Functional characterization of FOXP3+ T cells in healthy donors.

<p><b>A.</b> Cell sorting strategy to isolate rTregs, aTregs, FOXP3+ non-Tregs, naive and memory T cells as live cells. <b>Top Panel.</b> A representative histogram showing purified CD4+ T cells isolated from a healthy donor by negative selection. Cells were stained before sorting with anti-CD4, anti-CD25 and anti-CD45RA antibodies. rTreg cells were identified as CD25^lowCD45RA+, aTreg cells as CD25^highCD45RA-, FOXP3+ non-Tregs as CD25^lowCD45RA-, naive T cells as CD25-CD45RA+ and memory T cells as CD25-CD45RA-. <b>Bottom panel.</b> Dot plots of each cellular subset after sorting. The purity of the sorted populations was >98% in all the experiments. The expression of FOXP3 in sorted cells was detected in >90% of aTregs and >80% of either rTregs or FOXP3+ non-Tregs. Only a marginal expression of FOXP3 (<0.5%) was observed in conventional naive and memory T cells. <b>B.</b> The ability of each population of FOXP3+ T cells to suppress the mixed lymphocyte reaction was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052580#s2" target="_blank">Materials and Methods</a>. Autologous response or allogeneic responses without the addition of isolated FOXP3+ T cells are included as controls. A representative experiment (n = 3) is shown. <b>C.</b> Determination of the levels of cytokines and chemokines secreted by each activated T cell subset by the Bioplex system. rTregs, aTregs, FOXP3+ non-Tregs, naive and memory T cells were purified from PBMCs by cell sorting. Then, they were stimulated by PMA/ionomycin for 5 h, and the supernatants were harvested. The results are expressed as the mean ± SEM of six independent experiments.