-resident T cells. (A) T cells remain responsive after 2 d of organ culture. Freshly isolated epidermal cells stimulated with PMA and ionomycin, and stained with IFN-γ for analysis by flow cytometry. IFN-γ expression was assessed on normal skin freshly harvested (day 0) and after being cultured for 2 d (day 2) on gelfoam in DMEM/10% FCS. Skin from the same donor was used for both day 0 and 2 analyses. Data are representative of three independent experiments. Percentages are shown. (B) Anti-CD3 stimulation activates skin-resident T cells in organ culture. The experiment was performed on skin cultured for 2 d on a gelfoam in DMEM/10% FCS in the presence or absence of anti-CD3 (5 µg/ml OKT3) antibody. Flow cytometry of freshly isolated αβ⁺ and Vδ1⁺ T cells stained with CD25 is shown. Data are representative of three independent experiments. Percentages are shown. (C) The addition of stimulating antibodies to CD3 increased the rate of wound closure in skin organ culture. Analysis of skin wound closure kinetics in the presence (dashed line) or absence (continuous line) of an mAb to CD3. Data are presented as the means ± SD of three independent experiments with an average of three foreskins per experiment, and are representative of seven total experiments (with a sum of 20 foreskins). (D) Blocking of T cell–mediated wound closure with IGF-1R antibody in skin organ culture. Data are the means ± SD of the slope of wound closure between days 0 and 2 of three independent experiments performed in triplicate. Data were compared using a one-tailed paired Student's t test.