Fig. 2.

Activin-A–induced human Tr1-like cells display strong suppressive functions. (A) Human naive CD4⁺ T cells were stimulated with allergen-loaded, mitomycin-treated APCs in the presence of PBS (Tconv) or activin-A (act-A-iTr1) cells, and T cells were then isolated and cocultured with autologous naive CD4⁺ T responders (at the indicated ratios) in the presence of allergen-loaded APCs for 4 d. ³[H]Thymidine incorporation is shown. Data are representative of n = 3 independent experiments (n = 3 donors). TdR, tritiated thymidine. (B) Tconv or act-A–iTr1 cells were generated as in A, CFSE-labeled, and cocultured with Cell Trace Violet-labeled autologous naive CD4⁺ T responders and allergen-loaded, mitomycin-treated APCs. Representative FACS plots showing T-cell proliferation (Left) and cumulative data (Right), shown as mean ± SEM, are pooled from n = 6–8 separate experiments (n = 8 donors). (C) Th2 cytokines in culture supernatants are shown. Data are expressed as mean ± SEM of triplicate wells and are pooled from n = 6–8 separate experiments (n = 8 donors). (D) Tconv or act-A–iTr1 cells and allergen-loaded APCs were cultured in a transwell system across from cocultures of autologous naive CD4⁺ T cells and APCs. Suppression of T-responder proliferation is shown. Data are expressed as mean ± SEM of triplicate wells and are pooled from n = 3 independent experiments (n = 3 donors). (E) CM from Tconv or act-A–iTr1 culture supernatants was added to naive CD4⁺ T-cell/APC cocultures. ³[H]Thymidine incorporation and IL-10 release are depicted. Data are expressed as mean ± SEM of triplicate wells and are pooled from n = 4–6 separate experiments (n = 4 donors). (F) CM from Tconv or act-A–iTr1 culture supernatants was added to naive CD4⁺ T-cell/APC cocultures, as indicated. ³[H]Thymidine incorporation is depicted, and data are expressed as mean ± SEM of triplicate wells and pooled from n = 3–4 experiments (n = 4 donors). Statistical analysis was performed by the Student’s t test (A–C and E) and a nonparametric (Mann–Whitney) unpaired Student's t test (D and F) (*P < 0.05; **P < 0.01; ***P < 0.001).