IL-7Rα levels, as measured by mean RFI, were not significantly changed in the post-selection DP and CD8SP populations, although in the CD4SP (p=<0.05), and CD4+CD8lo thymocytes (p<0.002), mean RFI values were lower in Egr2f/fCD4Cre mice

relative to Egr2f/f X-396 littermates (Fig. 6A and Supporting Information Fig. 3). Therefore, IL-7R is present on post-selection cells, and there is a partial defect in its upregulation post-selection in some Egr2f/fCD4Cre subsets, perhaps attenuating the survival signal. Although the loss of high-level IL-7R signaling in post-selection CD4+CD8lo cells may be of importance, regulation of survival during selection itself is accomplished by suppressor of cytokine signaling 1 (Socs1), a protein that functions downstream of cytokine receptors, and takes part in a negative feedback loop to attenuate cytokine signaling. Pre-selection DP are susceptible to apoptosis because cytokine signal transduction is suppressed by high levels of Socs1, and cytokine responsiveness, and hence protection from apoptosis is only restored when

Socs1 is downregulated in response to TCR signaling during selection 30. Loss of Socs1 during thymocyte development results in an increase in the Nivolumab concentration CD8SP subset 33, as also observed here in Egr2-Tg mice. To determine whether Egr2 was able to influence Socs1 expression, thymocytes from Egr2-Tg and Egr2f/fCD4Cre knockout mice and littermate controls were sorted as shown in Fig. 1A, and Socs1 mRNA levels determined by qRT-PCR. Figure 6B demonstrates that relative to controls, levels of Socs1 were higher in both pre- and post-selection DP in Egr2f/fCD4Cre knockout mice, and lower in the same populations from Egr2-Tg animals. In CD8 and CD4SP thymocytes, Socs1 was downregulated normally irrespective of genotype. Therefore, Egr2 is able to regulate Socs1 expression during selection, but does not thereafter. Socs1 prevents cytokine signaling through inhibition of Stat5 phosphorylation 34, and so pStat5 levels following cytokine stimulation Cediranib (AZD2171) should be lowered in Egr2f/fCD4Cre mice, where Socs1 is increased. CD69+ post-selection thymocytes

were purified from Egr2f/fCD4Cre and Egr2f/f thymuses, and stimulated with IL-7 for 0, 10 and 20 min, after which pStat5 induction was assessed by Western blotting. As shown in Fig. 6C, relative to total Stat5 protein, pStat5 was reduced in the absence of Egr2. We then went on to assess whether IL-7-mediated survival was impaired. Although we did not observe gross changes in the survival profile of total thymocytes in the presence of IL-7 (data not shown), Fig. 6D shows that loss of Egr2 resulted in impaired survival of purified post-commitment CD4+CD8lo cells when cultured in the presence of IL-7 over a 3-day period. Therefore, loss of Egr2 results in Socs1 de-repression, and this, perhaps combined with the defect in IL-7R upregulation, causes a decrease in Stat5 phosphorylation and survival.