Indirect allo-recognition by Tregs. (A) Tregs were cultured with γ-irradiated autologous monocyte-derived DCs (10:1) pulsed overnight with allogeneic or autologous whole-cell lysate in the presence of cytokines and rapamycin. Results shown are average CPM of triplicates measured by the incorporation of ³H-thymidine in cocultures on day 6. (B) LDA showing frequency of allo-reactive purified CD4⁺CD25⁺CD127⁻ Tregs or CD4⁺CD25⁻CD127⁺ non-Tregs stimulated with self-DCs and pulsed with allogeneic whole-cell PBMC lysate in the presence of rapamycin, IL-2, and IL-15. Box plot represents the frequency estimate and the error bar shows 95% CI calculated by extreme LDA. Dot plot shows the proliferation of CFSE-labeled Tregs with indirect allospecificity after 14 days in culture (C) and the expression of CD25, Foxp3, and BCL-2 after 27 days in culture (D-E). (F) Expansion kinetics of allo-specific Tregs when antigen was presented indirectly. Tregs were seeded at 7.7 × 10⁵/well in a 48-well plate with irradiated allogeneic cell lysate–loaded self-DCs (7.7 × 10⁴), rapamycin (100 ng/mL), IL-2 (10 U/mL), and IL-15 (10 ng/mL). Cells were cultured for 27 days with APC restimulation on day 14. The total Treg numbers at each time point were calculated by flow cytometry using timed event acquisition. The frequency of allo-specific Tregs at time 0 was calculated by LDA. The frequencies of allo-specific Tregs at subsequent times were calculated by flow microfluorometry of Ki-67⁺ Tregs. Results shown are from 1 representative experiment of 3. **P < .01; ***P < .001.