FOXP3 Induction in T Cell Subsets
It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation. However, it is not known if all CD4+ T cells have the same capacity to express FOXP3. To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1). The CD4+CD25-, CD45RA+, CD45RO+, and CD4+CD45RO+CD25- were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-beta. Th1 cells showed only a 10-fold increase. In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression. The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel). 
Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-beta signaling and thus prevent FOXP3 induction. However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data). These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B). The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A). Only iTreg cells expressed FOXP3 in resting conditions. Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression. Repeated exposure to TGF-beta did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells. Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4-producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B). Furthermore TGF-beta-mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B). Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS). IL-4-expressing cells were most abundant among CD45RO+CD25- cells, which did not co-express FOXP3 (Figure 3A, left panel). In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel). As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background. The IL-4+ cells were confined to the FOXP3- cells, as shown for the CD45RO+CD25-, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B). In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C). Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap. Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells. 
