The role of sphingosine-1-phosphate (S1P) and its receptors in Notch1-mediated T-cell development in the thymus: TEC-dependent and -independent signaling pathways

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S1P has important immunological, inflammatory and cardiovascular functions. In the immune system, S1P regulates the circulation, infiltration and local positioning of lymphocytes through S1P receptors (S1PR). This has been the basis for the approval of the functional S1PR1 antagonist FTY720 for the treatment of multiple sclerosis. Acute S1PR1 antagonism inhibits the exit of single-positive (SP) thymocytes, whereas chronic FTY720 administration leads to a ~20% reduction of all lymphocyte content. Furthermore, deletion of the S1P degrading enzyme S1P lyase causes thymus atrophy with massively reduced output. The reasons, mechanisms and consequences of this dramatic reduction of the cellular adaptive immune system are largely unknown. The thymus is indispensable for the functional T-cell repertoire, and its age-dependent involution impairs the recovery of the immune system after therapeutic immune depletion. Our focus lies on the roles of S1P in cardiovascular and metabolic diseases and their consequences for the immune system. Our preliminary data show that both pharmacological inhibition and genetic deletion of the S1P lyase lead to thymus atrophy caused by a so far unknown developmental T-cell defect impacting mainly on double-positive thymocytes (DPs). Lineage tracing experiments identified defects during the generation of double-negative DN2/DN3 thymocytes as well as during the differentiation of SPs from DPs. The key mechanistic hints were delivered by observations of: a) much less DNs expressing Notch1 as the crucial receptor for T-cell development, and b) a clearly altered phenotype of thymic epithelial cells (TECs) under S1P lyase inhibition. In this proposal, we will address the underlying mechanisms and characterize the role of S1P and S1PR in the early, TEC-dependent phase of T-cell development.We will first examine which S1PR on DNs mediate the inhibitory effect of S1P on Notch1 expression and how. We will address it in a co-culture system of DNs with Dll4-OP9 stroma cells, where S1P agonists, antagonists, S1P lyase inhibitors as well as DNs from S1PR and S1P lyase kos, respectively, will be employed. We will then study thymus development in global and hematopoetic S1PR kos as well as bone marrow chimera with and without S1P lyase inhibition. Furthermore, we will address the TEC phenotypic changes as a second, non-redundant cause for the thymocyte development defects. For this, we will analyze the Notch1 ligand Dll4 and the key cytokines SCF, CXCL12, CCL25 and IL-7 as well as FoxN1-regulated transcription with S1P lyase inhibition, deletion and in S1PR kos, respectively. We will also generate TEC-specific kos to identify the role of S1PR on TECs for differentiation, maturation and function. Finally, we will investigate direct, thymus cross talk-independent effects of S1P on TECs using a 3D cell culture model. The results will elucidate the role of S1P and S1PR in early thymus homeostasis and elicit possible therapeutic implications.