(A and B) Western blots detecting SPL expression in homogenates of SPLF and SPLTECKO mouse thymuses (A) and spleens (B). mature T cells through positive and negative selection. The population of mature T cells egressing from the thymus exhibits a diverse repertoire of antigen recognition capable of mounting effective protective immunity, yet lacking autoreactivity. Tight regulation of thymic egress ensures full maturation and prevents potentially dangerous autoreactive T cells from entering the circulation (Gr?ler et al., 2005). Although the vast majority of thymocytes are eventually culled through the processes of positive and negative selection, 2% reach the final stage of maturity, exiting from the thymus and entering into the circulation (Berzins et al., 1999). Thymic egress is an actively regulated process. Mature T cells egress from the thymus by chemotaxis in response to a sphingosine-1-phosphate (S1P) gradient (Schwab et al., 2005). S1P levels are highest in plasma and lowest in the lymphoid organs PF-04418948 (Rivera et al., 2008). S1P is a ubiquitous bioactive sphingolipid that regulates diverse immunological functions including hematopoietic cell trafficking, vascular PF-04418948 permeability, and mast cell activation (Spiegel and Milstien, 2011). S1P mediates many of its actions by signaling through its five cognate G proteinCcoupled receptors, S1P1C5. In the final stages of their maturation, thymocytes up-regulate the transcription factor Krppel-like factor 2 and its target gene S1P1 (Carlson et al., 2006). S1P1 expression on mature single-positive (SP) cells enables their entry into the circulation after encountering extracellular S1P produced by neural crestCderived perivascular cells located at the corticomedullary junction (Matloubian et al., 2004; Zachariah and Cyster, 2010). There is evidence that activation of thymocytes such as by antigen challenge, infection, and cytokines is capable of modulating T cell export from the thymus (Nunes-Alves PF-04418948 et al., 2013). However, the mechanisms responsible for this phenomenon are poorly understood. Two sphingosine kinases are capable of phosphorylating sphingosine to form S1P, and five lipid phosphatases are capable of dephosphorylating S1P, thereby regenerating sphingosine (Pyne et al., 2009). In contrast to this reversible reaction, the enzyme S1P lyase (SPL), a resident protein of the ER membrane, degrades S1P irreversibly, providing global control MYO5C over circulating and tissue S1P levels (Pyne et al., 2009). SPL expression is robust in mouse thymus starting early in development and continuing through adult life (Borowsky et al., 2012; Newbigging et al., 2013). A critical role for SPL in lymphocyte egress was revealed when the food additive tetrahydroxybutylimidazole was shown to cause lymphopenia via SPL inhibition (Schwab et al., 2005). Similarly, genetically modified mice globally deficient in SPL are lymphopenic (Vogel et al., 2009). The lymphopenia associated with SPL suppression is presumed to result from disruption of the S1P gradient maintained by thymic SPL activity (Schwab et al., 2005). Both S1P1 antagonism and SPL inhibition have been explored as therapeutic strategies for treatment of autoimmune disease by blocking lymphocyte egress from the thymus and peripheral lymphoid organs (Kappos et al., 2006; Bagdanoff et al., 2010; Weiler et al., 2014). Despite the importance of S1P signaling in lymphocyte trafficking, little is known about the compartmentalization of S1P metabolism in the thymus and the cell types responsible for producing the S1P gradient. Thymic stromal cells provide the matrix and signaling cues necessary to foster proper thymocyte development. PF-04418948 The stroma contains thymic epithelial cells (TECs) and vascular and perivascular cells, as well as BM-derived antigen-presenting cell types including macrophages, B cells, and DCs (Rodewald, 2008). B cells and DCs make up a small percentage of the stroma and are located mainly in PF-04418948 the medulla and corticomedullary region (Perera et al., 2013). Thymic DCs have been shown to cross-present self-antigens acquired from medullary TECs to developing thymocytes and to facilitate the generation of regulatory T cells (Hubert et al., 2011; Lei et al., 2011)..