No abstract

CD4 + CD25 + regulatory T cells (Treg) are potent suppressors, and play important roles in autoimmunity and transplantation. Recent reports suggest that CD4 + CD25 + Treg are not a homogeneous cell population, but the differences in phenotype, function, and mechanisms among different subsets are unknown. Here, we demonstrate CD4 + CD25 + Treg cells can be divided into subsets according to cell-surface expression of CD62L. While both subsets express foxp3 and are anergic, the CD62L + population is more potent on a per cell basis, and proliferates and maintains suppressive function far better than the CD62L-population and unseparated CD4 + CD25 + Treg. The CD62L + population preferentially migrates to CCL19, MCP-1 and FTY720. Both CD62L + and CD62L-subsets prevent the development of autoimmune gastritis and colitis induced by CD4 + CD25-CD45RB high cells in severe combined immunodeficiency (SCID) mice. Overall, these results suggest CD4 + CD25 + Treg are not a homogenous cell population, but can be divided into at least two subsets according to CD62L expression. The CD62L + subset is a more potent suppressor than the CD62L-population or unfractionated CD4 + CD25 + Treg cells, can be expanded far more easily in culture, and is more responsive to chemokine-driven migration to secondary lymphoid organs. These properties may have significant implications for the clinical manipulation of the CD4 + CD25 + CD62L + cells. Tolerance is a feature of the immune system that is intimately related to discrimination between self and nonself. Clonal deletion of self-reactive T cells in the thymus is a primary tolerance mechanism, while induction of unresponsiveness or anergy in post thymic T cells may be required for the establishment of peripheral tolerance. Recent data show that Treg may play a critical role in the induction and maintenance of immune tolerance (1-5). Various types of Treg cells have been described, including Tr1 cells, Th3 cells, CD4 + CD25 + Treg cells, and others (6). CD4 + CD25 + Treg cells were first described by Sakaguchi (7). Five to 10% of CD4 + T cells constitutively expressed the a chain of IL-2-receptor (CD25) and were crucial for the control of autoreactive T cells in vivo. These cells are generated in the thymus of naïve mice, perhaps via altered negative selection by self-antigen (8). Subsequent in vitro studies showed that CD4 + CD25 + cells are typically anergic, unresponsive to TCR stimulation alone, but proliferate after addition of exogenous IL-2 (9). These cells suppress the proliferation of other CD4 + and CD8+ T cells in an antigen-nonspecific manner via a cell contact-dependent, cytokine-independent mechanism (10,11). A similar population of CD4 + CD25 + Treg cells has been defined in humans, with identical phenotypic and functional properties (12-15).CD4 + CD25 + Treg are potent suppressors in a number of in vivo models of autoimmunity, including gastritis, thyroiditis, inflammatory bowel disease and insulin-dependent diabetes (16)(17)(18)(19). Regulation of disease activity in vivo seems...

IntroductionFTY720 is a synthetic sphingosine immunosuppressant that prolongs the survival of allografts without impairing normal T and B cell activation (1). In vitro studies have proposed that FTY720, like other sphingosine derivatives, may cause apoptotic death of the T cell, although the concentrations needed for this effect are greater than the normal therapeutic dose (2). FTY720 may also prolong graft survival by decreasing T cell infiltration into allografts or otherwise altering normal T cell trafficking. Recent studies demonstrate the disappearance of lymphocytes, especially T cells, from peripheral blood and spleen within 3-24 hours after a single oral dose of the drug. Concurrently, there is a marked increase in the number of lymphocytes in peripheral LNs, mesenteric LNs, and Peyer's patches (3). The LN sequestration of lymphocytes may be involved in the immunosuppressive effects of FTY720, as regulated trafficking of lymphocytes is necessary for systemic immune responses and allograft rejection (4, 5).A recent report demonstrates that FTY720 becomes phosphorylated in vivo and then acts as a potent agonist for multiple sphingosine-1-phosphate (S1P) receptors (S1PRs) (6). S1PR activation seems responsible for downstream events that lead to lymphocyte homing. However, the cellular and molecular mechanisms are not understood. Recent studies demonstrate a novel role for lipid mediators in LN homing of DCs. A blind screening of neutralizing mAb's led to the discovery that the lipid transporter ABCB1 (previously called MDR-1 and P-glycoprotein; for new nomenclature, see http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html) regulates human DC migration to afferent lymphatics (7). Coupled with the hydrolysis of ATP, ABCB1 transports a wide range of compounds with various structures and targets, including ceramide analogs (8) and platelet-activating factor (9, 10), from the cytosol to the extracellular environment. A subsequent study demonstrated a role for another lipid transporter, Abcc1 (previously called Mrp1), in the migration of DCs to lymphatics. Abcc1 physiologically transports sphingolipid analogs and cysteinyl leukotriene C 4 (LTC 4 ), which is metabolized in the extracellular environment to LTD 4 and LTE 4 (11, 12). Mice lacking the Abcc1 gene show markedly decreased migration of Langerhans cells to LNs. Administration of exogenous cysteinyl leukotrienes (cysLTs) restores DC migration in these mice (13). These studies also demonstrate that both transporters are required for human DC migratory activity. However, whether the two transporters function in an interactive manner remains unclear (13,14).The mechanism by which Abcc1 causes LN homing of DCs was further shown to be chemokine-mediated. FTY720 is a sphingosine-derived immunosuppressant. Phosphorylated FTY720 promotes T cell homing from spleen and peripheral blood to LNs by acting as an agonist for sphingosine-1-phosphate (S1P) receptors. Here we demonstrate that FTY720 enhances the activity of the sphingosine transporter Abcb1 (Mdr1) and ...

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.

IntroductionFTY720 is a synthetic sphingosine immunosuppressant that prolongs the survival of allografts without impairing normal T and B cell activation (1). In vitro studies have proposed that FTY720, like other sphingosine derivatives, may cause apoptotic death of the T cell, although the concentrations needed for this effect are greater than the normal therapeutic dose (2). FTY720 may also prolong graft survival by decreasing T cell infiltration into allografts or otherwise altering normal T cell trafficking. Recent studies demonstrate the disappearance of lymphocytes, especially T cells, from peripheral blood and spleen within 3-24 hours after a single oral dose of the drug. Concurrently, there is a marked increase in the number of lymphocytes in peripheral LNs, mesenteric LNs, and Peyer's patches (3). The LN sequestration of lymphocytes may be involved in the immunosuppressive effects of FTY720, as regulated trafficking of lymphocytes is necessary for systemic immune responses and allograft rejection (4, 5).A recent report demonstrates that FTY720 becomes phosphorylated in vivo and then acts as a potent agonist for multiple sphingosine-1-phosphate (S1P) receptors (S1PRs) (6). S1PR activation seems responsible for downstream events that lead to lymphocyte homing. However, the cellular and molecular mechanisms are not understood. Recent studies demonstrate a novel role for lipid mediators in LN homing of DCs. A blind screening of neutralizing mAb's led to the discovery that the lipid transporter ABCB1 (previously called MDR-1 and P-glycoprotein; for new nomenclature, see http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html) regulates human DC migration to afferent lymphatics (7). Coupled with the hydrolysis of ATP, ABCB1 transports a wide range of compounds with various structures and targets, including ceramide analogs (8) and platelet-activating factor (9, 10), from the cytosol to the extracellular environment. A subsequent study demonstrated a role for another lipid transporter, Abcc1 (previously called Mrp1), in the migration of DCs to lymphatics. Abcc1 physiologically transports sphingolipid analogs and cysteinyl leukotriene C 4 (LTC 4 ), which is metabolized in the extracellular environment to LTD 4 and LTE 4 (11, 12). Mice lacking the Abcc1 gene show markedly decreased migration of Langerhans cells to LNs. Administration of exogenous cysteinyl leukotrienes (cysLTs) restores DC migration in these mice (13). These studies also demonstrate that both transporters are required for human DC migratory activity. However, whether the two transporters function in an interactive manner remains unclear (13,14).The mechanism by which Abcc1 causes LN homing of DCs was further shown to be chemokine-mediated. FTY720 is a sphingosine-derived immunosuppressant. Phosphorylated FTY720 promotes T cell homing from spleen and peripheral blood to LNs by acting as an agonist for sphingosine-1-phosphate (S1P) receptors. Here we demonstrate that FTY720 enhances the activity of the sphingosine transporter Abcb1 (Mdr1) and ...