mHA-specific Treg proliferative response. (A) Tregs were purified from a normal donor and stimulated with γ-irradiated dendritic cells from an HLA-matched sibling in the presence of rapamycin (100 ng/mL), IL-2 (10 U/mL), and IL-15 (10 ng/mL). Each bar represents mean + SD of 3H-thymidine uptake (in CPM) on day 6 of culture. The inset shows Foxp3 expression of DBY-specific (bold line) and UTY-specific (thin line) Treg clones by flow cytometry. (B,C) Identification of mHA-specific T-cell clones. Treg (B) and Tconv (C) cells were obtained from a female, purified, and stimulated with γ-irradiated dendritic cells from an HLA-matched brother; wells positive for TGF-β release were pooled and cloned by limiting dilution. After 14 days, wells containing Tconv or Treg clones were split into 2 and restimulated with allogeneic (male) or self- (female) DCs for 24 hr. Each bar represents TGF-β or TNF released by mHA-specific T-cell clones in the culture supernatant after restimulation with allogeneic DCs or self-DCs. (D) mHA-specific Treg clones were tested for proliferative response to allogeneic LCL, self-LCL, or self-LCL transduced with DBY, UTY, DFFRY-2, or SMCY. Results shown here are CPM and are representative of 2 experiments. (E) The DBY-specific Treg clone was cultured with a different ratio of autologous-DBY–specific Tconvs in the presence of DBY-transduced autologous LCL, with autologous-UTY–specific Tconvs in the presence of UTY-transduced auto-LCL as specificity control. Mean CPM of DBY- or UTY-specific Tconvs with the stimulators alone was 3919 and 4914, respectively. (F) The UTY-specific autologous Treg clone was cultured with a different ratio of autologous-UTY–specific Tregs in the presence of UTY-transduced autologous LCL, with autologous-DBY–specific Tconvs in the presence of DBY-transduced autologous LCL as specificity control. Mean CPM of UTY- or DBY-specific Tconvs with the stimulators alone was 4906 and 4053, respectively. P values refer to the difference in suppression of specific vs control Tconvs by the ANOVA model.
