PMC:2222968 / 11403-13786 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2222968","sourcedb":"PMC","sourceid":"2222968","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2222968","text":"FOXP3 and GATA3 Kinetics in Differentiating Cells\nThe limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs. Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-γ, and anti IL-12), Th2, and iTreg phenotypes. After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18]. Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel). Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B). Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process. The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B). On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B). Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.\nFigure 4 FOXP3 Induction During the Differentiation Process\n(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β (5 ng/ml) or IL-4 (25 ng/ml). The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression. Bars show the mean ± SD of three independent experiments. \n(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure. 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