importance of identifying nTreg from iTreg, we decided to investigate the role of buy 1462249-75-7 Helios in mice and human T cells using well-characterized molecules of naive/effector/memory phenotypes, as well as Treg-associated markers. Results Helios co-expression with T cell and Treg-associated markers We first assessed Helios expression by flow cytometric analysis of human and murine peripheral blood mononuclear cells, plus cells from murine lymph nodes and spleens. August 2011 | Volume 6 | Issue 8 | e24226 Helios Is a Marker of T Cell Activation Cell population % Helios+ murine cells Blood LN 12.361.2 74.863.5 2.860.6 4.660.5 4.461.2 Spleen 11.260.6 76.763 3.861.3 7.460.9 3.260.4 % Helios + human cells Blood 12.661.2 63.461.2 3.560.7 21.863.5 16.162.8 CD4+cells CD4+ Foxp3+ cells CD4+ Foxp3- cells CD8+ cells CD4- CD8- cells 13.460.8 79.760.7 6.662 6.962.9 3.860.3 doi:10.1371/journal.pone.0024226.t001 CD4+, CD8+ and CD4-CD8- cells expressed Helios, with CD4+ Foxp3+ Treg showed the highest Helios expression in both species. There were no gender-based differences in Helios expression when tested using age-matched samples. In mice, Helios+ T cells from lymph nodes and spleen were more likely to co-express Foxp3 and CD25 than PBMC. In human CD4+ cells, the highest levels of Helios expression were associated with Foxp3, CD25, CD39, CTLA-4 and low levels of CD127, while intermediately positive Helios+ cells included non-Treg cells. Importantly, CD4+ Helios+ and CD4+ Helios- cells expressed CD31, a marker of recent thymic emigrant cells, almost equally. Together, these data “1656303 suggest that Helios might not be a specific marker of nTreg cells. Helios expression and T-cell maturation Flow cytometric analysis showed murine CD4+ Helios- T cells were mostly naive CD62L+ CD44- cells, whereas CD4+ Helios+ T cells were enriched for memory or effector phenotypes. These data suggested that Helios might be induced by T cell activation. Indeed, Helios+ cells largely lacked expression of CD45RB, a CD45 isoform of naive cells. Since iTreg undergo T cell receptor stimulation during conversion, they are less naive in phenotype than nTregs. Assessment of markers of cell maturation using murine CD4+Foxp3+ Tregs gated into Helios+ or Heliossubsets showed that murine CD4+ Foxp3+ Helios- Tregs had twice as many naive CD45RB+ cells, and only half as many CD44+ effectors/memory cells, as Helios+ Tregs. 2 August 2011 | Volume 6 | Issue 8 | e24226 Helios Is a Marker of T Cell Activation Human PBMC showed a similar pattern with Helios+ Tregs or conventional CD4+ Foxp3- T cells and CD8+ T cells being enriched for mature CD45RO+ CD45RA- cells, whereas Helios- populations exhibited the more naive CD45ROCD45RA+ phenotype. These data support the concept that Helios may be a marker of T cell activation. Helios expression upon T cell activation We tested whether Helios expression is a marker of T 
cell activation by studying Helios expression in Tregs and T-effector cells during standard in vitro Treg suppression assays. In these assays, Teffs are stimulated using CD3 mAb and antigenpresenting cells, in the presence of varying proportions of Tregs, for 3 days or 4 days. To monitor their divisions during each assay, Teff cells were labeled with CFSE. Numerous murine CFSE+ CD4+ Teffs acquired a Helios+ phenotype, and by day 3, 2530% of mouse Teffs were Helios+; “2987731 this was an,6-fold increase in Helios expression compared to freshly isolated CD4+CD25- cells. As bead-isolated CD4+ CD25- Teffs coul
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