Human thymic MR1-restricted MAIT cells are innate pathogen-reactive effectors that adapt following thymic egress.

Human mucosal-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor (TCR) Vα7.2 and are restricted by the major histocompatibility complex-Ib molecule MR1. While MAIT cells share similarities with other innate T cells, the extent to which MAIT cells are innate and their capacity to adapt is unknown. We evaluated the function of Vα7.2(+) T cells from the thymus, cord blood, and peripheral blood. Although antigen-inexperienced MAIT cells displayed a naïve phenotype, these had intrinsic effector capacity in response to Mycobacterium tuberculosis (Mtb)-infected cells. Vα7.2(+) effector thymocytes contained signal joint TCR gene excision circles (sjTRECs) suggesting limited replication and thymic origin. In evaluating the capacity of Mtb-reactive MAIT cells to adapt, we found that those from the peripheral blood demonstrated a memory phenotype and had undergone substantial expansion, suggesting that they responded to antigenic stimulation. MAIT cells, an evolutionarily conserved T-cell subset that detects a variety of intracellular infections, share features of innate and adaptive immunity.

Transcriptomes of the major human pancreatic cell types.

AIMS/HYPOTHESIS: We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors. METHODS: Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor-ligand representation in these populations. RESULTS: Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand-receptor interactions suggested that EPH receptor-ephrin communication between exocrine and endocrine cells contributes to pancreatic function. CONCLUSIONS/INTERPRETATION: This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types-including beta cells-and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.

Immune evasion by neocartilage-derived chondrocytes: Implications for biologic repair of joint articular cartilage.

Degeneration of joint articular cartilage is a leading cause of disability worldwide, and is due in large part to the fact that adult articular cartilage is unable to undergo effective intrinsic repair. To overcome this barrier, we have developed a tissue engineering strategy which harnesses the superior anabolic activity of juvenile chondrocytes to produce a scaffold-independent, living neocartilage graft. Preclinical studies demonstrate that bioengineered neocartilage survives allogeneic and xenogeneic transplantation, suggesting the utility of universal donor-derived neocartilage for joint repair. However, the mechanism underlying neocartilage transplant tolerance remains poorly understood. We show here that neocartilage-derived chondrocytes are unable to stimulate allogeneic T cells in vitro, and they do not constitutively express cell surface molecules required for induction of T cell immune responses, including major histocompatibility complex (MHC) Class II antigens and costimulatory molecules B7-1 and B7-2. Additionally, chondrocytes suppress, in a contact-dependent manner, the proliferation of activated T cells, with suppression associated with chondrocyte expression of multiple negative regulators of immune responses, including B7 family members (B7-H1, B7-DC, B7-H2, B7-H3, and B7-H4), chondromodulin-I and indoleamine 2,3-dioxygenase. Thus, the survival of transplanted bioengineered neocartilage may depend on both passive and active mechanisms of immune evasion.

Pre-clinical safety testing supporting clinical use of allogeneic multipotent adult progenitor cells.

BACKGROUND: Successful clinical development of novel cellular therapeutics requires the evaluation of clinical acute toxicity endpoints in scoring patient adverse events (AE) contributing to dose-limiting toxicity (DLT) for establishment of the maximum-tolerated dose (MTD). However, many clinical pathology parameters are not routinely evaluated in pre-clinical safety testing. The objective of this pre-clinical study was to investigate thoroughly the acute toxicity of single- and multiple-dose administrations of allogeneic multipotent adult progenitor cells (MultiStem), which represent a class of stromal stem cells with therapeutic potential. METHODS: MultiStem were tested as an adjunct treatment in a rat myeloablative hematopoietic stem cell transplantation (HSCT) model for impact on clinical parameters, clinical chemistry, hematology, immunology and histopathology parameters. Animals received MultiStem in a single dose of 12.5 million cells/kg on day 2 after HSCT or in five infusions at this dose on days 2, 9, 16, 23 and 30. Controls received phosphate-buffered saline injections and all animals were killed on day 37. RESULTS: There were no significant differences between tests and controls regarding evaluation of respiratory distress upon infusion, clinical assessment and hematology and clinical chemistry analysis. Gross necropsy and histopathology analysis showed no organ profile alterations. There was no significant evidence for allogeneic antibody production or T-cell sensitization upon MultiStem infusion. DISCUSSION: These studies demonstrate the safety of administration of allogeneic stromal stem cells in repeat dosing regimens in bone marrow transplant settings, and define pre-clinical safety testing standards relevant to the development of cellular therapeutics using allogeneic adherent adult stem cells.

Modification of the Woodruff-Stamper assay demonstrates binding of Toxoplasma gondii tachyzoites to retinal vascular endothelium.

In the human host, infection with the protozoan parasite, Toxoplasma gondii, most commonly involves the eye and/or the brain. Previous work indicates a relative susceptibility of the human retinal vascular endothelium to infection with the T. gondii tachyzoite, which may contribute to this tissue localization. To facilitate the investigation of potential adhesive interactions between parasite and endothelium in the retina, we have modified the Woodruff-Stamper assay, originally described to study lymphocytic-endothelial binding. Vascular endothelium was identified in sections of human retina by Alexa Fluor 594-tagged anti-von Willebrand factor antibody. Binding of yellow fluorescent protein-expressing tachyzoites to endothelium under conditions of flow, simulated by rotation on an orbital shaker, was quantified in a masked fashion using imaging software. We observed multiple yellow spots in contact with Alexa Fluor 594-positive retina, indicating binding of T. gondii tachyzoites to retinal vascular endothelium. This modification of the Woodruff-Stamper assay provides an opportunity to evaluate potential host receptors for T. gondii on the retinal vascular endothelium. In addition, the assay suggests a methodology that could be used to examine adhesion of other microbes to microvasculature in different tissues.

Progenipoietin-1: a multifunctional agonist of the granulocyte colony-stimulating factor receptor and fetal liver tyrosine kinase-3 is a potent mobilizer of hematopoietic stem cells.

OBJECTIVE: Progenipoietin-1 is an agonist of both the granulocyte colony-stimulating factor and fetal liver tyrosine kinase-3 receptors capable of inducing the proliferation of multiple hematopoietic cell lineages. The potential of progenipoietin-1 to mobilize transplantable hematopoietic stem cells into the peripheral blood was evaluated. METHODS: Cohorts of donor mice were treated with either progenipoietin-1, fetal liver tyrosine kinase-3 ligand, granulocyte colony-stimulating factor, or a vehicle control. Hematopoietic progenitor/stem-cell activity in donor blood was assayed by radioprotection, multilineage reconstitution, secondary transplantation, and competitive repopulation. RESULTS: Only 1 microL of peripheral blood from progenipoietin-1-treated donors was required to protect 80% of lethally irradiated mice, while in contrast 1 microL of peripheral blood from granulocyte colony-stimulating factor-treated donors failed to protect any recipients. The radioprotected recipients of progenipoietin-1-treated donor cells showed donor-derived (Ly5.2) multilineage hematopoietic reconstitution for up to 6 months. Serial transplantation studies using bone marrow from radioprotected, chimeric recipients demonstrated long-term donor-derived hematopoiesis, indicating the successful transplantation of multipotent hematopoietic stem cells. The engraftment potential of progenipoietin-1 donor-derived cells was directly compared with donors treated with granulocyte colony-stimulating factor or fetal liver tyrosine kinase-3 ligand alone or in combination. Both spleen colony-forming activity and competitive repopulating activity was highest in the blood from progenipoietin-1-treated donors. CONCLUSIONS: These studies demonstrate that progenipoietin-1 is a potent mobilizer of transplantable hematopoietic stem cells and indicate that this dual-receptor agonist has greater biologic activity than its constituent molecules.

Dendritic cells generated in vivo by a chimeric hematopoietic growth factor, progenipoietin-4, demonstrate potent immunological function.

Recently, a dual receptor agonist for human Flt3 and G-CSF receptors, progenipoietin-4 (ProGP-4), was shown to be highly effective in expanding DC in vivo. In this study, we examined the immunological activity of ProGP-4-generated dendritic cell (DC) in an HLA-A2.1 transgenic mouse system. ProGP-4 DC were found to be approximately equivalent in presenting a cytotoxic T lymphocyte (CTL) peptide to a CTL line in vitro compared with bone marrow (BM)-derived DC and >20-fold more efficient than macrophages or B cells, and >100-fold better than BM-DC, macrophages, or B cells at presenting PADRE, a universal helper T cell epitope, to a T cell clone. The heightened epitope presentation by ProGP-4 DC was paralleled in vivo inasmuch as a >6-fold increase in CTL induction was observed compared with other APC populations following ex vivo loading with peptide. The in vitro and in vivo CTL responses stimulated by ProGP-4 DC could be further augmented by either culturing with tumor necrosis factor-alpha (TNF-alpha) or co-loading with PADRE. Collectively, our results indicate that peptide-loaded ProGP-4-generated DC demonstrate potent antigenicity and immunogenicity for CTL, making them an attractive component of epitope-based vaccines.

Progenipoietins: biological characterization of a family of dual agonists of fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor.

The progenipoietins, a class of engineered proteins containing both fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor receptor agonist activities, were functionally characterized in vitro and in vivo. Four representative progenipoietins were evaluated for receptor binding, receptor-dependent cell proliferation, colony-forming unit activity, and their effects on hematopoiesis in the C57BL/6 mouse.The progenipoietins bound to fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor with affinities within twofold to threefold of the native ligands, and each progenipoietin bound simultaneously to both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. The progenipoietins exhibited different levels of activity in receptor-dependent cell proliferation assays. The fetal liver tyrosine kinase-3-dependent cell proliferation activity of three of four progenipoietins was decreased sixfold to 33-fold relative to native fetal liver tyrosine kinase-3 ligand, while granulocyte colony-stimulating factor receptor-dependent activity of the progenipoietins was within twofold to threefold of native granulocyte colony-stimulating factor. At nonsaturating concentrations, the progenipoietins stimulated colony formation to a greater extent than the equimolar combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor. Treatment of mice with the progenipoietins yielded dramatic increases in peripheral blood and splenic white blood cells, polymorphonuclear leukocytes, and dendritic cells. These preclinical results demonstrate that the progenipoietins are potent hematopoietic growth factors that stimulate cells in a receptor-dependent manner. When administered in vivo, the progenipoietins effectively promote the generation of multiple cell lineages. Thus, in both in vitro and in vivo settings, the progenipoietins as single molecules exhibit the synergistic activity of the combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor.

Murine isograft studies of gut immunity: recirculation and homing of mononuclear cells.

BACKGROUND & AIMS: Recirculation of lymphocytes requires appropriate signals on the lymphocytes as well as the vascular endothelium. The aim of this report was to study the expression of the mucosal addressin required for Peyer's patch-selective lymphocyte homing during ontogeny of the murine intestine and investigate the role of this addressin in recirculation of mononuclear cells. METHODS: Immunohistochemistry and murine intestinal isografts were used in which the small intestine from neonatal mice is implanted subcutaneously in mice with severe combined immunodeficiency disease. RESULTS: Expression of the mucosal addressin cell adhesion molecule 1 during murine intestinal ontogeny was detected from embryonic day 15 in developing gut and in adult Peyer's patches and lamina propria. Isografted intestine developed normally; it was not exposed to luminal contents but expressed the mucosal addressin cell adhesion molecule 1. Mononuclear cells recirculated to native and isografted intestine and recirculation was inhibited by monoclonal antibody to mucosal addressin in a regionally specific manner with greatest inhibition in the distal intestine. CONCLUSIONS: The mucosal vascular addressin is expressed early in ontogeny and can be detected in lamina propria in addition to Peyer's patches of the gut. The isograft not only developed into a morphologically normal intestine but also expressed differentiation antigens required for normal lymphoid homing to gut without exposure to luminal contents or lymphocytes.

Sulfation and sialylation requirements for a glycoform of CD34, a major endothelial ligand for L-selectin in porcine peripheral lymph nodes.

Leukocyte recruitment from blood into peripheral lymph nodes is controlled in part by a specific interaction of lymphocyte-associated L-selectin with endothelial cell receptors known as peripheral addressins. In murine lymph nodes, two peripheral addressins have been identified, Gly-CAM-1, a 50 kDa molecule that also appears as a secreted form in plasma, and CD34, a 90 kDa membrane-associated sialomucin. A predominant 105 kDa CD34 mucin-like protein has also been identified in human tonsil as peripheral addressin. We have identified a 120 kDa sialomucin as the predominant peripheral addressin in porcine lymph nodes. Validation of the 120 kDa porcine molecule as a peripheral addressins was based on its ability to bind MECA-79, a monoclonal antibody previously used to isolate peripheral addressins from mouse and human tissues, and to bind an L-selectin-Fc chimera (LS-Fc). The binding with LS-Fc was abolished in the presence of fucoidin, a sulfated polysaccharide known to inhibit L-selectin-receptor interactions. To address the possibility that the 120 kDa ligand may contain common recognition determinants for MECA-79 and L-selectin, the requirements for sialylation and sulfation were compared. Whereas desialylation of 120 kDa ligand drastically reduced its binding to LS-Fc, this treatment appeared to enhance the binding of 120 kDa ligand to MECA-79. In contrast, the binding of both MECA-79 and LS-Fc to 120 kDa ligand was drastically reduced when de novo sulfation of this ligand was reduced by including chlorate, a metabolic inhibitor of sulfation, in the culture media. N-Terminal amino acid sequences of the porcine 120 kDa protein revealed homology with human CD34. Taken together, these findings suggest that the porcine 120 kDa peripheral addressin is an L-selectin-binding glycoform of CD34.

A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3- cells to colonize lymph nodes.

IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

Expression of the mucosal vascular addressin, MAdCAM-1, on sinus-lining cells in the spleen.

The MAdCAM-1 adhesion molecule is involved in lymphocyte homing into mucosal sites and is expressed on high endothelial venules of Peyer's patches and mesenteric lymph nodes. In the spleen, where high endothelial venules are absent, expression can be found on cells in the marginal zone between red and white pulp. By immunohistochemistry and electron microscopy it was demonstrated that in the spleen cells expressing MAdCAM-1 belong to the population of sinus-lining cells and that the expression is restricted to the sinus-lining cells closest to the lymphoid white pulp. Lymphocytes that migrate from the blood into this white pulp area will have to pass through the rim of cells expressing MAdCAM-1. A functional role for MAdCAM-1 or its lymphocyte ligand, the alpha 4 beta 7 integrin complex, was investigated by in vivo short-term homing experiments with anti-MAdCAM-1 and anti-alpha 4 beta 7 antibodies, but no direct role for this receptor-ligand interaction could be demonstrated.

L-selectin and alpha 4 beta 7 integrin homing receptor pathways mediate peripheral lymphocyte traffic to AKR mouse hyperplastic thymus.

Before the development of thymic lymphoma, AKR mice undergo a striking lymphoid hyperplasia of the thymic medulla. We have previously shown that there is a marked increase in traffic of B and T lymphocytes from the periphery into the preneoplastic, hyperplastic thymuses of these mice, in contrast to the scant traffic of such cells to normal thymuses. The traffic of lymphocytes to lymph nodes and Peyer's patches is controlled in part by the interaction of lymphocyte adhesion molecules called homing receptors with their tissue-selective endothelial ligands known as vascular addressins. We have investigated the roles of homing receptors and vascular addressins in the traffic of lymphocytes to the AKR hyperplastic thymus. We demonstrate that development of hyperplasia is accompanied by an increase in the number of thymic medullary blood vessels with high endothelial venule morphology and expression of the peripheral node addressin (PNAd) and the mucosal addressin (MAdCAM-1). In vitro and in vivo functional assays show that the addressin/homing receptor pairs PNAd/L-selectin and MAdCAM-1/alpha 4 beta 7 are involved in lymphocyte traffic to the hyperplastic thymus. These results indicate that molecular adhesion mechanisms involved in tissue-selective migration of lymphocytes to peripheral lymph node and to mucosal lymphoid tissues play a role in the recruitment of B and T lymphocytes to the AKR thymus and thus in the pathogenesis of thymic hyperplasia.

Enzymatic synthesis of a 6'-sulphated sialyl-Lewisx which is an inhibitor of L-selectin binding to peripheral addressin.

A sulphated form of sialyl-Lewisx, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc6OSO3 beta 1-3Gal, was synthesized enzymatically from a precursor disaccharide, GlcNAc6OSO3 beta 1-3Gal, using sequential steps involving beta 1,4-galactosyltransferase, alpha 2,3-trans-sialidase and recombinant alpha 1,3-fucosyltransferase, respectively. Successful enzymatic fucosylation at the 3 position of the GlcNAc6OSO3 residue demonstrated that fucosyltransferase are capable of generating, in situ, sulphated sialyl Lewisx structures containing sulphate at the 6 position of GlcNAc. The sulphated sialyl-Lewisx pentasaccharide produced by this procedure inhibited binding of a soluble form of L-selectin to 35SO4-labelled peripheral addressin with an IC50 of 0.8 mM, whereas sialyl-Lewisx tetrasaccharide was a weaker inhibitor, displaying an IC50 of 3.2 mM. Hemmerich and Rosen (Biochemistry, 33, 4820-4829, 1994) recently reported the presence of Gal beta 1-4GlcNAcO6SO3 structures on murine peripheral addressin Sgp50, in addition to sialyl Lewisx structures sulphated at the 6-O-galactose position. Based on our data, we suggest that sialyl Lewisx sulphated at the 6-O-GlcNAc position may also exist on receptors and function as a ligand for L-selectin.

Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin.

Mice rendered deficient in lymphotoxin (LT) by gene targeting in embryonic stem cells have no morphologically detectable lymph nodes or Peyer's patches, although development of the thymus appears normal. Within the white pulp of the spleen, there is failure of normal segregation of B and T cells. Spleen and peripheral blood contain CD4+CD8- and CD4-CD8+ T cells in a normal ratio, and both T cells subsets have an apparently normal lytic function. Lymphocytes positive for immunoglobulin M are present in increased numbers in both the spleen and peripheral blood. These data suggest an essential role for LT in the normal development of peripheral lymphoid organs.

The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing.

The extravasation of blood-borne lymphocytes into organized lymphoid tissues and sites of chronic inflammation is directed in part by interactions of lymphocyte surface adhesion molecules, known as homing receptors, with tissue-selective endothelial ligands called vascular addressins. In mice and humans, lymphocyte L-selectin and the peripheral lymph node addressin (PNAd) form a homing receptor-endothelial ligand pair involved in lymphocyte traffic to peripheral lymph node (PLN). We have examined the tissue distribution and function of human PNAd, using monoclonal antibody MECA-79 and in vitro assays of L-selectin-dependent lymphocyte binding. We demonstrate that PNAd is expressed by human high endothelial venules (HEV) in lymphoid tissues which support lymphocyte adhesion via a PLN-associated recognition system. MECA-79 inhibits adhesion to these HEV of a cell line that binds predominantly via the PLN-homing receptor, L-selectin, but has no effect on adhesion by a mucosal HEV-binding cell line. Furthermore, MECA-79 blocks binding of human peripheral blood mononuclear cells to both PLN and tonsil HEV, but not significantly to HEV in the appendix. In addition, we demonstrate PNAd induction on venules at chronic inflammatory sites in humans, particularly sites with severe or long-standing chronic inflammatory involvement. These results confirm that PNAd functions as a PLN vascular addressin in humans, and that in addition to directing normal lymphocyte recirculation to lymph nodes and tonsils, this addressin likely participates in lymphocyte recruitment to sites of chronic inflammation.

Vascular addressins are induced on islet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid cell binding to islet endothelium.

In the nonobese diabetic (NOD) mouse, lymphocytic and monocytic infiltration of the pancreatic islets leads to beta cell destruction. To investigate the mechanisms by which lymphocytes enter the NOD pancreas, pancreata were immunostained using monoclonal antibodies to a variety of adhesion molecules known to be involved in lymphocyte binding to vascular endothelium, an initial step in the migration of lymphocytes from blood into organized lymphoid and inflamed tissues. These adhesion molecules include: lymphocyte homing receptors involved in tissue-selective binding of lymphocytes to peripheral lymph node (L-selectin) or mucosal lymphoid tissue (LPAM-1, alpha 4 beta 7-integrin) high-endothelial venules (HEV); and HEV ligands peripheral vascular addressin (PNAd) and mucosal vascular addressin (MAdCAM-1). In NOD pancreata, alpha 4 beta 7 is expressed on most infiltrating cells at all stages of insulitis, whereas L-selectin expression is more pronounced on cells in the islets at later stages. During the development of insulitis, MAdCAM-1 and to a lesser extent PNAd became detectable on vascular endothelium adjacent to and within the inflamed islets. The Stamper-Woodruff in vitro assay was used to examine lymphoid cell binding to such vessels. These functional assays show that both the mucosal (MAdCAM-1/alpha 4 beta 7) and the peripheral (PNAd/L-selectin) recognition systems are involved in this binding. Our findings demonstrate that expression of peripheral and mucosal vascular addressins is induced on endothelium in inflamed islets in NOD pancreas, and that these addressins participate in binding lymphoid cells via their homing receptors. This suggests that these adhesion molecules play a role in the pathogenesis of diabetes in these mice by being involved in the migration of lymphocytes from blood into the inflamed pancreas.

Developmental regulation of vascular addressin expression: a possible role for site-associated environments.

Tissue selective traffic of lymphocytes into different lymphoid organs is mediated by adherence of blood borne lymphocytes to specialized endothelial cells lining the high endothelial venules (HEV) in lymphoid organs. Lymphocytes discriminate between HEV in peripheral lymph nodes and in mucosal lymphoid tissues by means of membrane associated lymphocyte homing receptors adhering to their putative HEV ligands, the vascular addressins. The expression of particular vascular addressins on HEV is site- or tissue-selective and may be directed by factors unique to a specific location or lymph node environment. In this study we investigated the impact of regional environments on lymph node HEV differentiation and function. Experimentally, this problem was approached by the transplantation of lymph nodes from one region to a second region. The sites selected for receipt of tissues were the mesentery, a mucosal site, and the popliteal fossa, a peripheral site. We found that the phenotype of lymph node HEV following transplantation was influenced by both donor age and transplantation site. The transplantation site could influence vascular addressin expression, when tissues were obtained from late fetal or early neonatal donors and not when obtained from adult donors. Transplanted adult tissues retained their pre-transplantation vascular addressin expression phenotype regardless of transplantation site. Thus the endothelium within adult lymph nodes may be committed to expression of a particular addressin or addressins during lymph node development. It is also possible that regulatory cells or structures present within lymph nodes at the time of transplantation direct vascular addressin expression following tissue engraftment.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical evaluation of human hematolymphoid function in the SCID-hu mouse.

Work in the SCID-hu system has progressed significantly over the past 3-4 years. Reliable SCID-hu constructs can now be created for the evaluation of long-term multilineage human hematopoiesis, human immune function, and HIV infection of human hematolymphoid organs in vivo. Given these constructs, a variety of therapeutically important modalities can now be discovered and developed with a relevant in vivo model, prior to the time that they are moved into the clinic. With further progress in this and other preclinical models, useful therapies for important human disease states will hopefully be forthcoming at an even faster pace.

The influence of afferent lymphatic vessel interruption on vascular addressin expression.

Tissue-selective lymphocyte homing is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. These vessels, the post capillary high endothelial venules (HEV), are found in organized lymphoid tissues, and at sites of chronic inflammation. Lymphocytes bearing specific receptors, called homing receptors, recognize and adhere to their putative ligands on high endothelial cells, the vascular addressins. After adhesion, lymphocytes enter organized lymphoid tissues by migrating through the endothelial cell wall. Cells and/or soluble factors arriving in lymph nodes by way of the afferent lymph supply have been implicated in the maintenance of HEV morphology and efficient lymphocyte homing. In the study reported here, we assessed the influence of afferent lymphatic vessel interruption on lymph node composition, organization of cellular elements; and on expression of vascular addressins. At 1 wk after occlusion of afferent lymphatic vessels, HEV became flat walled and expression of the peripheral lymph node addressin disappeared from the luminal aspect of most vessels, while being retained on the abluminal side. In addition, an HEV-specific differentiation marker, defined by mAb MECA-325, was undetectable at 7-d postocclusion. In vivo homing studies revealed that these modified vessels support minimal lymphocyte traffic from the blood. After occlusion, we observed dramatic changes in lymphocyte populations and at 7-d postsurgery, lymph nodes were populated predominantly by cells lacking the peripheral lymph node homing receptor LECAM-1. In addition, effects on nonlymphoid cells were observed: subcapsular sinus macrophages, defined by mAb MOMA-1, disappeared; and interdigitating dendritic cells, defined by mAb NLDC-145, were dramatically reduced. These data reveal that functioning afferent lymphatics are centrally involved in maintaining normal lymph node homeostasis.