Suppressive activity of expanded allo-specific Tregs. (A-C) The suppressive activity of cultured CFSE⁻ versus CFSE ⁺ (A), CFSE⁻ (replicated) versus naive Tregs (B), or allo-specific versus polyspecific Tregs (C) were tested at different ratios to self-CD4⁺CD25⁻ T-cell responders in the presence of original allogeneic DCs. (D) Allo-specific suppressive function of cultured CFSE⁻ Tregs was tested against the original stimulator or a HLA class II–mismatched third party stimulator. Results are shown as average CPM of triplicates measured by the incorporation of ³H-thymidine in cocultures at day 6 after subtracting the CPM of background wells without Tregs. Results shown are representative of 3 individual experiments for each of the panels.