Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P 1 knock-out and the unavailability of selective agonists or antagonists. A potent, S1P 1 -receptor selective agonist structurally unrelated to S1P was found to activate multiple signals triggered by S1P, including guanosine 5 -3-O-(thio)triphosphate binding, calcium flux, Akt and ERK1/2 phosphorylation, and stimulation of migration of S1P 1 -but not S1P 3 -expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S1P 3 receptor reveals that agonism of S1P 1 receptor alone is sufficient to control lymphocyte recirculation. Moreover, S1P 1 receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S1P 3 , and not S1P 1 , is directly implicated in sinus bradycardia. The sustained bradycardia induced by S1P receptor nonselective immunosuppressive agonists in wild-type mice is abolished in S1P 3 ؊/؊ mice, whereas S1P 1 -selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S1P 1 receptor agonists with an enhanced therapeutic window. S1P 1 -selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P 1 suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.Sphingosine 1-phosphate (S1P), 1 through its high affinity G-protein-coupled receptors, is a physiological mediator with widespread effects upon multiple physiological systems (1). It regulates heart rate (2), coronary artery blood flow (3), blood pressure (4), endothelial integrity in lung (5, 6) and most recently has been shown to regulate the recirculation of lymphocytes (7-11). Many of the physiologically relevant functions occur in the low nanomolar range, including activation of endothelial nitric oxide synthase (12, 13), vasorelaxation (14), and inhibition of thymic egress and lymphocyte recirculation (11). Free plasma levels of S1P are tightly regulated by protein binding to albumin and high density lipoprotein to avoid the deleterious effects of systemic S1P receptor subtype activation at high concentrations of ligand, such as bradycardia and coronary artery vasospasm (3, 15). The choice of S1P, through its receptors, as an acute regulator of the number of blood lymphocytes may represent an interesting evolutionary choice by the immun...

Processing of proteins for major histocompatibility complex (MHC) class II-restricted presentation to CD4-positive T lymphocytes occurs after they are internalized by antigen-presenting cells (APCs). Antigenic proteins frequently contain disulfide bonds, and their reduction in the endocytic pathway facilitates processing. In humans, a gamma interferon-inducible lysosomal thiol reductase (GILT) is constitutively present in late endocytic compartments of APCs. Here, we identified the mouse homolog of GILT and generated a GILT knockout mouse. GILT facilitated the processing and presentation to antigen-specific T cells of protein antigens containing disulfide bonds. The response to hen egg lysozyme, a model antigen with a compact structure containing four disulfide bonds, was examined in detail.

Major histocompatibility complex (MHC) class II molecules are cell surface proteins that present peptides to CD4(+) T cells. In addition to these wellcharacterized molecules, two other class II-like proteins are produced from the class II region of the MHC, HLA-DM (DM) and HLA-DO (DO) (called H2-M, or H2-DM and H2-O in the mouse). The function of DM is well established; it promotes peptide loading of class II molecules in the endosomal/lysosomal system by catalyzing the release of CLIP peptides (derived from the class II-associated invariant chain) in exchange for more stably binding peptides. While DM is present in all class II- expressing antigen presenting cells, DO is expressed mainly in B cells. In this cell type the majority of DM molecules are not present as free heterodimers but are instead associated with DO in tight heterotetrameric complexes. The association with DM is essential for the intracellular transport of DO, and the two molecules remain associated in the endosomal system. DO can clearly modify the peptide exchange activity of DM both in vitro and in vivo, but the physiological relevance of this interaction is still only partly understood.

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). ...

Blood lymphocyte numbers, which are maintained by recirculation through secondary lymphoid organs, are essential for the efficient development of immune responses. Recirculating populations of B and T lymphocytes are regulated by the sphingosine-1-phosphate (S1P) receptor-dependent control of lymphocyte egress. T-cell egress from thymus into blood, egress from lymph node and Peyer's patch into lymph, and B-cell egress into lymph are rapidly and completely inhibited by agonism of S1P receptors. Mesenteric lymph nodes show log-jamming of lymphocytes subjacent to sinus-lining endothelium. Agonism of S1P receptors produces rapid peripheral blood lymphopenia, which is maintained in the presence of receptor agonist. Effector CD4+ and CD8+ T cells, produced by clonal expansion in draining lymph node in response to antigen, are sequestered in lymph node and fail to reach the peripheral blood. The S1P receptor system may represent an early physiological link between the non-specific inflammatory response and the alteration of lymphocyte traffic through draining lymph nodes. Pharmacological subversion of the S1P receptor system, through systemic S1P agonist-induced inhibition of lymphocyte egress, suppresses antigenic responses to peripheral, but not to systemically, delivered antigen. This inhibition induces significant immunosuppression in models of transplantation and autoimmune tissue damage that may prove to be of clinical benefit.

To address the role of different proteases in degradation of antigen destined for MHC class II‐restricted presentation, we generated cathepsin‐deficient mice carrying a transgenic B cell receptor (BCR) specific for hen egg lysozyme (HEL). We demonstrate that degradation of HEL in B lymphocytes is highly processive and does not result in discrete processing intermediates. Moreover, degradation of HEL does not require initial unlocking of the antigen by any of the cathepsins tested. Using mass spectrometry and microsequencing, we show that all major cathepsins (CatS, CatL, CatB, and CatD) digest HEL in vitro with considerable redundancy, although some preferential cleavages are evident. These observations have a functional correlate: when triggered by cathepsin S‐deficient antigen‐presenting cells, T cells that recognize different HEL epitopes fail to present two HEL‐derived epitopes, while a third epitope is presented independently of the activity of cysteine proteases. We conclude that the proteolytic processing machinery is redundant, and that several proteases can substitute for each other to degrade a given antigen. However, a certain degree of proteolytic specificity is demonstrable for the generation of particular epitopes, notably by CatS.

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.

HLA-DM (DM; in mouse H2-DM) promotes the exchange of MHC class II-associated peptides, resulting in the accumulation of stable MHC class II-peptide complexes. In naive (but not germinal center) B cells, a large part of DM is tightly associated with HLA-DO (DO; in mouse H2-O), but the functional consequence of this association for Ag presentation is debated. Here, we have extended previous studies by examining the presentation of multiple epitopes after Ag internalization by fluid phase endocytosis or receptor-mediated uptake by membrane Ig (mIg) receptors. We find that the effects of H2-O are more complex than previously appreciated; thus, while only minor influences on Ag presentation could be detected after fluid phase uptake, many epitopes were substantially affected after mIg-mediated uptake. Unexpectedly, the presentation of different epitopes was found to be enhanced, diminished, or unaffected in the absence of H2-O, depending on the specificity of the mIg used for Ag internalization. Interestingly, epitopes from the same Ag did not necessarily show the same H2-O dependency. This finding suggests that H2-O may control the repertoire of peptides presented by B cells depending on the mIg-Ag interaction. The absence of DO/H2-O from germinal center B cells suggests that this control may be released during B cell maturation.