Suppression of T-cell proliferation by CD4⁺CD25^brightFoxp3⁺ Tregs involves both contact-dependent and contact-independent mechanisms. (A) Autologous T cells stimulated by IDO-expressing, mature moDCs were sorted cytofluorographically to collect CD4⁺CD25^bright candidate Tregs. These were then added 1:1 with either (A left panel) new allogeneic responder T cells stimulated by freshly thawed, mature moDCs, autologous to the candidate Tregs, or (A right panel) new responder T cells, autologous to the candidate Tregs, stimulated by new allogeneic, mature moDCs. The candidate Tregs were in contact (□) or separated in transwell inserts (▩) from the responder T cells, compared with control MLRs containing no candidate Tregs (■). After 4 to 5 days in culture, responder T-cell proliferation was measured by [³H]TdR incorporation (average of quadruplicate means ± SEM, n = 2 independent experiments; P value indicated on each graph). (B) Neutralizing anti–TGF-β and anti–IL-10 antibodies, or their respective isotype-matched, nonreactive antibodies (mouse and rat IgG1), were added to MLRs containing CD4⁺CD25^bright candidate Tregs added 1:1 with new allogeneic responder T cells stimulated by mature moDCs, autologous to the candidate Tregs. After 4 to 5 days in culture, responder T-cell proliferation was measured by [³H]TdR incorporation and compared with control MLRs containing no candidate Tregs (■) and control MLRs containing candidate Tregs without neutralizing antibodies (□; average of triplicate means ± SEM, n = 2 independent experiments; P values indicated on graph).