Figure 1

Gamma chain cytokines rescue T _reg cells from apoptosis in vitro. (a) Viability of T_con or T_reg cells stimulated for four days with soluble anti-CD3 and anti-CD28. IL-2 was added at indicated concentrations in T_reg cultures at the beginning of stimulation (left panel). Percentages of events in the live gates (PI^neg and FSC^high) in flow cytometric analyses are shown. Viability of T_reg cells stimulated as in 'a'. Indicated cytokines were added at 20 ng/ml in T_reg cultures at the beginning of stimulation (right panel). (b) Proliferation of CFSE labeled T_con or T_reg cells that were isolated ex vivo or stimulated as in 'a' with or without indicated cytokines, each at 20 ng/ml concentration. (c) Viability of CFSE labeled T_reg cells that were stimulated as in 'a' and cultured without or with indicated numbers of conventional CD4⁺ T cells. CFSE labeling was done to distinguish T_reg cells and conventional CD4⁺ T cells in the cultures. IL-2 and IL-2 receptors were blocked using blocking antibodies added at 10 μg/ml each, in the beginning of the stimulation. Data from (a-c) represent 3 independent experiments (d) Proliferation of CFSE labeled Foxp3⁺ T_reg cells that were stimulated as in 'a' and cultured without or with CD4⁺ T cells at indicated ratios. Contaminating Foxp3^- population in CFSE labeled T_reg population is excluded in the analyses. Electron micrographs (e) or confocal microscopy analyses (f) of 2 or 3-day stimulated T_con or T_reg cells showing apoptotic T_reg cells at different stages as defined by condensed, shrunken nuclei. (Blue = nuclei stained by 6-diamidino-2-phenylindole (DAPI) in (f).