T-cell Transendothelial Migration Research Articles

D-2-hydroxyglutarate regulates human brain vascular endothelial cell proliferation and barrier function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) genes result in excessive production of (D)-2-hydroxyglutarate (D-2HG) which intrinsically modifies tumor cell epigenetics and impacts surrounding noncancerous cells through nonepigenetic pathways. However, whether D-2HG has a paracrine effect on endothelial cells in the tumor microenvironment needs further clarification. We quantified microvessel density by immunohistochemistry using tissue sections from 60 high-grade astrocytic gliomas with or without IDH mutation. Microvessel density was found to be reduced in tumors carrying an IDH mutation. Ex vivo experiments showed that D-2HG inhibited endothelial cell migration, wound healing, and tube formation by suppressing cell proliferation but not viability, possibly through reduced activation of the mTOR/STAT3 pathway. Further, D-2HG reduced fluorescent dextran permeability and decreased paracellular T-cell transendothelial migration by augmenting expression of junctional proteins thereby collectively increasing endothelial barrier function. These results indicate that D-2HG may influence the tumor vascular microenvironment by reducing the intratumoral vasculature density and by inhibiting the transport of metabolites and extravasation of circulating cells into the astrocytoma microenvironment. These observations provide a rationale for combining IDH inhibition with antitumor immunological/angiogenic approaches and suggest a molecular basis for resistance to antiangiogenic drugs in patients whose tumors express a mutant IDH allele.

"Armed" CEA CAR-T with a SIRPγ-CD28 chimeric co-receptor to exhibit the enhanced antitumor activity in preclinical study of colorectal cancer.

e14530 Background: Nowadays, CAR-T cell therapy still faces limited therapeutic efficacy for solid tumors. SIRPγ is known as a ligand for a well-known immune checkpoint molecule CD47. It is distinguished from other members of its family in that it has a very short intracytoplasmic tail and is incapable of transducing signals on its own. Of note, the role of SIRPγ still largely unknown to date. Preliminary data indicated SIRPγ plays a key role in T-cell transendothelial migration and promotes antigen-specific T-cell proliferation. We thus designed a novel SIRPγ-CD28 chimeric receptor comprising the extracellular part of SIRPγ, the transmembrane and intracellular domains of CD28 and used as the co-receptor for a CEA-targeting CAR. Here, we report on the activity of this "armed" CAR-T with the SIRPγ-CD28 co-receptor in colorectal tumor (CRC) and its anti-tumor efficacy in mice xenograft model. Methods: In vitro luciferase-based cytotoxicity assay were used by coculturing CAR-T and DLD1-CEA CRC cells to assess the activities of CAR-T w/wo the co-receptor. The incubation supernatants were collected for detecting the release of cytokines. In vivo evaluation of the anti-tumor efficacy of CAR-T with the SIRPγ-CD28 co-receptor versus control T cell, and control CAR-T was performed in NOG mice CRC xenograft model. DLD1-CEA cells expressing luciferase reporter were s.c. implanted and T cells were i.v. injected. Anti-tumor efficacy was assessed by in vivo system. Results: CAR-T with the SIRPγ-CD28 co-receptor exhibited specific cytotoxicity only to CEA+ CRC cells, and the efficacies of CAR-T w/wo the co-receptor were comparable (88.6% vs 87.8%, p > 0.05). However, cytokines were significantly higher in the CAR-T group with the co-receptor. The levels of IFNγ, IL2 and TNFα in groups of CAR-T w/wo the co-receptor were 72,793.33 vs 19,013.33 pg/ml (p < 0.05), 1834.67 vs 25.15 pg/ml (p < 0.05) and 79.65 vs 0 pg/ml (p < 0.05), respectively. In mice CRC xenografts model, the tumors were totally eliminated in the CAR-T group with the co-receptor at 21 days and lasted until the end of experiment, while the tumor growth was suppressed in the control CAR-T group at first and out-of-control in the end (Table). Conclusions: These preclinical studies demonstrated the potential of SIRPγ-CD28 co-receptor as a novel T cellular activation signals for CAR-T. The "armed" CAR-T exhibits a fabulous anti-tumor efficacy in CRC and has great potential for applications in other solid tumors. [Table: see text]

Red light-green light: T-cell trafficking in cardiac and vascular inflammation.

Extravasation of T cells from the bloodstream into inflamed tissues requires interactions between T cells and vascular endothelial cells, a necessary step that allows T cells to exert their effector function during the immune response to pathogens and to sterile insults. This cellular cross talk involves adhesion molecules on both the vascular endothelium and the T cells themselves that function as receptor-ligand pairs to slow down circulating T cells. These will eventually extravasate into sites of inflammation when they receive the correct chemokine signals. Accumulation of T cells within the vascular wall can lead to vessel thickening and vascular disease, whereas T-cell extravasation into the myocardium often leads to cardiac chronic inflammation and adverse cardiac remodeling, hallmarks of heart failure. On the flip side, T-cell trafficking is required for pathogen clearance and to promote tissue repair after injury resulting from cardiac ischemia. Thus, a better understanding of the central players mediating these interactions may help develop novel therapeutics to modulate vascular and cardiac inflammation. Here, we review the most recent literature on pathways that regulate T-cell transendothelial migration, the last step leading to T-cell infiltration into tissues and organs in the context of vascular and cardiac inflammation. We discuss new potential avenues to therapeutically modulate these pathways to enhance or prevent immune cell infiltration in cardiovascular disease.

Research Highlights

Research Highlights

HLA in Alzheimer's Disease: Genetic Association and Possible Pathogenic Roles.

Alzheimer's disease (AD) is commonly considered as the most prominent dementing disorder globally and is characterized by the deposition of misfolded amyloid-β (Aβ) peptide and the aggregation of neurofibrillary tangles. Immunological disturbances and neuroinflammation, which result from abnormal immunological reactivations, are believed to be the primary stimulating factors triggering AD-like neuropathy. It has been suggested by multiple previous studies that a bunch of AD key influencing factors might be attributed to genes encoding human leukocyte antigen (HLA), whose variety is an essential part of human adaptive immunity. A wide range of activities involved in immune responses may be determined by HLA genes, including inflammation mediated by the immune response, T-cell transendothelial migration, infection, brain development and plasticity in AD pathogenesis, and so on. The goal of this article is to review the recent epidemiological findings of HLA (mainly HLA class I and II) associated with AD and investigate to what extent the genetic variations of HLA were clinically significant as pathogenic factors for AD. Depending on the degree of contribution of HLA in AD pathogenesis, targeted research towards HLA may propel AD therapeutic strategies into a new era of development.

Regulatory T cells suppress the cytotoxic phenotype of T cells in intestinal tumors of APCMin/+ mice

Abstract The presence of activated T cells in colorectal cancer (CRC) tissues is a strong predictor of patient survival. Our previous research have shown that regulatory T cells (Treg) are able to reduce T-cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo in a murine model for intestinal adenomas (APCMin/+). In this study we investigated the effect of Treg depletion on the cytotoxic potential of conventional αβ T cells and γδ T cells in intestinal adenomas. We used the APCMin/+ \DEREG mouse model, which harbours a high affinity diphtheria toxin receptor under the control of the FOXP3 promoter, to deplete Treg in tumor bearing mice. We found that the number of CD8αβ T cells per mg tissue in the lamina propria was significantly increased in the Treg depleted intestinal adenomas in comparison to the non-depleted tumors. We could confirm this finding with IHC staining in tissue sections. The number of CD8αα T cells and γδ T cells remained unchanged. Furthermore, ex vivo frequencies in the lamina propria of Granzyme B+ CD8αβ T cells were increased in the Treg depleted intestinal adenomas. Following in vitro stimulation of lymphocytes from lamina propria with PMA/ionomycin, there was a trend towards a higher frequency of IFNγ+ CD8αβ T cells in Treg depleted adenomas, but frequencies of CD107a+ CD8αβ T cells were decreased. The frequency of TNF+CD8αβ T cells was elevated in the tumor in comparison to the unaffected tissue regardless of Treg depletion. We are investigating the combined effect of Treg depletion and PD-1 immunotherapy on the cytotoxic phenotype of T cells in the intestinal adenomas. The results indicate that the modulation of Treg in colorectal cancer could have apositive effect on T cell cytotoxicity.

Regulatory T cells control endothelial chemokine production and migration of T cells into intestinal tumors of APCmin/+ mice

Tumor-infiltrating lymphocytes are crucial for anti-tumor immunity. We have previously shown that regulatory T cells (Treg) are able to reduce T-cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo. Treg depletion also resulted in increased levels of the chemokines CXCL9 and CXCL10 specifically in the tumors. In this study, we investigated the mechanisms for Treg mediated suppression of T-cell migration into intestinal tumors in the APCmin/+ mouse model. By breeding APCmin/+ mice with DEREG mice, which harbour a high affinity diphtheria toxin receptor under the control of the FOXP3 promoter, we were able to deplete Treg in tumor-bearing mice. Using adoptive transfer experiments, we could document a markedly increased migration of T cells specifically into Treg depleted tumors, and that Treg depletion results in increased production of the CXCR3 ligand CXCL10 from endothelial cells in the tumors. Furthermore, we were able to demonstrate that T cells use CXCR3 to migrate into intestinal tumors. In addition, human colon adenocarcinomas express high levels of mRNA CXCR3 ligands and tumor endothelial cells produce CXCL9 and CXCL10 ex vivo. In conclusion, this study demonstrates that Treg reduce endothelial CXCL10 production, inhibit T-cell migration into tumors and that CXCR3 mediated signalling is crucial for lymphocyte accumulation in intestinal tumors. Thus, immunotherapy aimed at Treg depletion may be effective by increasing not only T effector cell activity, but also their accumulation in tumors.

Regulatory T Cells from Colon Cancer Patients Inhibit Effector T-cell Migration through an Adenosine-Dependent Mechanism.

T cell-mediated immunity is a major component of antitumor immunity. In order to be efficient, effector T cells must leave the circulation and enter into the tumor tissue. Regulatory T cells (Treg) from gastric cancer patients, but not from healthy volunteers, potently inhibit migration of conventional T cells through activated endothelium. In this study, we compared T cells from colon cancer patients and healthy donors to determine the mechanisms used by Tregs from cancer patients to inhibit conventional T-cell migration. Our results showed that circulating Tregs from cancer patients expressed high levels of CD39, an ectoenzyme mediating hydrolysis of ATP to AMP, as a rate-determining first step in the generation of immunosuppressive adenosine. Tumor-associated Tregs expressed even more CD39, and we therefore examined the importance of adenosine in Treg-mediated inhibition of T-cell transendothelial migration in vitro. Exogenous adenosine significantly reduced migration of conventional T cells from healthy volunteers, and blocking either adenosine receptors or CD39 enzymatic activity during transmigration restored the ability of conventional T cells from cancer patients to migrate. Adenosine did not directly affect T cells or endothelial cells, but reduced the ability of monocytes to activate the endothelium. Taken together, our results indicate that Treg-derived adenosine acts on monocytes and contributes to reduced transendothelial migration of effector T cells into tumors. This effect of Tregs is specific for cancer patients, and our results indicate that Tregs may affect not only T-cell effector functions but also their migration into tumors.

A Novel Th17-Prone CD146+CCR5+ T-Cell Population As an Early Marker of Intestinal Graft-Versus-Host Disease

GVHD of the gastrointestinal tract (GI) is associated with high mortality. We have identified systemic, skin- and GI-specific plasma biomarkers present at clinical GVHD onset, and more recently biomarkers for treatment responsiveness. However, in order to identify early GI-specific biomarkers prior to GVHD onset, we performed state-of-the-art proteomics on plasma samples taken 14 days prior to clinical manifestation of GI GVHD. We selected candidates that were increased at least 1.5 fold in plasma from GI GVHD patients compared to HSCT patients without GVHD at matched time points. We identified two lead proteins: CD146, a cell adhesion and trafficking molecule expressed on a subset of CD4+ T cells and endothelial cells, and the chemokine (C-C motif) ligand 14 that binds to the chemokine receptor CCR5 on T cells. As these proteins have not been previously identified in proteomics experiments and antibodies for their corresponding receptors on T cells were available, we analyzed their expression profiles on PB cells from 214 HSCT patients (71 GI GVHD, 48 no GVHD, 33 non-GVHD enteritis, 22 skin first GVHD, 40 isolated skin GVHD) at the onset of symptoms. The frequency of CD146+CCR5+ T cells was significantly increased in GI GVHD patients compared to patients without GVHD, non-GVHD enteritis, or with isolated skin GVHD, as well as increased in patients who first experienced skin and then GI GVHD (Fig. 1). When using the median % of CD146+CCR5+ T cells detected in GI GVHD patients to classify patients into low and high-risk groups, patients in the high-risk group had higher 6-month non-relapse mortality (42 vs. 20%, p = 0.02). Importantly, CD146+CCR5+ T cells at onset of clinical symptoms were not correlated with GI histologic severity, suggesting that these cells are not mucosa damage products but rather systemic effectors. Thus, we measured their frequencies in samples taken at a median of 19 days post-transplant and with a median interval of 14 days prior to clinical symptoms and found that CD146+CCR5+ T cells circulate in patients before GI GVHD clinical onset.In GI GVHD patients, these cells expressed a Th1 and Th17 phenotype and expressed high levels of the activation marker ICOS known to be critical for the development of human Th17 cells. To test the hypothesis that CD146+ T cells are Th17-prone, we next investigated whether in vitro polarization of CD4 T cells with defined stimulation conditions will increase the CD146+CCR5+ expression as well as the production of Th1 and Th17 cytokines. Fig. 2A demonstrates that CD4 T cells differentiated with both Th17-inducing cytokines and ICOS co-stimulation had a significantly higher percentage of CD146+CCR5+ T cells and co-expressed more IL-17A+IFNγ+ than T-cells stimulated with Th1-inducing cytokines or by CD28.We then analyzed colonic mucosa biopsies from patients with GI GVHD (N = 18) and non-GVHD enteritis (N = 10) for the expression of CD146 by immunohistochemistry. CD146 expression was detected on CD3 lymphoid cells and was strongly present on the endothelium. The CD146+ vessel count in GI GVHD tissues was significantly higher than in non-GVHD enteritis tissues (p < 0.001).Th17 cells migrated more efficiently through endothelial cell monolayers than their Th1 counterparts (Fig. 2B). Lentivirus-mediated shRNA knock-down of CD146 in either cell type demonstrated that reduced CD146 expression on CD4+ T cells, but not on endothelial cells significantly reduced the T-cell transendothelial migration (Fig. 2C), suggesting that CD146 on T cells is paramount for promoting infiltration of pathogenic T cells into GVHD target organs. As proof of principle for this hypothesis, we tested donor CD146-/- T cells in an allogeneic murine GVHD model and did not find any difference in GVHD severity when compared to wild type CD146+/+ T cells. Finally, we used a xenogeneic GVHD mouse model with injection of human CD4+ T cells lentivirally transduced with CD146 or control shRNA. In comparison to the vector control group, mice transplanted with CD146 shRNA transduced T cells did not lose weight (Fig. 3A), had similar human T cells engraftment (B), had less splenic CD146+CCR5+ T cells (C), and expressed less TBET 53 days after transplant (D).In conclusion, early quantification of a novel CD146+CCR5+ Th17-prone and ICOS-induced population may allow identification of patients at risk for GI GVHD development and subsequent mortality. Targeting CD146 may represent a new avenue to treat GVHD. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Novel Role of CD47 in Rat Microvascular Endothelium

Objective—Although endothelial CD47, a member of the immunoglobulin superfamily, has been implicated in leukocyte diapedesis, its capacity for intracellular signaling and physical localization during this process has not been addressed in detail. This study examined endothelial CD47 spatiotemporal behavior and signaling pathways involved in regulating T-cell transendothelial migration.Approach and Results—By biochemical methods, transmigration assays, and live-cell microscopy techniques, we show that endothelial CD47 engagement results in intracellular calcium mobilization, increased permeability, and activation of Src and AKT1/phosphoinositide 3-kinase in brain microvascular endothelial cells. These signaling pathways converge to induce cytoskeleton remodeling and vascular endothelial cadherin phosphorylation, which are necessary steps during T-cell transendothelial migration. In addition, during T-cell migration, transmigratory cups and podo-prints enriched in CD47 appear on the surface of the endothelium, indicating that the spatial distribution of CD47 changes after its engagement. Consistent with previous findings of intercellular adhesion molecule 1, blockade of CD47 results in decreased T-cell transmigration across microvascular endothelium. The overlapping effect of intercellular adhesion molecule 1 and CD47 suggests their involvement in different steps of the diapedesis process.Conclusions—These data reveal a novel role for CD47-mediated signaling in the control of the molecular network governing endothelial-dependent T-cell diapedesis.

EphB and Ephrin-B interactions mediate human mesenchymal stem cell suppression of activated T-cells.

Mesenchymal stromal/stem cells (MSC) express the contact-dependent erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinase family and their cognate ephrin ligands, which are known to regulate thymocyte maturation and selection, T-cell transendothelial migration, activation, co-stimulation, and proliferation. However, the contribution of Eph/ephrin molecules in mediating human MSC suppression of activated T-cells remains to be determined. In the present study, we showed that EphB2 and ephrin-B2 are expressed by ex vivo expanded MSC, while the corresponding ligands, ephrin-B1 and EphB4, respectively, are highly expressed by T-cells. Initial studies demonstrated that EphB2-Fc and ephrin-B2-Fc molecules suppressed T-cell proliferation in allogeneic mixed lymphocyte reaction (MLR) assays compared with human IgG-treated controls. While the addition of a third-party MSC population demonstrated dramatic suppression of T-cell proliferation responses in the MLR, blocking the function of EphB2 or EphB4 receptors using inhibitor binding peptides significantly increased T-cell proliferation. Consistent with these observations, shRNA EphB2 or ephrin-B2 knockdown expression in MSC reduced their ability to inhibit T-cell proliferation. Importantly, the expression of immunosuppressive factors, indoleamine 2, 3-dioxygenase, transforming growth factor-β1, and inducible nitric oxide synthase expressed by MSC, was up-regulated after stimulation with EphB4 and ephrin-B1 in the presence of interferon (IFN)-γ, compared with untreated controls. Conversely, key factors involved in T-cell activation and proliferation, such as interleukin (IL)-2, IFN-γ, tumor necrosis factor-α, and IL-17, were down-regulated by T-cells treated with EphB2 or ephrin-B2 compared with untreated controls. Studies utilizing signaling inhibitors revealed that inhibition of T-cell proliferation is partly mediated through EphB2-induced ephrin-B1 reverse signaling or ephrin-B2-mediated EphB4 forward signaling by activating Src, PI3Kinase, Abl, and JNK kinase pathways, activated by tyrosine phosphorylation. Taken together, these observations suggest that EphB/ephrin-B interactions play an important role in mediating human MSC inhibition of activated T cells.

Anti‐inflammatory effects of tobramycin and a copper–tobramycin complex with superoxide dismutase‐like activity

Airway inflammation in cystic fibrosis (CF) patients is characterized by accumulations of neutrophils in the airway and T cells in bronchial tissue, with activation of platelets in the circulation. CF patients are routinely treated with systemic or inhaled tobramycin for airway infection with Pseudomonas aeruginosa. Clinical trials have indicated an anti-inflammatory effect of tobramycin beyond its bactericidal activity. Here, we investigate the anti-inflammatory properties of tobramycin in vitro and consider if these relate to the ability of tobramycin to bind copper, which is elevated in blood and sputum in CF. A copper-tobramycin complex was synthesized. The effect of tobramycin and copper-tobramycin on neutrophil activation and migration of T cells and neutrophils across human lung microvascular endothelial cells in response to thrombin-activated platelets were investigated in vitro. Tobramycin uptake was detected by immunocytochemistry. Intracellular reactive oxygen species were detected using the fluorescent indicator, 2',7'-dichlorofluorescein diacetate (DCFDA). Neutrophil superoxide, hydrogen peroxide and neutrophil elastase activity were measured using specific substrates. Copper was measured using atomic absorption spectroscopy. Tobramycin and copper-tobramycin were taken up by endothelial cells via a heparan sulphate-dependent mechanism and significantly inhibited T-cell and neutrophil transendothelial migration respectively. Copper-tobramycin has intracellular and extracellular superoxide dismutase-like activity. Neutrophil elastase inhibition by α1-antitrypsin is enhanced in the presence of copper-tobramycin. Tobramycin and copper-tobramycin are equally effective anti-pseudomonal antibiotics. Anti-inflammatory effects of tobramycin in vivo may relate to the spontaneous formation of a copper-tobramycin complex, implying that copper-tobramycin may be more effective therapy.

Vδ1 T lymphocytes producing IFN-γ and IL-17 are expanded in HIV-1–infected patients and respond to Candida albicans

AbstractIn early HIV-1 infection, Vδ1 T lymphocytes are increased in peripheral blood and this is related to chemokine receptor expression, chemokine response, and recirculation. Herein we show that, at variance with healthy donors, in HIV-1–infected patients ex vivo–isolated Vδ1 T cells display cytoplasmic interferon-γ (IFN-γ). Interestingly, these cells coexpress cytoplasmic interleukin-17 (IL-17), and bear the CD27 surface marker of the memory T-cell subset. Vδ1 T cells, isolated from either patients or healthy donors, can proliferate and produce IFN-γ and IL-17 in response to Candida albicans in vitro, whereas Vδ2 T cells respond with proliferation and IFN-γ/IL-17 production to mycobacterial or phosphate antigens. These IFN-γ/IL-17 double-producer γδ T cells express the Th17 RORC and the Th1 TXB21 transcription factors and bear the CCR7 homing receptor and the CD161 molecule that are involved in γδ T-cell transendothelial migration. Moreover, Vδ1 T cells responding to C albicans express the chemokine receptors CCR4 and CCR6. This specifically equipped circulating memory γδ T-cell population might play an important role in the control of HIV-1 spreading and in the defense against opportunistic infections, possibly contributing to compensate for the impairment of CD4+ T cells.

The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration

AbstractMigration toward chemoattractants is a hallmark of T-cell trafficking and is essential to produce an efficient immune response. Here, we have analyzed the function of the Rac activator Tiam1 in the control of T-cell trafficking and transendothelial migration. We found that Tiam1 is required for chemokine- and S1P-induced Rac activation and subsequent cell migration. As a result, Tiam1-deficient T cells show reduced chemotaxis in vitro, and impaired homing, egress, and contact hypersensitivity in vivo. Analysis of the T-cell transendothelial migration cascade revealed that PKCζ/Tiam1/Rac signaling is dispensable for T-cell arrest but is essential for the stabilization of polarization and efficient crawling of T cells on endothelial cells. T cells that lack Tiam1 predominantly transmigrate through individual endothelial cells (transcellular migration) rather than at endothelial junctions (paracellular migration), suggesting that T cells are able to change their route of transendothelial migration according to their polarization status and crawling capacity.

Endothelial CD47 interaction with SIRPγ is required for human T-cell transendothelial migration under shear flow conditions in vitro

AbstractLeukocyte transendothelial migration (TEM) is a critical event during inflammation. CD47 has been implicated in myeloid cell migration across endothelium and epithelium. CD47 binds to signal regulatory protein (SIRP), SIRPα and SIRPγ. So far, little is known about the role of endothelial CD47 in T-cell TEM in vivo or under flow conditions in vitro. Fluorescence-activated cell sorting and biochemical analysis show that CD3+ T cells express SIRPγ but not SIRPα, and fluorescence microscopy showed that CD47 was enriched at endothelial junctions. These expression patterns suggested that CD47 plays a role in T-cell TEM through binding interactions with SIRPγ. We tested, therefore, whether CD47-SIRPγ interactions affect T-cell transmigration using blocking mAb against CD47 or SIRPγ in an in vitro flow model. These antibodies inhibited T-cell TEM by 70% plus or minus 6% and 82% plus or minus 1%, respectively, but had no effect on adhesion. In agreement with human mAb studies, transmigration of murine wild-type T helper type 1 cells across TNF-α–activated murine CD47−/− endothelium was reduced by 75% plus or minus 2% even though murine T cells appear to lack SIRPγ. Nonetheless, these findings suggest endothelial cell CD47 interacting with T-cell ligands, such as SIRPγ, play an important role in T-cell transendothelial migration.

Physiologic and aberrant regulation of memory T-cell trafficking by the costimulatory molecule CD28

AbstractProductive T-cell immunity requires both the activation and the migration of specific T cells to the antigenic tissue. The costimulatory molecule CD28 plays an essential role in the initiation of T-cell–mediated immunity. We investigated the possibility that CD28 may also regulate migration of primed T cells to target tissue. In vitro, CD28-mediated signals enhanced T-cell transendothelial migration, integrin clustering, and integrin-mediated migration. In vivo, T cells bearing a mutation in the CD28 cytoplasmic domain, which abrogates PI3K activation, displayed normal clonal expansion but defective localization to antigenic sites following antigenic rechallenge. Importantly, antibody-mediated CD28 stimulation led to unregulated memory T-cell migration to extra-lymphoid tissue, which occurred independently of T-cell receptor (TCR)–derived signals and homing-receptor expression. Finally, we provide evidence that CD28- and CTLA-4–mediated signals exert opposite effects on T-cell trafficking in vivo. These findings highlight a novel physiologic function of CD28 that has crucial implications for the therapeutic manipulation of this and other costimulatory molecules.

Differential Effects of Immunosuppressive Drugs on T-Cell Motility

The best-characterized mechanism of the action of immunosuppressive drugs is to prevent T-cell clonal expansion, thus containing the magnitude of the ensuing immune response. As T-cell recruitment to the inflammatory site is another key step in the development of T-cell-mediated inflammation, we analyzed and compared the effects of two commonly used immunosuppressants, cyclosporin A (CsA) and the rapamycin-related compound SDZ-RAD, on the motility of human CD4+ T cells. We show that CsA, but not SDZ-RAD, inhibits T-cell transendothelial migration in vitro. CsA selectively impaired chemokine-induced T-cell chemotaxis while integrin-mediated migration was unaffected. The inhibition of T-cell chemotaxis correlated with reduced AKT/PKB but not ERK activation following exposure to the chemokine CXCL-12/SDF-1. In addition, CsA, but not SDZ-RAD, prevents some T-cell receptor-mediated effects on T-cell motility. Finally, we show that CsA, but not SDZ-RAD inhibits tissue infiltration by T cells in vivo. Our data suggest a prominent antiinflammatory role for CsA in T-cell-mediated tissue damage, by inhibiting T-cell trafficking into tissues in addition to containing clonal expansion.

Endothelial cell expression of galectin-1 induced by prostate cancer cells inhibits T-cell transendothelial migration

A critical control point in the immune response to tumors or to pathogens is the egress of lymphocytes from blood into damaged or infected tissue. While several specific endothelial cell proteins promote lymphocyte adhesion to and migration across endothelium, little is known about endothelial cell surface proteins that negatively regulate transendothelial migration of lymphocytes. Galectin-1 is a mammalian lectin expressed by a variety of cell types, including endothelial cells, that has pleiotropic anti-inflammatory effects. Galectin-1 is known to alter T-cell cytokine production and to trigger T-cell death. We now demonstrate that galectin-1 inhibits T-cell migration across endothelial cells, identifying a novel anti-inflammatory effect of galectin-1. We observed reduced T-cell migration across endothelial cells induced to increase galectin-1 expression by exposure to prostate cancer cell conditioned medium, compared to T-cell migration across control-treated endothelial cells, and the inhibitory effect of galectin-1 on T-cell migration was reversed by specific antiserum. Decreased T-cell migration was not due to decreased adhesion to galectin-1 expressing endothelial cells, nor to death of T cells, as T cells lacking core 2 O-glycans and thus resistant to galectin-1 death displayed reduced migration across endothelial cells. Galectin-1 on the surface of extracellular matrix also reduced the ability of T cells to migrate through the matrix. T cells bound to galectin-1-coated matrix demonstrated enhanced clustering of CD43, including at the T-cell:matrix interface, compared to CD43 on T cells bound to matrix in the absence of galectin-1. As translocation of CD43 to the trailing edge is essential for polarized T-cell migration, these data indicate that galectin-1-mediated clustering of CD43 contributes to the inhibitory effect on T-cell migration. Inhibition of T-cell migration is a novel anti-inflammatory activity of galectin-1.

Vav1 and Rac Control Chemokine-promoted T Lymphocyte Adhesion Mediated by the Integrin α4β1

The chemokine CXCL12 promotes T lymphocyte adhesion mediated by the integrin alpha4beta1. CXCL12 activates the GTPase Rac, as well as Vav1, a guanine-nucleotide exchange factor for Rac, concomitant with up-regulation of alpha4beta1-dependent adhesion. Inhibition of CXCL12-promoted Rac and Vav1 activation by transfection of dominant negative Rac or Vav1 forms, or by transfection of their siRNA, remarkably impaired the increase in T lymphocyte attachment to alpha4beta1 ligands in response to this chemokine. Importantly, inhibition of Vav1 expression by RNA interference resulted in a blockade of Rac activation in response to CXCL12. Adhesions in flow chambers and soluble binding assays using these transfectants indicated that initial ligand binding and adhesion strengthening mediated by alpha4beta1 were dependent on Vav1 and Rac activation by CXCL12. Finally, CXCL12-promoted T-cell transendothelial migration involving alpha4beta1-mediated adhesion was notably inhibited by expression of dominant negative Vav1 and Rac. These results indicate that activation of Vav1-Rac signaling pathway by CXCL12 represents an important inside-out event controlling efficient up-regulation of alpha4beta1-dependent T lymphocyte adhesion.

Helicobacter pyloriInduces Transendothelial Migration of Activated Memory T Cells

Helicobacter pylori infection is associated with pronounced infiltration of granulocytes and lymphocytes into the gastric mucosa, resulting in active chronic gastritis that may develop into duodenal ulcer disease or gastric adenocarcinoma. Infiltrating T cells play a major role in the pathology of these diseases, but the signals involved in recruitment of T cells from blood to H. pylori-infected tissues are not well understood. We therefore examined H. pylori-induced T-cell transendothelial migration (TEM). The Transwell system, employing a monolayer of human umbilical vein endothelial cells, was used as a model to study TEM. H. pylori induced a significant T-cell migration, compared to spontaneous migration. CD4+ and CD8+ T cells migrated to the same extent in response to H. pylori, whereas there was significantly larger transmigration of memory T cells compared to naive T cells. Both H. pylori culture filtrate and urease induced migration, and the presence of the H. pylori cag pathogenicity island increased TEM. T-cell TEM was mediated by LFA-1-ICAM-1 interactions in accordance with an increased ICAM-1 expression on the endothelial cells after contact with H. pylori. Migrating T cells had increased expression of activation marker CD69 and chemokine receptors CXCR3, CCR4, and CCR9. Furthermore, T cells migrating in response to H. pylori secreted Th1 but not Th2 cytokines upon stimulation. In conclusion, our data indicate that live H. pylori and its secreted products contribute to T-cell recruitment to the gastric mucosa and that the responding T cells have an activated memory Th1 phenotype.