CD46-induced Tr1-like cell supernatants do not suppress dendritic-cell (DC) maturation despite high IL-10 content. (A) General experimental approach. Purified human blood monocytes were cultured in GM-CSF/IL-4–containing media for 72 hours. The generated DC precursors were incubated for 24 hours with supernatants derived from either CD3-, CD3/CD28-, or CD3/CD46-activated T cells (Tr1) or maintained in GM-CSF/IL-4–containing control media. Maturation of these DC populations was induced by LPS addition for 24 hours. The maturation stage of the DCs was determined by their maturation marker expression profile and their potential to induce allogeneic T-cell proliferation in an MLR. (B) DCs exposed to supernatants from CD3/CD46-activated Tr1 cells up-regulate maturation markers. iDCs were generated and the media replaced with supernatants derived from CD3/CD46-induced Tr1 cells, CD3- and CD3/CD28-activated CD4⁺ T cells, or fresh media with 500 pg/mL recombinant human IL-10 (rec. IL-10). DC maturation was induced by LPS addition and surface expression of MHCII, and CD86 was analyzed by FACS after 24 hours. (C-D) DCs matured in control media (C) or in supernatants from CD3/CD46-activated Tr1 cells (D) demonstrate strong MLR potential. DCs were generated and treated as described in panel B, and their potential to induce allogeneic T-cell proliferation was measured in an MLR: DCs were seeded in serial 2-fold dilutions beginning with 50 × 10⁴ cells/well and irradiated. Purified allogeneic PBMCs were then added at 50 × 10⁴ cells/well, the cocultures were incubated for 4 days, and proliferation of allogeneic T cells was measured via [³H] thymidine incorporation. Shown is 1 representative of 8 independently performed experiments (with each activation condition in triplicate). (E) Summary and statistical evaluation of all proliferation experiments is shown as relative data ± standard error. n indicates the number of separate proliferation conditions (ie, 4 dilutions/experiment in duplicates or triplicates) considered for this evaluation. Mo indicates monocyte; Tr1 supern., supernatant derived from CD3/CD46-induced Tr1-like cells; and MLR, mixed lymphocyte reaction. F statistics and P values are shown for the comparison.