Egress Of Thymocytes Research Articles

Recent progress in the development of selective S1P1 receptor agonists for the treatment of inflammatory and autoimmune disorders

Background: The sphingosine-1-phosphate receptor 1 (S1P1) belongs to the endothelial differentiation gene family of G-protein-coupled receptors involved in embryonic development, cellular differentiation and survival, adherence and tight junction assembly, and leukocyte trafficking. In vivo S1P1 is required for egress of thymocytes into the blood and of naive lymphocytes from secondary lymphoid organs back into the lymph. Downregulation of S1P1 disrupts lymphocyte migration, tissue homing and recirculation. To explore the immunomodulatory potential of S1P1, pharma is developing S1P1-selective agonists for treating autoimmune and inflammatory disorders. FTY720 (fingolimod, Novartis AG), a nonselective S1P1 agonist that induces sustained lymphopenia, is a promising pseudo-lipid aminodiol prodrug that has been shown in preclinical and early clinical trials to be effective in suppressing various autoimmune conditions and to aid in acute organ transplant, and is currently in pivotal Phase III clinical trials for relapsing-remitting multiple sclerosis. Objective: In this review article, we outline recent advances made in the discovery and patenting of new small-molecule S1P1 selective agonists and summarize a rapidly growing body of intellectual property. Conclusion: Fingolimod-like S1P1 agonists with a novel mechanism of action have emerged as promising immunosuppressants. S1P1 agonists induce T-cell sequestration in lymph nodes and Peyer's patches, diminishing their ability to reach distant sites and cause pathology. Preclinical studies have already provided preliminary evidence that low-dose treatment with fingolimod in conjunction with subtherapeutic doses of ciclosporin A and tacrolimus enable potent immunosuppression in the absence of immediately serious side effects. Future efforts will determine whether S1P1 agonists do indeed synergize with existing DMAs to create new mutidrug paradigms with superior efficacy and safety for the treatment of autoimmune diseases and the prevention of organ transplant rejection.

The thymus is required for the ability of FTY720 to prolong skin allograft survival across different histocompatibility MHC barriers

The immunosuppressive effect of FTY720 is associated with the reversible sequestration of lymphocytes from the blood and the spleen into secondary lymphoid organs and reduced egress of mature thymocytes from the thymus. This work was designed to dissect the differential effect of FTY720 on CD4 and CD8 T cell-mediated mechanisms of skin graft rejection in the presence (euthymic) or absence (thymectomized) of thymic output. To that end, untreated and FTY720-treated euthymic (Euthy) and thymectomized (ATX) mice received skin allografts across a full, class II or class I major histocompatibility complex (MHC) mismatched (MM) barriers and graft survival was monitored. We demonstrate that a short course of FTY720 treatment significantly augments the survival of full, class I and class II MHC MM skin grafts compared to the nontreated controls. Interestingly, FTY720-treated Euthy recipients showed a significantly prolonged skin allograft survival compared to FTY720-treated ATX mice. These results together show that FTY720 impairs both CD4 and CD8 T cell-mediated mechanisms of rejection and, more importantly, the presence of the thymus is necessary for the ability of FTY720 to modulate skin allograft rejection across different histocompatibility MHC barriers.

Kruppel-like factor 2 regulates thymocyte and T-cell migration

Mammalian Kruppel-like transcription factors are implicated in regulating terminal differentiation of several tissue types. Deficiency in Kruppel-like factor (KLF) 2 (also known as LKLF) leads to a massive loss of the peripheral T-cell pool, suggesting KLF2 regulates T-cell quiescence and survival. Here we show, however, that KLF2 is essential for T-cell trafficking. KLF2-deficient (Klf2-/-) thymocytes show impaired expression of several receptors required for thymocyte emigration and peripheral trafficking, including the sphingosine-1-phosphate (S1P) receptor S1P1, CD62L and beta7 integrin. Furthermore, KLF2 both binds and transactivates the promoter for S1P1--a receptor that is critical for thymocyte egress and recirculation through peripheral lymphoid organs. Our findings suggest that KLF2 serves to license mature T cells for trafficking from the thymus and recirculation through secondary lymphoid tissues.

CD69 down‐modulation and inhibition of thymic egress by short‐ and long‐term selective chemical agonism of sphingosine 1‐phosphate receptors

Thymic development requires proliferation, selection, maturation and release of mature single-positive CD4 and CD8 T cells into the periphery. In mice, non-selective sphingosine-1 phosphate (S1P) receptor agonists, active on four of the five known S1P receptors, alter thymocyte phenotype and egress. Here, we show that down-modulation of CD69 occurs acutely and transiently at a discrete and late stage of medullary development after a single-dose administration of S1P(1) receptor-selective agonist, which induces long-term tonic receptor activation in the absence of receptor degradation. In addition, agonist acutely inhibited egress of mature thymocytes into peripheral lymphoid organs, suggesting that both the phenotype and migration of medullary thymocytes are regulated simultaneously and coordinately by agonism of S1P(1) alone. Long-term dosing shifted the early/late medullary thymocyte ratio with an expansion of the late medullary compartment, as mature CD69(-) thymocytes were retained within the thymus. Therefore, chemical agonism of S1P(1) accelerates medullary phenotypic maturation and inhibits egress, leading to the expansion and accumulation of the recent thymocyte emigrant population in the medulla. However, chemical agonism fails to replicate the S1P(1)-null CD69(hi) late medullary phenotype, suggesting that agonism and gene deletion operate by distinct mechanisms, and that functional receptor antagonism may not be required for lymphocyte sequestration.

Cutting Edge: Foxj1 Protects against Autoimmunity and Inhibits Thymocyte Egress

Previous studies suggest that the forkhead transcription factor Foxj1 inhibits spontaneous autoimmunity in part by antagonizing NF-kappaB activation. To test this hypothesis, we ectopically expressed Foxj1 in the T cells of lupus-prone MRL/lpr mice by backcrossing a CD2-Foxj1 transgene against the MRL/lpr background. Strikingly, CD2-Foxj1-MRL/lpr animals showed a significant reduction in lymphadenopathy, pathogenic autoantibodies, and end-organ disease-but surprisingly, reversion of autoimmunity was not attributable to modulation of NF-kappaB. Instead, CD2-Foxj1 transgenic mice exhibited a peripheral T cell lymphopenia, associated with an accumulation of mature single-positive thymocytes. Transgenic thymocytes demonstrated unimpaired lymphoid organ entry in adoptive transfer studies but demonstrated impaired thymic exodus in response to CCL19, apparently independent of CCR7, S1P1, and NF-kappaB. These findings confirm the importance of Foxj1 in the regulation of T cell tolerance but furthermore suggest a novel and specific role for Foxj1 in regulating thymic egress.

A CXCR4-Dependent Chemorepellent Signal Contributes to the Emigration of Mature Single-Positive CD4 Cells from the Fetal Thymus

Developing thymocytes undergo maturation while migrating through the thymus and ultimately emigrate from the organ to populate peripheral lymphoid tissues. The process of thymic emigration is controlled in part via receptor-ligand interactions between the chemokine stromal-derived factor (SDF)-1, and its cognate receptor CXCR4, and sphingosine 1-phosphate (S1P) and its receptor S1PR. The precise mechanism by which S1P/S1PR and CXCR4/SDF-1 contribute to thymic emigration remains unclear. We proposed that S1P-dependent and -independent mechanisms might coexist and involve both S1P-induced chemoattraction and SDF-1-mediated chemorepulsion or fugetaxis of mature thymocytes. We examined thymocyte emigration in thymi from CXCR4-deficient C57BL/6 embryos in a modified assay, which allows the collection of CD62L(high) and CD69(low) recent thymic emigrants. We demonstrated that single-positive (SP) CD4 thymocytes, with the characteristics of recent thymic emigrants, failed to move away from CXCR4-deficient fetal thymus in vitro. We found that the defect in SP CD4 cell emigration that occurred in the absence of CXCR4 signaling was only partially overcome by the addition of the extrathymic chemoattractant S1P and was not associated with abnormalities in thymocyte maturation and proliferative capacity or integrin expression. Blockade of the CXCR4 receptor in normal thymocytes by AMD3100 led to the retention of mature T cells in the thymus in vitro and in vivo. The addition of extrathymic SDF-1 inhibited emigration of wild-type SP cells out of the thymus by nullifying the chemokine gradient. SDF-1 was also shown to elicit a CXCR4-dependent chemorepellent response from fetal SP thymocytes. These novel findings support the thesis that the CXCR4-mediated chemorepellent activity of intrathymic SDF-1 contributes to SP thymocyte egress from the fetal thymus.

Making and Breaking the Gradient

To combat infection, lymphocytes must first be "enticed" to leave their organ of residence and enter the circulation. The same is true for mature thymocytes that must exit the thymus to seed peripheral lymphoid organs. A dominant signal for lymphocyte egress comes from sphingosine 1-phosphate (S1P), although it remains to be established how this extracellular lipid mediator is itself regulated to control lymphocyte trafficking. Schwab et al. (see the Perspective by Hla) observe that a long-established inhibitor of thymocyte egress, the food colorant 2-acetyl-4-tetrahydroxybutylimidazole (THI), also inhibits lymphocyte migration and causes the accumulation of S1P in lymph nodes, which are otherwise low in S1P relative to the lymph and blood. This accumulation could be overcome with saturating levels of an essential cofactor for S1P lyase, an enzyme that degrades S1P, which suggests that THI works by preventing S1P breakdown. The maintenance of an S1P gradient between lymphoid tissues and the circulation by S1P lyase may provide a target for selective therapeutic immune suppression. S. R. Schwab, J. P. Pereira, M. Matloubian, Y. Xu, Y. Huang, J. G. Cyster, Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science 309 , 1735-1739 (2005). [Abstract] [Full Text] T. Hla, Dietary factors and immunological consequences. Science 309 , 1682-1683 (2005). [Summary] [Full Text]

Cutting Edge: Regulation of T Cell Trafficking and Primary Immune Responses by Sphingosine 1-Phosphate Receptor 1

Signaling by sphingosine 1-phosphate (S1P) through its receptor S1P(1) has recently been shown to promote thymocyte egress. In the periphery, S1P(1) is expressed on naive T cells but lost upon T cell activation. To determine the significance of S1P(1) down-regulation and function of S1P(1) in peripheral T cells, we developed transgenic mice that constitutively express S1P(1) in T cells. Mature T cells from these mice exhibited enhanced chemotactic response toward S1P, and preferentially distributed to the blood rather than secondary lymphoid organs. S1P(1)-transgenic mice showed significant delay in the onset of experimental autoimmune encephalomyelitis, and had defective contact hypersensitivity reaction and local Ag-induced responses. These impairments were associated with reduced numbers of Ag-activated T cells in the draining lymph nodes. Our studies demonstrate that S1P(1) signaling affects systemic trafficking of peripheral T cells and immune responses and highlight that levels of S1P(1) expression represent an important mechanism of immune regulation.

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

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.

Thymocyte emigration is mediated by active movement away from stroma-derived factors

T cells leave the thymus at a specific time during differentiation and do not return despite elaboration of known T cell chemoattractants by thymic stroma. We observed differentiation stage–restricted egress of thymocytes from an artificial thymus in which vascular structures or hemodynamics could not have been playing a role. Hypothesizing that active movement of cells away from a thymic product may be responsible, we demonstrated selective reduction in emigration from primary thymus by inhibitors of active movement down a concentration gradient (chemofugetaxis). Immature intrathymic precursors were insensitive to an emigration signal, whereas mature thymocytes and peripheral blood T cells were sensitive. Thymic stroma was noted to elaborate at least two proteins capable of inducing emigration, one of which was stromal cell–derived factor-1. Thymic emigration is mediated, at least in part, by specific fugetaxis-inducing factors to which only mature cells respond.

Thymocyte emigration is mediated by active movement away from stroma-derived factors.

T cells leave the thymus at a specific time during differentiation and do not return despite elaboration of known T cell chemoattractants by thymic stroma. We observed differentiation stage-restricted egress of thymocytes from an artificial thymus in which vascular structures or hemodynamics could not have been playing a role. Hypothesizing that active movement of cells away from a thymic product may be responsible, we demonstrated selective reduction in emigration from primary thymus by inhibitors of active movement down a concentration gradient (chemofugetaxis). Immature intrathymic precursors were insensitive to an emigration signal, whereas mature thymocytes and peripheral blood T cells were sensitive. Thymic stroma was noted to elaborate at least two proteins capable of inducing emigration, one of which was stromal cell-derived factor-1. Thymic emigration is mediated, at least in part, by specific fugetaxis-inducing factors to which only mature cells respond.