To investigate the role of E proteins in regulatory T cell development and Foxp3 expression, we utilized inducible E protein conditional KO mice in which both E2A and HEB (E2Af/fHEBf/f ER-Cre) can be deleted by treatment with tamoxifen for 6-8 weeks. To determine thymic development of Treg cells in E protein-deficient mice, we subjected thymocytes from mice with single KO (E2A or HEB deletion only), double KO (both E2A and HEB deletion) or mice with deletion of three alleles of the four E2A/HEB alleles to intracellular staining for Foxp3+ cells. In all of these KO mice, the Foxp3+ population constituted approximately 15-30% of the total CD4+SP thymocyte population compared to 3-5% in the WT mice. The finding that E protein levels in the thymus is inversely related to thymic Treg cell differentiation prompted us to investigate if E protein expression affects the generation of Treg cells in peripheral lymphoid organs. Indeed, we found a small but significant increase of the percentage of CD4+Foxp3+cells in peripheral lymphoid tissues of E protein KO mice as compared to WT mice In further studies we determined the functional (suppressor) capacity of FACS-sorted splenic Foxp3+CD4+ cells of E protein KO mice. To examine the function of these cells in vitro we employed standard T suppressor assay and found that splenic E protein KO CD4+ T cells had a similar capacity as WT CD4+ T cells to inhibit proliferation of conventional CD4+ T cells stimulated with anti-CD3/CD28 plus IL-2. To examine the function of splenic Treg cells from E protein KO mice in vivo, we determined the capacity of the cells to inhibit cell transfer colitis. Here, we transferred wild-type nave CD45RBhi CD4+ T cells alone or in combination with WT or E protein KO Foxp3+ Treg cells into recipient mice deficient in recombination-activating gene 2(Rag2-/-) and then monitored body weight and the development of colitis in recipient mice. As expected, mice administered nave CD4+ T cells alone developed colitis within 12 weeks;however, E protein KO and WT Tregs had a similar capacity to inhibit nave CD4 T cell-triggered body-weight loss and colitis. These finding demonstrate that although E protein regulates the number of Foxp3+ Treg cells, it does not regulate Treg cell function. We next turned our attention to the mechanism by which E protein deletion could lead to increased induction of Foxp3+ Tregs. It is well established that IL-2 and IL-7 signaling dependent on common gamma chain receptor (gc receptor) signaling plays an indispensable role in nTreg development ( );we therefore focused initially on the possibility that deletion of E protein promotes increased Foxp3 expression because such deletion results in increased gc receptor expression and/or sensitivity. In a first set of studies along these lines we determined the expression of IL-2Ra, IL-2Rb, IL-7Ra on CD4+ SP and CD8+ SP thymocytes by flow cytometry. Both IL-2Ra (CD25) and IL-2Rb (CD122) were up-regulated in the thymocytes of E protein KO mice as compared to WT mice. Previous studies have shown that both IL-2 and IL-7 can induce Foxp3 expression in thymic CD4+CD25+Foxp3- Treg precursor cells without TCR engagement. We therefore explored the capacity of IL-2 and IL-7 to induce Foxp3 expression in thymocytes in the absence of TCR stimulation. To this end, we obtained purified CD4+CD25+Foxp3- thymocyte populations by flow cytometric sorting from E protein KO and WT mice and then cultured the sorted cells with IL-2 or IL-7. We found that the extent of conversion of thymocytes to Foxp3+ cells is much higher in thymocytes exposed to either IL-2 or IL-7 from E protein KO mice than WT mice. Thus, down-regulation of E protein not is only associated with increased common gamma chain receptor expression, but also with a lower threshold of response to IL-2 and IL-7 signaling. The above observations relating to increased IL-2 and IL-7 receptor expression and sensitivity in thymocytes of E protein deleted mice could be associated with increased activation of STAT5, a transcription factor activated by these receptors that has been found to be essential for Foxp3 expression and Treg cell development. Indeed, we found that thymocytes from E protein KO mice exhibited greatly increased spontaneous STAT5 phosphorylation in vivo by both flow cytometry and immunoblot. Such increased STAT5 phosphorylation was noted in CD4+ SP and CD8+ SP cells, but not DP cells, consistent with the fact that CD25 was not up-regulated in DP cells. Taken together, these various findings relating to IL-2 signaling provides strong support for the idea that E protein regulates Foxp3 expression, at least in part, by modulating IL-2 signaling via the common gamma chain.
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