Figure 1. The phenotype and sensitivity to apoptosis of CD4⁺CD25^high T cells expanding±RAPA.

A. Gating strategy used to designate CD25^high and CD25^inter/low T cells within the CD3⁺CD4⁺ T cell population. Note that after 6 weeks of culture+RAPA, nearly all cells are CD25^high. B. Left panel, percentages of expanded CD4⁺CD25^neg or CD4⁺CD25⁺ T cells±RAPA after 3 weeks in culture. Data are means±SD cells in 10 cultures initiated with MACS-purified CD4⁺ T-cell subsets. Right panels, ANX V binding to fresh, uncultured MACS-isolated CD4⁺CD25⁻ and CD4⁺CD25^high T cells. A representative dot blot is shown. C. ANX V binding to CD4⁺CD25⁻ T cells in 3 week cultures±RAPA. The data are mean percentages±SD of ANX V⁺ cells in 10 cultures. Note that only few CD4⁺CD25^neg cells expanding in cultures with no RAPA bind ANX V, while all CD4⁺CD25^neg cells present in +RAPA cultures bind ANX V. D. ANX V binding to CD4⁺CD25^neg and CD4⁺CD25^high T cells after 3 weeks of culture+1 nM RAPA. A representative dot blot is shown. E. Western blots of CD4⁺CD25^high and CD4⁺CD25^neg T cells±RAPA showing caspase-3 activation only in the CD4⁺CD25^neg T-cell subset +RAPA.