Increased donor T-cell apoptosis in lymphoid organs and perforin- and FasL-mediated reduction of donor Tcons in the presence of donor NK cells. (A) FACS-based TUNEL staining on reisolated donor Tcons on day 4 after transplantation. Histograms indicate CD8⁺ and CD4⁺ reisolated from pLNs and spleens from Tcon + NK animals (gray filled histograms) stained significantly higher for TUNEL than the Tcon group (white filled histograms) group. TUNEL staining on freshly isolated T cells is shown as a control (black filled histograms). Quantification of the mean fluorescence intensity (right panels) shows significantly increased TUNEL stain in the Tcon + NK group (*P < .001). Error bars indicate SE. Data are pooled from 3 mice and are representative of 3 independent experiments. (B) Average photons emitted from animals receiving luc⁺ Tcon together with NK cells from wild-type donors or donors deficient for either IFNγ (IFN-γ KO), perforin (perf KO), or FasL (FasL KO) on day 4 after T-cell transplantation. Bioluminescent signal was significantly greater in animals receiving perforin^−/− or FasL^−/− NK cells compared with wild-type NK cells, implicating these molecules in the mechanism of NK cell–mediated reduction of donor Tcons (*P < .05, **P < .01). One-way analysis of variance was also significant (P = .001) and Dunnett multiple comparison test showed that only the Tcon + WT NK and Tcon + perf KO NK groups were significantly different from control. Data are an average of 5 mice per group, and are representative of 2 experiments.