Rapid induction of medullary thymocyte phenotypic maturation and egress inhibition by nanomolar sphingosine 1-phosphate receptor agonist

Abstract

Only a small number of T cells generated in the thymus each day are selected to replenish the peripheral T cell pool. Much is known about thymic selection; however, little is known of the mechanisms regulating medullary maturation and the release of mature T cells into the blood. Here we demonstrate a rapid acceleration of medullary thymocyte phenotypic maturation through loss of CD69 induced by sphingosine 1-phosphate (S1P) receptor agonist. Low nanomolar agonist concentrations selectively induce changes in CD69 int CD62L high single positive T cells, resulting in down-modulation of CD69 within 2 h. While CD69 loss is accelerated, egress of mature T cells into blood is inhibited >95% within 2 h. Both processes exhibit parallel sensitivities and dose-responses. Together, these data reveal a potent means for rapidly regulating thymic export where S1P receptor agonism alters both phenotypic maturation and egress of thymocytes into blood during late thymic maturation. The S1P system is now shown to acutely regulate both thymic and lymph node egress. Inhibition of lymphocyte egress from thymus and lymph node can contribute synergistically to clinically useful immunosupression by disrupting recirculation of peripheral T cells. S phingosine 1-phosphate (S1P) receptor agonists, such as the phosphate-ester metabolite of FTY720 (1, 2), are clinically useful immunosuppressants for transplantation rejection (3) and have recently been shown to regulate egress of naïve lymphocytes (4) and both CD4 (5) and CD8 (6) effector cells by retention of lymphocytes on the abluminal side of sinus-lining endothelium in lymph nodes but not spleen (4, 7). Long-term studies have also demonstrated effects on the emigration of thymocytes from thymus into blood and secondary lymphoid organs (8), suggesting that an important step in thymic egress is also affected. FTY720 and its chiral analog 2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol [AAL-(R)] are full agonists for four of five G protein-coupled receptors for S1P. Originally described as endothelial differentiation genes, or edg receptors, five cloned G protein-coupled high-affinity receptors (9) have been described for S1P that show low nanomolar binding affinities (10-14). They are closely related structurally to the receptors edg2͞lpa1, edg4͞lpa2, and edg7͞lpa3 for lysophophatidic acid. They are expressed on endothelial cells (S1P 1 , S1P 3 ), and mRNA can be detected in lymphocytes (S1P 4 , S1P 5 ) (15, 16) and on vascular smooth muscle and myocardium (S1P 2 and S1P 1 ). Some investigators have suggested that S1P 1 is also expressed in lymphocytes, although the presence of active receptor on uncultured lymphocytes remains controversial (17)(18)(19). Of these receptors, only S1P 2 is not activated by the phosphoryl metabolite of FTY720 (1). S1P 1 , S1P 3 , S1P 5 , and S1P 4 receptors activate multiple cellular responses, through pertussis toxin (PTX)-sensitive i.e., G icoupled events, and PTX-independent transduction steps (activation of rac and rho small GTP-binding proteins) (20-23). ...

Expression of the Sphingosine 1-Phosphate Receptor, S1P1, on T-cells Controls Thymic Emigration

“…In the thymus, FTY720 at low doses and within a short period of time triggers a maturation of single positive cells to a late medullary phenotype and, ultimately, causes an inhibition of T-cell emigration without changes in the total single positive cell numbers (26). With prolonged FTY720 treatment, mice show an increase in mature single positive cells in the thymus and a block in emigration of T-cells to the periphery (25).…”

“…FTY720, a sphingosine analogue, is phosphorylated by sphingosine kinase type I and more efficiently by sphingosine kinase type II (19 -21) and functions as an agonist ligand for S1P 1 , S1P 3 , S1P 4 , and S1P 5 receptors (19,22). FTY720 causes lymphopenia through sequestration of circulating lymphocytes within lymph nodes and Peyer's patches (19,(22)(23)(24) and also blocks the egress of T-cells from the thymus (25,26). However, it is not known at which S1P receptor and cell type FTY720 exerts its effects.…”

The Apoptotic Pathway Contributing to the Deletion of Naive CD8 T Cells during the Induction of Peripheral Tolerance to a Cross-Presented Self-Antigen

“…The sphingosine-1-phosphate receptor-1 agonist (FTY720) was generously provided by Dr. Hugh Rosen (The Scripps Research Institute) (33)(34)(35)(36) To measure CD8 T cell apoptosis in vivo, single-cell suspensions were prepared from the peripheral and pancreatic LN and Annexin V staining was measured by flow cytometry using Annexin V-allophycocyanin staining Ab (BD Biosciences) according to the manufacturer's instructions. Briefly, cells were stained with Pacific Blue-conjugated anti-CD8 mAb (Caltag Laboratories) and PE-conjugated anti-Thy1.1 mAb (BD Pharmingen) followed by washing twice with cold PBS and then re-suspended in 1ϫ binding buffer at a concentration of 1 ϫ 10 6 cells/tube.…”

The role of apoptosis in the development and function of T lymphocytes

“…After successful positive selection, DP thymocytes further differentiate into CD4 + or CD8 + single positive thymocytes and undergo several maturation steps including the downregulation of HSA and upregulation of CCR7 [136]. SP thymocytes migrate from the cortex into the medulla of the thymus.…”