(A) DSA were not detected after allograft rejection following Foxp3+ cell depletion. DBA/2 thymocytes were incubated with serum from recipients in each group or with serum from naïve B6 mice and subsequently stained with anti-mouse IgG secondary antibody. The graphs show the average delta mean fluorescence intensity values from histograms of anti-mouse IgG between thymocytes incubated with each recipient serum and those incubated with naïve B6 serum. Results represent mean values ± s.e.m. of 12, 6 and 5 recipients in Groups A, B and C, respectively. *p < 0.05 (t-test). (B) Enzyme-linked immunospot assays to measure recipient T cell reactivity to donor (DBA/2), self (B6) or third-party (C3H). WT1 and WT2 represent nonchimeric WT B6 recipients of DBA/2 allografts, sacrificed 6 weeks after transplant. A1-4, B1-2 and C1-3 represent recipients in Groups A, B and C, respectively, sacrificed 6 weeks after diphtheria toxin treatment. Little or no reactivity to donor splenocytes (DBA/2) was detected in Groups A, B or C, while Group A recipients had rejected skin and heart allografts. All recipients had strong responses to Con A and variable responses to third-party (C3H). In contrast, control WT recipients that had rejected DBA/2 cardiac allografts had strong responses to DBA/2 splenocytes. Results represent mean values of triplicates ± s.e.m. of interferon gamma producing clones/106 recipient T cells.