Functional distribution and further characterization of human endothelial ligand for cellular L-selectin.

In the present study we examine the functional distribution of the human endothelial L-selectin ligand, which determines the sites of extravasation of L-selectin-positive cells. A murine cell line transfected with human L-selectin adhered preferentially to the high endothelial venules (HEV) of human peripheral lymph nodes compared to the HEV of mucosal lymphoid tissues (mean of 0.83 compared to a mean of 0.07 cells per HEV respectively). In addition, an antibody against L-selectin differentially inhibited the adhesion of human lymphocytes to peripheral lymphoid tissue versus mucosal lymphoid tissue HEV (mean 41 and 5% inhibition respectively). Although both sulfoglucuronyl-containing glycolipids and sialyl-Lewis X have been proposed as endothelial ligands for L-selectin, an antibody against the former did not bind to peripheral lymph node endothelium, and an antibody against the latter did not block adhesion of L-selectin-expressing cells. The enzyme O-sialoglycoprotein endopeptidase caused up to an 84% reduction in L-selectin-dependent binding, indicating that sialylated glycoproteins containing O-linked glycans are essential for a large majority of adhesion via L-selectin.     

Evolutionary conservation of tissue-specific lymphocyte-endothelial cell recognition mechanisms involved in lymphocyte homing

Tissue-specific interactions with specialized high endothelial venules (HEV) direct the homing of lymphocytes from the blood into peripheral lymph nodes, mucosal lymphoid organs, and tissue sites of chronic inflammation. These interactions have been demonstrated in all mammalian species examined and thus appear highly conserved. To assess the degree of evolutionary divergence in lymphocyte-HEV recognition mechanisms, we have studied the ability of lymphocytes to interact with HEV across species barriers. By using an in vitro assay of lymphocyte binding to HEV in frozen sections of lymphoid tissues, we confirm that mouse, guinea pig, and human lymphocytes bind to xenogeneic as well as homologous HEV. In addition, we show that mouse and human lymphoid cell lines that bind selectively to either peripheral lymph node or mucosal vessels (Peyer's patches, appendix) in homologous lymphoid tissues exhibit the same organ specificity in binding to xenogeneic HEV. Furthermore, monoclonal antibodies that recognize lymphocyte "homing receptors" and block homologous lymphocyte binding to peripheral lymph node or to mucosal HEV, also inhibit lymphocyte interactions with xenogeneic HEV in a tissue-specific fashion. Similarly, anti-HEV antibodies against organ-specific mouse high endothelial cell "addressins" involved in lymphocyte homing to peripheral lymph node or mucosal lymphoid organs, not only block the adhesion of mouse lymphocytes but also of human lymphocytes to target mouse HEV. The results illustrate a remarkable degree of functional conservation of elements mediating these cell-cell recognition events involved in organ-specific lymphocyte homing.     

Lymphocyte adhesion to cultured Peyer's patch high endothelial venule cells is mediated by organ-specific homing receptors and can be regulated by cytokines

Adhesion of lymphocytes to high endothelial venule (HEV) cells is the first step in the migration of these cells from blood into lymph nodes and Peyer's patches (PP). In the present study, we isolated and cultured HEV cells from PP of the rat and assessed their capacity to interact with lymphocytes. Flow cytometric analysis with a rat HEV-specific mAb KJ-4 revealed that greater than 90% of the cultured cells were stained by the antibody. Furthermore, confluent monolayers of PP HEV cells retained the capacity to support the adhesion of lymphocytes from spleen, thoracic duct, and lymph nodes but not binding of immature cells from thymus and bone marrow, which are deficient in cells capable of binding to HEV in vivo. In addition, intraepithelial lymphocytes that preferentially migrated into mucosal lymphoid tissues were also enriched in cells that adhered to the endothelial monolayers. The binding process required energy, was calcium-dependent, and could be inhibited by cytochalasin D, trypsin, and mixed glycosidase. Interestingly, pretreatment of PP HEV cells with rTNF, IFN-gamma, or granulocyte-macrophage CSF significantly increased the endothelial adhesiveness for thoracic duct lymphocytes in a time- and dose-dependent manner. In contrast, stimulation of lymphocytes with phorbol ester or TNF resulted in the rapid modulation of the surface expression of the PP homing receptor and decrease in lymphocyte binding to normal or TNF-stimulated HEV cells. The adhesion of lymphocytes to normal or cytokine-stimulated HEV cells can be blocked by pretreatment of lymphocytes, but not HEV cells, with the PP homing receptor-specific 1B.2.6 antibody. Taken together, these experiments provide strong evidence that the interaction between lymphocytes and cultured HEV cells are mediated by adhesive mechanisms that regulate lymphocyte entry into PP in vivo and that cytokines can promote HEV adhesiveness for lymphocytes through increased expression of organ-specific ligands on HEV cells.     

Evidence for an accessory role of LFA-1 in lymphocyte-high endothelium interaction during homing

In a variety of lymphocyte interactions, lymphocyte function-associated antigen-1 (LFA-1) plays an important role as an accessory mechanism mediating cell adhesion. We tested the possibility that LFA-1 could also be involved in the specific binding of lymphocytes to high endothelial venules (HEV) during homing. Antibodies against LFA-1 but not against various other cell surface molecules (except the putative gp90 homing receptor defined by the MEL-14 antibody) were found to inhibit in vitro adherence of lymphocytes to HEV in frozen sections of lymph nodes. Binding of T cell lines to HEV was also inhibited by anti-LFA-1 antibody. Using sublines selected for differential expression of the MEL-14 antigen, MEL-14 high cells (which bind well to HEV) were less susceptible to inhibition by anti-LFA-1 than poor binders with low levels of the homing receptor, supporting the model of LFA-1 being an accessory mechanism strengthening weak interactions between cells. Parallel results were found in vivo where anti-LFA-1 antibodies reduced the migration of normal lymphocytes into lymph nodes and Peyer's patches by 40 to 60%. Localization in the lung, especially of activated lymphocytes, was also impaired, although to a lesser extent. These findings suggest that LFA-1 plays an accessory role in cellular interactions relevant for lymphocyte migration.     

Migration patterns of dendritic cells in the rat: comparison of the effects of gamma and UV-B irradiation on the migration of dendritic cells and Lymphocytes

To further define the underlying mechanisms of immune suppression induced by UV-B irradiation, we have examined the kinetics of homing patterns of in vitro UV-B-irradiated and gamma-irradiated-thoracic duct lymphocytes (TDL) compared to dendritic cells (DC). Our findings show that 111In-oxine-labeled TDL specifically home to the spleen, liver, lymph nodes, and bone marrow with subsequent recirculation of a large number of cells from the spleen to lymph nodes. In contrast, DC preferentially migrate to the spleen and liver with a relatively insignificant distribution to lymph nodes and an absence of subsequent recirculation. Splenectomy prior to cell injection significantly diverts the spleen-seeking DC to the liver but not to the lymph nodes, while the homing of TDL to lymph nodes is significantly increased. In vitro exposure of 111In-oxine labeled TDL to gamma irradiation does not significantly impair immediate homing to lymphoid tissues but inhibits cell recirculation between 3 and 24 hr. In contrast, gamma irradiation does not affect the tissue distribution of labeled DC, suggesting that DC are more radioresistant to gamma irradiation than TDL. Unlike the findings in animals injected with gamma-irradiated cells, UV-B irradiation virtually abolished the homing of TDL to lymph nodes and significantly reduced the homing of the spleen-seeking DC to the splenic compartment while a large number of cells were sequestered in the liver. The results of in vitro cell binding assay show that TDL, unlike DC, have the capacity to bind to high endothelial venules (HEV) within lymph node frozen sections while gamma and UV-B irradiation significantly inhibit the binding of TDL to lymph node HEV. These findings suggest that: (i) DC, unlike TDL, are unable to recirculate from blood to lymph nodes through HEV; (ii) although gamma irradiation impairs TDL recirculation, it does not affect DC tissue distribution; and (iii) UV-B irradiation impairs both TDL and DC migration patterns. We conclude that the lack of capacity of irradiated TDL to home to lymph nodes is due to damage to cell surface homing receptors and that the failure of DC to home to the lymph node microenvironment is related to the absence of HEV homing receptors on their cell surface.     

Differences in in vivo distribution and homing of T cell subsets to mucosal vs nonmucosal lymphoid organs

The migratory properties of Lyt-2- and Lyt-2+ T cells in the mouse have been investigated. In short-term in vivo homing studies, Lyt-2- T cells localized consistently more efficiently than Lyt-2+ T cells in Peyer's patches (about 1.5 times as well), whereas both populations localized roughly equivalently in peripheral lymph nodes. These homing characteristics of Lyt-2- and Lyt-2+ subsets are largely independent of their organ source. The specificity of migration appears to be determined by selective recognition of organ-specific determinants on the endothelial cells of high endothelial venules (HEV), specialized venules that mediate the exit of migrating lymphocytes from the blood: In an in vitro assay of lymphocyte binding to HEV in lymphoid organ frozen sections, Lyt-2- cells constituted a significantly and consistently greater proportion of T cells binding to Peyer's patch HEV than of those binding to peripheral node HEV. The homing and HEV recognition preferences of the Lyt subsets are reflected in differences in their in situ representation in mucosal vs nonmucosal lymphoid organs, which suggests that the selective migration of these populations may be an important factor in determining the character of local immune responses.     

The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor

The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin.     

CD8+ T-lymphocyte response to major immunodominant epitopes after vaginal exposure to simian immunodeficiency virus: too late and too little

In the acute stage of infection following sexual transmission of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), virus-specific CD8+ T-lymphocyte responses partially control but do not eradicate infection from the lymphatic tissues (LTs) or prevent the particularly massive depletion of CD4+ T lymphocytes in gut-associated lymphatic tissue (GALT). We explored hypothetical explanations for this failure to clear infection and prevent CD4+ T-lymphocyte loss in the SIV/rhesus macaque model of intravaginal transmission. We examined the relationship between the timing and magnitude of the CD8+ T-lymphocyte response to immunodominant SIV epitopes and viral replication, and we show first that the failure to contain infection is not because the female reproductive tract is a poor inductive site. We documented robust responses in cervicovaginal tissues and uterus, but only several days after the peak of virus production. Second, while we also documented a modest response in the draining genital and peripheral lymph nodes, the response at these sites also lagged behind peak virus production in these LT compartments. Third, we found that the response in GALT was surprisingly low or undetectable, possibly contributing to the severe and sustained depletion of CD4+ T lymphocytes in the GALT. Thus, the virus-specific CD8+ T-lymphocyte response is "too late and too little" to clear infection and prevent CD4+ T-lymphocyte loss. However, the robust response in female reproductive tissues may be an encouraging sign that vaccines that rapidly induce high-frequency CD8+ T-lymphocyte responses might be able to prevent acquisition of HIV-1 infection by the most common route of transmission.     

Lymphocyte entry into inflammatory tissues in vivo. Qualitative differences of high endothelial venule-like vessels in sponge matrix allografts vs isografts

Sponge matrix allografts and isografts become extensively encapsulated and neovascularized after s.c. implantation. Sponge allografts acquire alloantigen-reactive T lymphocytes, whereas sponge isografts fail to do so, even though these T cells are continuously circulating in the peripheral blood. We have investigated the possibility that the vascular endothelia regulates lymphocytic accumulation in sponge matrix implants. In normal lymph nodes, specialized high endothelial venules (HEV) regulate lymphocyte extravasation from the blood. We have now identified HEV-like vessels in sponge matrix allografts. These vessels are operationally defined as "HEV-like" in that they react with mAb MECA 325 which identifies murine HEV, and bind lymphocytes in ex vivo adhesion assays. In contrast, sponge isografts contain MECA 325 reactive vessels that are significantly smaller than those found in allografts. Further, vessels of sponge isografts do not readily bind lymphocytes in ex vivo adhesion assays. Immunohistologic analysis also revealed that the small MECA 325+ vessels present in sponge isografts are consistently found in close proximity to nerve bundles. Although this MECA 325 reactive vessel-nerve bundle association is also observed in sponge allografts, large MECA 325 reactive vessels are widely distributed in allografts. Our data suggest that small, poorly adhesive MECA 325 reactive vessels develop in sponge isografts and allografts, possibly under the influence of local nerve tissue. These vessels respond to regional alloimmune responses by developing into the larger HEV-like vessels capable of binding lymphocytes in sponge allografts. The value of this experimental system as an in vivo model to evaluate mechanisms involved in neovascularization and endothelial differentiation is discussed.     

Phosphomannosyl-derivatized beads detect a receptor involved in lymphocyte homing

Recirculating lymphocytes initiate extravasation from the blood stream by binding to specialized high endothelial venules (HEV) within peripheral lymph nodes (PN) and other secondary lymphoid organs. We have previously reported that lymphocyte attachment to PN HEV is selectively inhibited by mannose-6-phosphate (M6P) and related carbohydrates (Stoolman, L. M., T. S. Tenforde, and S. D. Rosen, 1984, J. Cell Biol., 99:1535-1540). In the present study, we employ a novel cell-surface probe consisting of fluorescent beads derivatized with PPME, a M6P-rich polysaccharide. PPME beads directly identify a carbohydrate-binding receptor on the surface of mouse lymphocytes. In every way examined, lymphocyte attachment to PPME beads (measured by flow cytofluorometry) mimics the interaction of lymphocytes with PN HEV (measured in the Stamper-Woodruff in vitro assay): both interactions are selectively inhibited by the same panel of structurally related carbohydrates, are calcium-dependent, and are sensitive to mild treatment of the lymphocytes with trypsin. In addition, thymocytes and a thymic lymphoma, S49, bind poorly to PPME beads in correspondence to their weak ability to bind to HEV. When the S49 cell line was subjected to a selection procedure with PPME beads, the ability of the cells to bind PPME beads, as well as their ability to bind to PN HEV, increased six- to eightfold. We conclude that a carbohydrate-binding receptor on mouse lymphocytes, detected by PPME beads, is involved in lymphocyte attachment to PN HEV.     

Ultrastructural analysis of HNK-1+ cells in human peripheral blood and lymph nodes

HNK-1 positive (HNK-1+) cells in human peripheral blood and lymph nodes were comparatively analysed by means of immunohistochemistry and immunoelectron microscopy. In peripheral blood, the HNK-1+ cells were grouped into large granular lymphocytes (LGLs), small lymphocytes and intermediate forms, all of which had many fine cytoplasmic processes. Except for smooth-surfaced lymphocytes, they could not be distinguished from helper/inducer T (OKT4/Leu3a) cells and suppressor/cytotoxic T (OKT8/Leu2a) cells. In double staining, HNK-1+T3- cells and HNK-1+T3+ cells could not be clearly distinguished in terms of morphology, although the former contained many LGLs. The HNK-1+ cells in the lymph nodes accumulated in the light zones of the germinal centers (GCs). These cells were small to medium-sized lymphocytes with few electron-dense granules and exclusively co-expressed helper/inducer T cell antigens (HNK-1+T4+). Their cytoplasmic projections were interwoven with those of the follicular dendritic cells which trap immune complexes for a long duration. These configurations suggest that HNK-1+T4+ cells in GCs are engaged in an immunological regulation of germinal center cells. On the other hand, large blastic HNK-1+ cells were scattered outside the GCs and some of them were in the process of mitosis. Furthermore, HNK-1+LGL-like cells with a few large electron-dense granules were rarely seen. These observations indicate that the HNK-1+ cells in the lymph nodes may proliferate outside GCs and differentiate into LGLs with a strong natural killer function.     

Influence of aging on intracellular free calcium and proliferation of mouse T-cell subsets from various lymphoid organs.

The influence of aging on T-cell activation and proliferation was examined in lymphocytes derived from peripheral blood, spleen, and lymph nodes of WBB6F1 C57B1/6J x WB/Re) mice. Following activation with anti-CD3 monoclonal antibodies, the greatest age-related changes were seen in CD4+ cells derived from spleens of 27- to 30-month-old mice. These CD4+ lymphocytes showed reduced [Ca2+]i signaling and decreased proliferation in the presence of exogenous interleukin 2. CD8+ cells from spleens of old animals showed reduced [Ca2+]i but not altered proliferation. Both CD4+ and CD8+ cells derived from peripheral blood of old mice showed decreased peak [Ca2+]i, but no defect in cell proliferation. In contrast, age-related deficits in either [Ca2+]i or proliferation were not observed in CD4+ and CD8+ cells from lymph nodes. Additionally, the percentage of CD4+ cells was decreased in all lymphoid organs from old mice, while the percentage of CD8+ cells was similar in lymphoid organs of old and young mice. Old mice had a significant increase in expression of Pgp-1 in CD4+ cells from spleen and peripheral blood and CD8+ cells derived from lymph node. Our studies indicate that there are differential effects of aging in T lymphocytes derived from different lymphoid organs in mice. Among the cell sources and subsets examined, the age-related changes noted in CD4+ cells from mouse peripheral blood were the most similar to those previously observed in the corresponding peripheral blood lymphocyte subset in humans.     

Migration pathways of recirculating murine B cells and CD4+ and CD8+ T lymphocytes

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.     

Protein malnutrition up-regulates growth hormone receptor expression in rat splenic B lymphocytes

The reciprocal interaction between the endocrine and immune systems has been the subject of active research during the last decade, and an important body of evidence has accumulated supporting the role of the GH/IGF axis in immune function. More recently, the GH/IGF axis has been postulated as playing an important role in the modulation of stress conditions, such as catabolic stages, aging-related disorders, immunodeficient aids patients and malnutrition. Whether these effects are exerted through endocrine, autocrine or paracrine mechanisms remains to be determined for different immune cell types and tissues. The aim of the current study was to define which specific subsets of lymphocytes are the primary targets for GH action. In addition, the regulatory role of stress induced by protein restriction was investigated with respect to the relative distribution of GH receptor positive lymphoid cells. Normal growing rats were fed isocaloric diets with variable protein content (0, 4, 8, 12 and 20%) for a period of 14 days. The lymphoid cells were then separated from spleen, lymph nodes and peripheral blood lymphocytes. Flow cytometry analysis measured the binding characteristics of Fluos-rrGH to lymphocytes together with specific PE-labelled mAbs defining CD4+ and CD8+ T cells and B lymphocytes. The pattern of expression of the GH receptor differed among the lymphoid tissues and cell subsets. Spleen was the most responsive organ to protein deprivation with highest GH receptor expression in B lymphocytes, followed by CD4+ T cells. As the protein intake was decreased from 20% to 0%, the percentage of GHR positive cells increased from 12% to 52% in splenic B lymphocytes and from 8% to 17% in CD4+ T cells. In contrast, only 10%-13% of lymphocytes in lymph nodes and 2%-4% in circulation, showed binding sites to GH associated with protein deprivation. In conclusion, the increase in GH receptors on lymphocytes under catabolic stress induced by protein malnutrition gives support to the hypothesis of a modulatory role of the GH/IGF axis in preserving the homeostasis of immune tissues.     

Expression of low levels of peripheral lymph node-associated vascular addressin in mucosal lymphoid tissues: possible relevance to the dissemination of passaged AKR lymphomas

Lymphoid tumors display a wide variety of growth patterns in vivo, from that of a solitary extralymphoid tumor, to a general involvement of all lymphoid organs. Normal lymphocytes are uniquely mobile cells continuously recirculating between blood and lymph throughout much of their life cycle. Therefore, it is reasonable to propose that disseminating malignant lymphocytes may express recirculation characteristics or homing properties consistent with that of their normal lymphoid counterparts. Trafficking of lymphocytes involves the expression and recognition of both lymphocyte homing receptors and their opposing receptors on endothelium, the vascular addressins. These cell surface elements direct the tissue-selective localization of lymphocyte subsets in vivo into organized lymphoid organs and sites of chronic inflammation where specific binding events occur between lymphocytes and the endothelium of specialized high endothelial venules (HEV). In a recent murine study of 13 lymphoma lines, we found that lymphomas that bind well to high endothelial venules, in the Stamper-Woodruff in vitro assay (an assay of lymphocyte binding to venules in frozen sections of peripheral lymph nodes or Peyer's patches), spread hematogenously to all high endothelial venule bearing lymphoid organs, whereas non-binding lymphomas did not. In some cases lymphomas that bound with a high degree of selectivity to peripheral lymph node (PLN) high endothelial venules exhibited only limited organ preference of metastasis, involving the mucosal lymphoid organs Peyer's patches (PP) in addition to the peripheral lymph nodes of adoptive recipients. Here we demonstrate that Peyer's patch high endothelial venules express a low but functional level of peripheral lymph node addressin (MECA-79) that can be recognized by lymphomas expressing the peripheral lymph node homing receptor (MEL-14 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cellular immune response against autologous human melanoma. II. Mechanism of induction and specificity of suppression

The cytotoxic immune response in the peripheral blood lymphocytes (PBL) against an autologous malignant melanoma cell line, PJ-M, was found to be down-regulated in in vitro co-culture (IVC) selectively by unfractionated resident lymph node lymphocytes (derived from a lymph node infiltrated with the PJ-M melanoma cells) and T4+ as well as T8+ fractions of the resident lymph node-derived lymphocytes. In this study, the mechanism involved in, and the specificities of, cytotoxic immune response in this autologous system were examined at population and clonal levels. Resident lymph node lymphocytes were isolated from both involved and uninvolved lymph nodes from the same patient. Resident lymphocytes from both sources regulated the generation of cytotoxic immune response when both types of resident lymph node lymphocytes were further sensitized against the PJ-M cells in IVC and were expanded in interleukin 2 (IL 2). An IL 2-dependent homogeneous lymphocyte line (I-10:1) bearing the phenotype of a helper T cell (T4+) and a T4+ clone (I-10.3) of the I-10:1 line, established by limiting dilution culture, also down-regulated the generation of cytotoxic immune effector cells in the PBL in IVC against the PJ-M targets. The IL 2-dependent T4+ inducer line I-10:1 generated a functionally differentiated T8+ suppressor population(s) that, in turn, could abrogate cytotoxic response in fresh PBL in IVC against PJ-M cells. The inducer line I-10:1 and its subclone I-10.3 suppressed the generation of cytotoxic effector cells in the PBL in IVC selectively against the autologous PJ-M cells. Generation of cytotoxic allo-response in IVC was unaffected by the inducer lines. These results provide further evidence for the involvement of the regulatory network in cytotoxic immune response in an autologous human tumor system, and suggest a potential explanation for cytotoxic unresponsiveness against autologous melanoma cells.     

Signaling through L-selectin mediates enhanced chemotaxis of lymphocyte subsets to secondary lymphoid tissue chemokine

L-selectin functions as an important adhesion molecule that mediates tethering and rolling of lymphocytes by binding to high endothelial venule (HEV)-expressed ligands during recirculation. Subsequent lymphocyte arrest and transmigration require activation through binding of HEV-decorated homeostatic chemokines such as secondary lymphoid tissue chemokine (SLC; CCL21) to its counterreceptor, CCR7. Importantly, L-selectin also functions as a signaling molecule. In this study, signaling induced by ligation of L-selectin using mAb or endothelial cell-expressed ligand significantly enhanced the chemotaxis of murine T cells and B cells to SLC but not to other homeostatic chemokines. Consistent with the expression levels of L-selectin in different lymphocyte subsets, L-selectin-mediated enhancement of chemotaxis to SLC was observed for all naive lymphocytes and effector/memory CD8(+) T cells, whereas only a subpopulation of effector/memory CD4(+) T cells responded. During in vivo mesenteric lymph node migration assays, the absence of L-selectin on lymphocytes significantly attenuated both their ability to migrate out of the HEV and their chemotaxis away from the vessel wall. Notably, ligation of L-selectin and/or CCR7 did not result in increased CCR7 expression levels, internalization, or re-expression. Pharmacologic inhibitor studies showed that L-selectin-mediated enhanced chemotaxis to SLC required intact intracellular kinase function. Furthermore, treatment of lymphocytes with the spleen tyrosine kinase family inhibitor piceatannol reduced their ability to migrate across the HEV in peripheral lymph nodes. Therefore, these results suggest that "cross-talk" in the signaling pathways initiated by L-selectin and CCR7 provides a novel mechanism for functional synergy between these two molecules during lymphocyte migration.     

Nonrandom migration of CD4+, CD8+, and gamma delta+T19+ lymphocyte subsets following in vivo stimulation with antigen

We report here the results of experiments in which the migration of three T cell subsets (CD4+, CD8+, and gamma delta+T19+ cells) through antigen-stimulated lymph nodes and subcutaneous granulomas has been compared with that through normal skin and resting lymph nodes. The percentage of gamma delta+T19+ lymphocytes was halved and the percentage of CD8+ lymphocytes was doubled in lymph draining stimulated compared with control tissues, and all lymphocyte subsets except gamma delta+T19+ lymphocytes had higher hourly outputs in lymph draining antigen-stimulated compared with control tissues. Antigen also resulted in a higher percentage of CD8+ lymphoblasts and a lower percentage of gamma delta+T19+ lymphoblasts in efferent lymph draining antigen-stimulated lymph nodes. The data indicate that lymphocyte subsets leave the blood with differing efficiencies in different vascular beds and raise the possibility that antigen can influence the rate at which tissues extract individual T cell subsets from the blood.     

Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85-95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells

The tissue-specific homing of lymphocytes is directed by specialized high endothelial venules (HEV). At least three functionally independent lymphocyte/HEV recognition systems exist, controlling the extravasation of circulating lymphocytes into peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches or appendix), and the synovium of inflamed joints. We report here that antibodies capable of inhibiting human lymphocyte binding to one or more HEV types recognize a common 85-95-kD lymphocyte surface glycoprotein antigen, defined by the non-blocking monoclonal antibody, Hermes-1. We demonstrate that MEL-14, a monoclonal antibody against putative lymph node "homing receptors" in the mouse, functionally inhibits human lymphocyte binding to lymph node HEV but not to mucosal or synovial HEV, and cross-reacts with the 85-95-kD Hermes-1 antigen. Furthermore, we show that Hermes-3, a novel antibody produced by immunization with Hermes-1 antigen isolated from a mucosal HEV-specific cell line, selectively blocks lymphocyte binding to mucosal HEV. Such tissue specificity of inhibition suggests that MEL-14 and Hermes-3 block the function of specific lymphocyte recognition elements for lymph node and mucosal HEV, respectively. Recognition of synovial HEV also involves the 85-95-kD Hermes-1 antigen, in that a polyclonal antiserum produced against the isolated antigen blocks all three classes of lymphocyte-HEV interaction. From these studies, it is likely that the Hermes-1-defined 85-95-kD glycoprotein class either comprises a family of related but functionally independent receptors for HEV, or associates both physically and functionally with such receptors. The findings imply that related molecular mechanisms are involved in several functionally independent cell-cell recognition events that direct lymphocyte traffic.