Abstract
T cell emigration from the thymus is essential for immunological homeostasis. While stromal cell-produced sphingosine-1-phosphate (S1P) has been shown to promote thymocyte egress via the S1P receptor, S1PR1, the significance of S1P/S1PR1 signaling in the thymic stromal cells that surround T cells remains unclear. To address this issue, we developed conditional knockout mice (Lyve1-CRE/S1pr1f/f mice) in which S1pr1 was selectively targeted in cells expressing the lymphatic endothelial cell marker, Lyve1. In these mice, T cells were significantly reduced in secondary lymphoid tissues, and CD62L+ mature CD4 and CD8 single-positive (SP) T cells accumulated in the medulla failed to undergo thymus egress. Using a Lyve1 reporter strain in which Lyve1 lineage cells expressed tdTomato fluorescent protein, we unexpectedly found that a considerable proportion of the thymocytes were fluorescently labeled, indicating that they belonged to the Lyve1 lineage. The CD4 and CD8 SP thymocytes in Lyve1-CRE/S1pr1f/f mice exhibited an egress-competent phenotype (HSAlow, CD62Lhigh, and Qa-2high), but were CD69high and lacked S1PR1 expression. In addition, CD4 SP thymocytes from these mice were unable to migrate to the periphery after their intrathymic injection into wild-type (WT) mice. In contrast, WT T cells could migrate to the periphery in both WT and Lyve1-CRE/S1pr1f/f thymuses. These results demonstrated that thymocyte egress is mediated by T cell-expressed, but not stromal cell-expressed, S1PR1 and caution against using the Lyve1-CRE system for selectively gene deletion in lymphatic endothelial cells.
Highlights
Sphingosine-1-phosphate (S1P) is a polar lipid mediator that is intracellularly generated from sphingomyelin by the successive actions of ceramidase and the sphingosine kinases, such as Sphk1 and Sphk2, and is transported out of the cells by S1P transporters, such as Spns2 [1]
Previous studies demonstrated that the thymocyte egress occurs via two different routes, blood vessels [10, 12] and lymphatics [12,13,14], and that thymocyte egress via blood vessels is critically regulated by lymphocyte-intrinsic S1PR1 and pericyte-derived S1P [10]
Our study showed that the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1)-CRE/S1pr1f/f mice exhibited marked increases in thymic CD4+ and CD8+ SP T cell subsets and enlarged thymic medullas compared with those of control S1pr1f/f mice or WT mice
Summary
Sphingosine-1-phosphate (S1P) is a polar lipid mediator that is intracellularly generated from sphingomyelin by the successive actions of ceramidase and the sphingosine kinases, such as Sphk and Sphk, and is transported out of the cells by S1P transporters, such as Spns2 [1]. S1PR1 couples mainly to Gi/o proteins to induce activation of the Ras–ERK, PI3K–Akt, and small GTPases (Rac and Rho) signaling pathways [5]. Both S1pr1-deficient mice [6] and Tie2-CRE/S1pr1f/f mice [7], in which S1pr is selectively disrupted in endothelial cells, die during embryogenesis due to vascular network abnormalities. S1PR1 is highly expressed in lymphocytes, and as described above, lymphocyte-intrinsic S1PR1 is thought to regulate lymphocyte egress from the thymus [8,9,10] as well as from secondary lymphoid tissues [9]
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