Figure 6.

Chimeras induced through recipient Treg treatment demonstrate donor-specific hyporesponsiveness in vitro and humoral tolerance. (a) Mixed lymphocyte reaction results from selected BMT recipients were obtained 28–37 weeks after BMT. Chimeras of recipient Treg treated mice (n = 4) showed specific hyporesponsiveness to donor antigens in vitro (p = 0.0247 SI anti-donor compared to third-party antigen; p = 0.0183 SI anti-donor compared to naïve B6 mice, n = 3). Donor reactivity was preserved in BMT recipients that were treated with donor (n = 3) or third-party (n = 3) type Tregs. SIs were calculated by dividing the mean cpm from responses against recipient (black column; B6), donor (white column; Balb/c), or third-party (grey column; C3H) stimulator cells by mean background cpm (i.e., cpm with no stimulator population). Error bars indicate standard deviation. (b) BMT recipients were analyzed for the existence of anti-donor antibodies in serum >3 months after BMT (i.e. ~1-2 months after skin grafting). Recipient Treg induced chimeras (n = 4) uniformly failed to develop detectable levels of anti-donor antibodies, whereas BMT recipients treated with donor (n = 4) or third-party (n = 3) Tregs and control mice without Treg treatment (but receiving BM, costimulation blockade, and rapamycin; n = 5) developed substantial antibody levels. The reactivity of sera with syngeneic (B6; grey filled area) and donor (Balb/c; black line) thymocytes is shown by flow cytometry through indirect staining with anti-mouse IgG. Representative histograms are shown.