Gamma chain cytokines rescue T
cells from apoptosis in vitro. (a) Viability of Tcon or Treg cells stimulated for four days with soluble anti-CD3 and anti-CD28. IL-2 was added at indicated concentrations in Treg cultures at the beginning of stimulation (left panel). Percentages of events in the live gates (PIneg and FSChigh) in flow cytometric analyses are shown. Viability of Treg cells stimulated as in 'a'. Indicated cytokines were added at 20 ng/ml in Treg cultures at the beginning of stimulation (right panel). (b) Proliferation of CFSE labeled Tcon or Treg 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 Treg 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 Treg 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+ Treg cells that were stimulated as in 'a' and cultured without or with CD4+ T cells at indicated ratios. Contaminating Foxp3- population in CFSE labeled Treg population is excluded in the analyses. Electron micrographs (e) or confocal microscopy analyses (f) of 2 or 3-day stimulated Tcon or Treg cells showing apoptotic Treg cells at different stages as defined by condensed, shrunken nuclei. (Blue = nuclei stained by 6-diamidino-2-phenylindole (DAPI) in (f).