<p>(A) Analysis of CD4+ gated murine Tregs and Teffs after 3 days of suppression assay showed that while Tregs gradually lose Foxp3 (top) and Helios (bottom) expression, a substantial proportion of Teff cells upregulate Helios (bottom), but not Foxp3 (top) expression; CFSE-negative gates show Tregs. (B) Treg division during suppression assays correlated with Helios and Foxp3 co-expression, while non-dividing Tregs lost both markers. Upper row: gating of dividing and non-dividing Tregs, with numbers showing % of Treg divisions at 1/1 (left), ½ (middle) and ¼ (right) ratios; arrows point to Helios and Foxp3 co-expression in corresponding divided and non-divided Tregs subsets through the same 1/1 to ¼ Treg/Teff ratios. (C) Tregs do not induce Helios expression in Teffs since CFSE+ CD4+ Teffs have similar Helios expression despite differing numbers of Tregs (1/1, ½ and ¼ ratios shown). (D) Analysis of Teffs gated into Helios+ and Helios- subsets showed Helios+ Teffs have a higher division rate and greater resistance to Treg suppression than Helios- mouse Teffs. Human Helios+ CD4+ (E) and Helios+ CD8 (F) Teffs showed higher divisions than Helios- cells regardless of Treg presence or absence, and Helios+ responder cells were more resistant to suppression than Helios- responders. Data are representative of 3 experiments.</p
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