Populations III and IV were further sorted into CD4SP and CD8SP subsets. qRT-PCR using cDNA from each sorted subset showed that Egr2 was upregulated between populations I and II, at the point when selection occurs, and that its expression

declined thereafter (Fig. 1B, left panel). When we performed the analogous sort, but using thymocytes purified from β2m−/− (centre panel) and MHC class II−/− (right panel) mice to exclusively generate CD4SP and CD8SP cells, respectively, a similar expression pattern was observed irrespective of genotype, suggesting Akt inhibitor that upregulation is dependent on selection, but is not lineage-specific. In primary DP cells, Egr2 is upregulated by the MAPK and calcineurin pathways following TCR ligation 15, 22, but the kinetics of its induction, and the interplay between these two pathways, has not been fully Epacadostat cell line explored. To address this issue, we cultured naïve MHC-null thymocytes directly ex vivo with PMA and ionomycin, with or without inhibitors of Erk or calcineurin signaling. As shown in Fig. 1C, maximal induction of Egr2 mRNA was achieved after 30 min of PMA/ionomycin stimulation. This rapid induction was inhibited by FK506 or cyclosporin A, inhibitors of calcium and hence calcineurin signaling, and was completely abrogated by

the inclusion of PD98059 or U0126 to inhibit Erk signaling. To further dissect Egr2′s induction by the MAPK pathway, we looked at Egr2 expression in mice deficient in the MAPK-activated transcription factor Sap-1 (Elk4), which is required for normal positive selection 23. Pre-selection TCR-βloCD69− and TCR signaled TCR-βloCD69+ thymocytes were sorted from Sap-1−/− mice and littermate controls, and Egr2 mRNA levels were measured by qRT-PCR. In both populations, there was a significant (p<0.02) reduction in the levels about of Egr2, compared with WT (Fig. 1D). Therefore, Egr2 is rapidly

upregulated by Erk and calcineurin signaling in primary thymocytes, and, like Egr1 23, its induction by Erk is dependent upon Sap-1. The timing and regulation of Egr2 expression are consistent with its having a role in positive selection. To study the role of Egr2 in thymocyte development, Tg mice overexpressing Egr2 were constructed. Mice carrying a Cre-inducible Egr2 Tg construct were bred to mice transgenic for CD4Cre recombinase 27, so that the effects of Egr2 overexpression specifically from the DP stage of development onwards could be examined. A schematic of the construct and verification that Egr2 is overexpressed in DP and SP cells, but not in the earlier DN stage, in the line presented in this paper, are shown in Supporting information Fig. 1A and B.