Movement towards or away from a given stimulus guides the directional migration of prokaryotes, simple eukaryotes and neurons. As bi-directional cues may influence entry and exit of immune effector cells from tissue sites, we evaluated the migratory responses of T-cell subsets to varying concentrations of the chemokine stromal cell derived factor-1 (SDF-1). There was selective repulsion of subpopulations of T cells at high concentrations of recombinant SDF-1 or naturally occurring bone marrow-derived SDF-1, which could be inhibited by pertussis toxin and antibody against the chemokine receptor CXCR4. Distinct sensitivity profiles to genistein, herbimycin and 8-Br-cAMP biochemically distinguished movement of cells towards or away from an SDF-1 gradient. In vivo, antigen-induced T-cell recruitment into the peritoneal cavity was reversed by high but not low concentrations of SDF-1. The phenomenon of movement away from a chemokine represents a previously unknown mechanism regulating the localization of mature T cells. It adds to the functional repertoire of chemokines that may participate in immune physiology and may be applied therapeutically to alter the immune response.

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Hematopoiesis in mammals undergoes a developmental shift in location from fetal liver to bone marrow accompanied by a gradual transition from highly proliferative to deeply quiescent stem cell populations. P2Y receptors are G-protein-coupled nucleotide receptors participating in vascular and immune responses to injury. We identified a P2Y-like receptor for UDP-conjugated sugars, GPR105 (P2Y 14 ), with restricted expression on primitive cells in the hematopoietic lineage. Anti-GPR105 antibody selectively isolated a subset of hematopoietic cells within the fetal bone marrow, but not in the fetal liver, that was enriched for G0 cell cycle status and for in vitro stem-cell-like multipotential long-term culture capability. Conditioned media from bone marrow stroma induced receptor activation and chemotaxis that was sensitive to G␣i and anti-receptor antibody inhibition. GPR105 is a G-protein-coupled receptor identifying a quiescent, primitive population of hematopoietic cells restricted to bone marrow. It mediates primitive cell responses to specific hematopoietic microenvironments and extends the known immune system functions of P2Y receptors to the stem cell level. These data suggest a new class of receptors participating in the regulation of the stem cell compartment. G-protein-coupled receptors (GPRs) have a broad repertoire of activating ligands ranging from photons to chemokines and induce an array of cellular events in virtually every physiologic system. Yet there remains a large proportion of GPR without known ligands or with limited known functions. A group of GPRs responsive to nucleotides (termed P2Y receptors) has been defined mediating cell-cell communication in the nervous system and in modulating vascular tone . A well-defined effect of nucleotides on platelet activation and vascular smooth muscle migration and growth has suggested participation of P2Y receptors in the response to injury. More recently, these receptors have been noted to affect cellular constituents of the innate immune system altering functional characteristics of monocytes, eosinophils, and dendritic cells and to play a critical role in terminating the inflammatory response in vivo (Mutini et al. 1999;Ferrari et al. 2000;Idzko et al. 2001;Santiago-Perez et al. 2001;Warny et al. 2001;Wilkin et al. 2001). The P2Y receptor specificity originally thought to be restricted to purine (adenine) nucleotides has been extended to pyrimidine nucleotides (uridine) and more recently to a receptor with specificity for UDP, but only when conjugated to glucose or related sugars (Chambers et al. 2000). This receptor, GPR105 (recently designated P2Y 14 ; Abbracchio et al. 2003), was originally noted to be expressed in rat brain tegmentum, but has no known function apart from being the presumed basis for UDP-glucose to induce diaphragmatic contraction or neural action potentials (Pastoris et al. 1979(Pastoris et al. , 1981. We provide evidence that this receptor participates in regulation of hematopoietic cells with stem cell characteristics.

The chemokine stroma-derived factor (SDF)-1, and its receptor, CXCR-4, have been shown to be essential for the translocation of hemopoietic stem cells from the fetal liver to the bone marrow (BM). We hypothesized that if CXCR-4 plays a crucial role in the localization of human hemopoiesis, stem cells from distinct tissue sources should demonstrate distinct CXCR-4 expression or signaling profiles. CD34+ cells from BM were compared with blood: either mobilized peripheral blood or umbilical cord blood. Unexpectedly, significantly higher levels of CXCR-4 surface expression on CD34+ cells from blood sources, mobilized peripheral blood, or cord blood were observed compared with BM (p = 0.0005 and p = 0.002, respectively). However, despite lower levels of CXCR-4, responsiveness of the cells to SDF-1 as measured by either calcium flux or transmigration was proportionally greatest in cells derived from BM. Further, internalization of CXCR-4 in response to ligand, associated with receptor desensitization, was significantly lower on BM-derived cells. Therefore, preserved chemokine receptor signaling was highly associated with marrow rather than blood localization. To test the functional effects of perturbing CXCR-4 signaling, adult mice were exposed to the methionine-SDF-1β analog that induces prolonged down-regulation/desensitization of CXCR-4 and observed mobilization of Lin−, Sca-1+, Thy-1low, and c-kit+ hemopoietic progenitor cells to the peripheral blood with a >30-fold increase compared with PBS control (p = 0.0007 day 1 and p = 0.004 day 2). These data demonstrate that CXCR-4 expression and function can be dissociated in progenitor cells and that desensitization of CXCR-4 induces stem cell entry into the circulation.

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

Biocompatible inorganic matrices have been used to enhance bone repair by integrating with endogenous bone architecture. Hypothesizing that a three-dimensional framework might support reconstruction of other tissues as well, we assessed the capacity of a tantalum-coated carbon matrix to support reconstitution of functioning thymic tissue. We engineered a thymic organoid by seeding matrices with murine thymic stroma. Co-culture of human bone marrow-derived hematopoietic progenitor cells within this xenogeneic environment generated mature functional T cells within 14 days. The proportionate T-cell yield from this system was highly reproducible, generating over 70% CD3+ T cells from either AC133+ or CD34+ progenitor cells. Cultured T cells expressed a high level of T-cell receptor excision circles (TREC), demonstrating de novo T lymphopoiesis, and function of fully mature T cells. This system not only facilitates analysis of the T-lymphopoietic potential of progenitor cell populations; it also permits ex vivo genesis of T cells for possible applications in treatment of immunodeficiency.

Chemotaxis or active movement of leukocytes toward a stimulus has been shown to occur in response to chemokinetic agents including members of the recently identified superfamily of proteins called chemokines. Leukocyte chemotaxis is thought to play a central role in a wide range of physiological and pathological processes including the homing of immune cells to lymph nodes and the accumulation of these cells at sites of tissue injury and pathogen or antigen challenge. We have recently identified a novel biological mechanism, which we term fugetaxis (fugere, to flee from; taxis, movement) or chemorepulsion, which describes the active movement of leukocytes away from chemokinetic agents including the chemokine, stromal cell derived factor-1, and the HIV-1 envelope protein, gp120. In this article, we review the evidence that supports the observation that leukocyte fugetaxis occurs in vitro and in vivo and suggestions that this novel mechanism can be exploited to modulate the immune response. We propose that leukocyte fugetaxis plays a critical role in both physiological and pathological processes in which leukocytes are either excluded or actively repelled from specific sites in vivo including thymic emigration, the establishment of immune privileged sites and immune evasion by viruses and cancer. We believe that current data support the thesis that a greater understanding of leukocyte fugetaxis will lead to the development of novel therapeutic approaches for a wide range of human diseases.

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