The Role of TGF-beta in T Cell Differentiation
Although TGF-beta-reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-beta-mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A). It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3- cells and thus decrease the relative percentage of FOXP3+ cells. However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3-. In fact both populations showed similarly enhanced proliferation (Figure 6A). Furthermore the TGF-beta-mediated induction of FOXP3 expression was not caused by overgrowth of a CD25-FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells. CD25 was down-regulated in TGF-beta-treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-beta and IL-4. 
The addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B). In line with the previous findings, the IL-4-producing cells and the FOXP3 expressing cells are nonoverlapping populations. Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression. The expression kinetic of naive T cells exposed to IL-4 and TGF-beta demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4-8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3. 
These results showed that IL-4 acts in vitro as an inhibitor of FOXP3 expression, without interfering with TGF-beta signaling, probably acting at the level of transcription factors, and possibly by a GATA3-dependent mechanism. 
