Characterization of expanded mHA-specific Tregs. LDA of mHA-specific blood Tregs or Tconvs stimulated by DCs from HLA-matched siblings. Plots represent (y-axis) the log-fraction of nonresponding Treg (A) or Tconv (B) responses against allogeneic or autologous DCs vs (x-axis) the number of T cells per well. The slopes represent frequency estimates validated by the likelihood ratio test of single-hit model. Not shown for clarity are the 95% confidence estimates. Each LDA was performed with a minimum of 10 replicates per cell number. (C) Tregs were purified from leukapheresis and cultured ex vivo for 12 days with HLA-identical sibling DCs, IL-2, IL-15, and rapamycin. LDA of allospecific Tregs was performed on day 0 and 12 of culture. Results shown are ³H-TdR incorporation on day 6 of LDA. Frequencies of Tregs against sibling DCs on day 0 are marked by a full circle and on day 12 by a full square. Tregs tested with autologous DCs on day 0 are marked by an empty circle and day 12 by an empty square. Each regression line significantly fit the data. (D) Cytokine release of mHA-specific Tregs in response to sibling DCs by LDA. TGF-β and TNF released by mHA-specific Tregs were measured in the culture supernatant after stimulation with allogeneic DCs. TGF-β levels by Tregs tested against allo-DCs on day 0 (full circle) and day 12 (full square). None of the wells stimulated with allogeneic DC showed positivity for TNF levels on day 0 (empty circle) or day 12 (data not shown). (E) mHA-specific suppressive function of cultured Tregs was tested at different ratios to self-Tconvs in the presence of DCs from the original sibling or an HLA-incompatible third party. Results shown are from 7 independent experiments. ***P < .001 by ANOVA. (F) Foxp3 gene promoter demethylation was assessed by PCR primer for methylated and demethylated Treg-specific Foxp3 promoter sequence in Tregs or Tconvs before and after expansion. Results are the representative of 3 individual experiments. (G,H) mHA-specific Treg proliferation and TGF-β release. The entire end-of-culture population and sorted Tregs were tested for proliferation by ³H-TdR incorporation (G) and TGF-β release (H) on day 6, in response to HLA-identical sibling DCs, self-DCs, or no stimulus in the presence or absence of IL-2. TGF-β and TNF levels in the culture supernatant were measured by ELISA. Each bar represents mean + SD of CPM or cytokine levels in response to stimulus. Results shown are 1 experiment representative of 3 independent experiments. *P < .05; **P < .01; ***P < .001. ns, nonsignificant.