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) Were deleted by treatment with tamoxifen for 6-8 weeks. To determine thymic development of Treg cells in such E-protein KO 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. We next turned our attention to the mechanism by which E-protein deletion leads to the above increased induction of Foxp3+ Tregs. It is well established that IL-2 signaling plays an indispensable role in nTreg development;we therefore focused on the possibility that deletion of E-protein promotes increased Foxp3 expression because such deletion results in increased IL-2 receptor expression and/or sensitivity. In a first set of studies along these lines we determined the expression of IL-2Ra, IL-2Rb and IL-2Rcommon gamma chain on CD4+ 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, whereas IL-2Rcommon gamma chain was not upregulated. Interestingly, the up-regulation of both CD25 and CD122 was noted to occur in Foxp3-negative thymocytes, i.e., cells that had not yet undergone differentiation into Foxp3+ cells. In a related set of studies we determined if the increased IL-2 receptor expression and sensitivity in thymocytes of E-protein KO mice could be associated with increased activation of STAT5, an activation event 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. As in the case of CD25, such up-regulation was also noted in Foxp3-negative thymocytes. In a final set of studies exploring the effect of effect of E-proteins on IL-2 signaling we conducted extensive molecular studies to determine if E-proteins affected IL-2Ra expression at the molecular level. In these studies we performed luciferase reporter assays with cells transfected with constructs containing a luciferase reporter driven by the IL-2Ra promoter and a conserved enhancer region and co-transfected with an E-protein expression plasmid. We found that E-protein expression inhibits the luciferase signal in cells with the enhancer-containing contruct but not in cells without the enhancer or a mutated enhancer. It was thus apparent that E-protein exerts a negative effect on the transcription of IL-2Ra via its capacity to bind to an IL-2Ra enhancer element. This negative effect thus provides one important reason why E-protein deletion is associated with increased expression of Foxp3+ cells. We next turned our attention to the relation of TCR stimulation to E-protein expression to determined if the intensity of TCR stimulation, a factor that regulates Foxp3 expression in the normal thymus, also regulates E-protein expression. Consistent with previous findings, we found that TCR stimulation led to dose-dependent E-protein down-regulation. These in vitro results established that TCR signaling causes E-protein down-regulation and thus that such signaling has the potential to regulate Treg cell differentiation via effects on E-protein levels;however, they did not address whether such regulation does in fact occur in vivo in association with the level of TCR signaling that is required for physiologic nTreg differentiation inn the thymus. To address this latter question we next determined E-protein levels in Nr4a1(Nur77)-GFP transgenic mice in which it has been shown: 1) that the TCR-induced expression of Nur77 (GFP) corresponds to the strength of TCR stimulation;2) that SP Foxp3+ thymocytes express higher levels of Nur77 than SP Foxp3- thymocytes indicating that differentiation into Foxp3+ cells is accompanied by more intense TCR stimulation (Moran et al., 2011). These mice thus offered a way of monitoring E-protein expression in cells before and after the physiological TCR stimulation that leads to Treg differentiation. Accordingly, we determined Nur77(GFP)expression in CD25+CD4SP and CD25-CD4SP thymocytes (i.e., cells equivalent to Foxp3+ and Foxp3- thymocytes, respectively) and showed that whereas CD25+ cells had higher Nur77-GFP than CD25- cells (Fig.7b, left panel) they expressed decreased E2A mRNA than CD25- cells as determined by quantitative RT-PCR. Thus, there was in fact a direct relation between strength of the TCR signal and the level of E-protein expression;in addition, the level of E-protein was decreased in association with the greater strength of the TCR stimulation occurring in association with differentiation of Tregs. To further studies to identify target genes directly regulated by E-protein activity, we performed gene profiling on sorted WT CD4+Foxp3+ and CD4SPFoxp3- thymocytes and sorted CD4+Foxp3- cell from tamoxifen-treated E2Af/fHEBf/f/ER-Cre/Foxp3KI mice. With this approach we could determine the E-protein effect distinct from the effect of Foxp3 induction by comparing gene expression profiles of CD4SPFoxp3- cells from WT and E-protein KO mice. On the other hand, we could also determine the Foxp3 differentiation effect by comparing gene expression profiles of WT CD4+Foxp3- cell and WT CD4+Foxp3+ cells. Such microarray analysis indicated that about 1250 genes exhibited mRNA transcript changes (significant at FDR 0.05) due to the E-protein effect, including 115 genes that have linear fold changes greater than 2.0, up- or down-regulated (Supp. Table I). Importantly, >50% (60 of 115) of these E-protein responsive genes were also responsive to Foxp3 differentiation. Hill et al. described a set of Treg signature genes, 331 of which were also assayed in the present study (Hill et al., 2007). Of the 118 genes common to both FoxP3+ differentiation and the Treg signature genes, 30% (37/118) were also observed to be responsive to E-protein. Thus, the E-protein effect (analogous to E-protein down-regulation caused by TCR signaling) accomplished many of the molecular changes accompanying and presumably necessary for Foxp3 differentiation. On the basis of the above data, we favor a model of TCR-induced Foxp3 induction in which one of the key initiating events is down-regulation of E-protein followed by de-repression of the transcription of many of the genes critically involved in Foxp3 expression such as the CD25 gene. This, in turn, is followed by the activation of the IL-2 signaling pathway and the elaboration of factors that underpin Treg cell development. Thus, in this view, modulation of E-protein activity is setting the threshold for TCR induction of Foxp3.
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