Fig. S3.

Human act-A–iTr1 cells are hyporesponsive to restimulation in vitro. (A) Tconv or act-A–iTr1 cells were isolated from primary stimulation cultures as above; labeled with CFSE; and restimulated with fresh allergen-loaded, mitomycin-treated APCs for another 7 d. Representative FACS plots are shown. (B) Cumulative data, shown as mean ± SEM, from n = 3 independent experiments (n = 3 donors) are depicted. (C) FACS plots showing annexin-V and 7-AAD expressing Tconv or act-A–iTr1 cells. Numbers in plots indicate percentages of annexin-V⁺7AAD⁺CD4⁺ T cells (data represent n = 2 donors). (D) Tconv or act-A–iTr1 cells were restimulated with allergen-loaded, mitomycin-treated APCs and recombinant human IL-2, as indicated. ³[H]Thymidine incorporation is depicted. Data shown are mean ± SEM of triplicate wells and are representative of n = 4 independent experiments (n = 4 donors). TdR, tritiated thymidine. (E) Naive CD4⁺ T cells were cultured with mitomycin-treated, allergen-loaded APCs in the presence of control (PBS) or activin-A for 14 d, and CD4⁺ T cells were then isolated and cultured with fresh APCs in the presence of tetanus toxoid for another 6 d. ³[H]Thymidine incorporation and IFN-γ release in secondary cultures are depicted. Data shown are mean ± SEM of triplicate wells and are pooled from n = 2 independent experiments (n = 2 donors). Statistical significance was obtained by the Student’s t test (**P < 0.01; ***P < 0.001).