Article

CD73 Engagement Promotes Lymphocyte Binding to Endothelial Cells Via a Lymphocyte Function-Associated Antigen-1-Dependent Mechanism

December 2000
The Journal of Immunology 165(10):5411-7

December 2000
165(10):5411-7

DOI:10.4049/jimmunol.165.10.5411

Source
PubMed

Authors:

Laura Airas

University of Turku and Turku University Hospital, Turku, Finland

Sirpa Jalkanen

University of Turku

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5'-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca(2+)-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.

Elevated ATP via enhanced miRNA-30b, 30c, and 30e downregulates the expression of CD73 in CD8+ T cells of HIV-infected individuals

Article

Full-text available

Mar 2022
PLOS PATHOG

CD8 ⁺ T cells play a crucial role against chronic viral infections, however, their effector functions are influenced by the expression of co-stimulatory/inhibitory receptors. For example, CD73 works with CD39 to convert highly inflammatory ATP to adenosine. However, its expression on T cells in the context of viral infections has not been well defined. Here, we analyzed the expression of CD73 on human T cells in a cohort of 102 HIV-infected individuals including those on antiretroviral therapy (ART), ART-naïve, and long-term non-progressors who were not on ART. We found that the frequency of CD73 ⁺ T cells was markedly lower among T cell subsets (e.g. naïve, effector or memory) in the peripheral blood of all HIV-infected individuals. Notably, CD73 was decreased at the cell surface, intracellular and gene levels. Functionally, CD8 ⁺ CD73 ⁺ T cells exhibited decreased cytokine expression (TNF-α, IFN-γ and IL-2) upon global or antigen-specific stimulation and impaired expression of cytolytic molecules at the gene and protein levels. In contrast, CD8 ⁺ CD73 ⁺ T cells expressed elevated levels of homing receptors such as CCR7, α4β7 integrin, which suggests a migratory advantage for these cells as observed in vitro . We also observed significant migration of CD73 ⁺ CD8 ⁺ T cells into the cerebrospinal fluids of multiple sclerosis (MS) patients at the time of disease relapse. Moreover, we found that elevated levels of ATP in the plasma of HIV-infected individuals upregulates the expression of miRNA30b-e in T cells in vitro . In turn, inhibition of miRNAs (30b, 30c and 30e) resulted in significant upregulation of CD73 mRNA in CD8 ⁺ T cells. Therefore, we provide a novel mechanism for the downregulation of CD73 via ATP-induced upregulation of miRNA30b, 30c and 30e in HIV infection. Finally, these observations imply that ATP-mediated downregulation of CD73 mainly occurs via its receptor, P2X1/P2RX1. Our results may in part explain why HIV-infected individuals have reduced risk of developing MS considering the role of CD73 for efficient T cell entry into the central nervous system.

CD73 expression and clinical significance in human metastatic melanoma

Article

Full-text available

Jun 2018

Background: CD73 is an ectoenzyme involved in the production of adenosine. It exerts immunosuppressive and protumoral roles and has emerged as a potential immuno-oncology target. Results: CD73 expression was detected in TC in 54% of melanoma metastases, involving < 50% TC in the majority of the cases, with variable intensity. CD73 expression was significantly associated with a lower Breslow's depth of the primary lesion and was more frequent in patients having received prior non-surgical therapies. In an adjusted analysis, CD73 expression in TC (H-score > 37.5 or intensity > 1) significantly correlated to decreased overall survival (OS) from biopsy. Of the samples containing TIMC, 35% presented CD73+ TIMC. Highly infiltrated tumors were more likely to contain CD73+ TIMC. CD73 expression in TIMC (percentage ≥1%) significantly correlated with improved OS from biopsy. Conclusions: Immunohistochemistry detected CD73 expression in more than half of metastatic melanomas. While CD73 expression in TC significantly correlated with decreased OS, CD73 expression in TIMC significantly associated with improved OS. These results encourage the study of anti-CD73 therapies for metastatic melanoma patients. Methods: CD73 expression was assessed by immunohistochemistry in metastatic melanomas from 114 patients. Immunostainings were evaluated in tumor cells (TC) (percentage, intensity (1-3) and H-score) and in tumor-infiltrating mononuclear cells (TIMC) (percentage).

HIGH INTENSITY RUNNING EXERCISE ALTERS BOTH THY-1 AND ECTO-NUCLEOTIDASE EXPRESSION ON LYMPHOCYTES SURFACE.

Article

Full-text available

Jun 2016

Acute exercise is a challenge to the homeostasis and after intense exercise there is a transient lymphopenia and immunosuppression. The cell adhesion molecules are expressed on the surface of leukocytes and endothelial cells, and play a fundamental role in lymphocyte-endothelial interactions. The aim of the present study was to evaluate the expression of CD73, CD90 and CD105 in leukocytes after a high intensity running exercise. Ten young men were enrolled in a 4 km race and blood was collected 60 min before, 5 minutes, 1, 3, 6 and 24 h after exercise to hematological measurements and cell surface markers determination by flow cytometry. One-way Analysis of Variance with repeated measures and Dunnet’s post hoc test were applied as statistical analysis. We set significant difference at p < 0.05. Leukocytes increased by 30% up to 6h after exercise and granulocytes increased 40-70% over the same period. Lymphopenia was observed 1h after exercise as expected. Monocytes, eosinophils and basophils, and other hematologic parameters did not change. T lymphocytes, T helper and cytotoxic cells increased in response to exercise. B lymphocytes and natural killer cells increased immediately after exercise. The CD73+ cells increased only immediately after exercise while CD90+cells decreased 24h after exercise. In conclusion, high intensity running induced a classic response of the endocrine-immune systems and was able to alter the number of CD73+ and CD90+ cells.

Anti-CD73 in Cancer Immunotherapy: Awakening New Opportunities

Article

Feb 2016

Over recent years, significant advances in cancer immunotherapy have been made due to a better understanding of the principles underlying tumor biology and immunology. In this context, ecto-5′-nucleotidase (CD73) is a key molecule, because the degradation of AMP into adenosine results in the generation of an immunosuppressed and pro-angiogenic niche within the tumor microenvironment that promotes the onset and progression of cancer. Targeting CD73 has resulted in favorable antitumor effects in preclinical models, and the combined treatment of CD73 blockade with other immune-modulating agents [i.e., anti-cytotoxic T lymphocyte antigen (CTLA)-4 monoclonal antibodies (mAb) or anti-programmed cell death protein (PD)-1 mAb] is a particularly attractive therapeutic option. Although there is still a long way to go, anti-CD73 therapy, through the development of CD73 mAb, could constitute a new biologic therapy for treating patients with cancer. In this review, we discuss the link between CD73 and the onset, development, and spread of tumors, highlighting the potential value of this molecule as a drug target and a novel biomarker in the context of personalized cancer therapy.

NT5E Mutations That Cause Human Disease Are Associated with Intracellular Mistrafficking of NT5E Protein

Article

Full-text available

Jun 2014
PLOS ONE

Ecto-5'-nucleotidase/CD73/NT5E, the product of the NT5E gene, is the dominant enzyme in the generation of adenosine from degradation of AMP in the extracellular environment. Nonsense (c.662C→A, p.S221X designated F1, c.1609dupA, p.V537fsX7 designated F3) and missense (c.1073G→A, p.C358Y designated F2) NT5E gene mutations in three distinct families have been shown recently to cause premature arterial calcification disease in human patients. However, the underlying mechanisms by which loss-of-function NT5E mutations cause human disease are unknown. We hypothesized that human NT5E gene mutations cause mistrafficking of the defective proteins within cells, ultimately blocking NT5E catalytic function. To test this hypothesis, plasmids encoding cDNAs of wild type and mutant human NT5E tagged with the fluorescent probe DsRed were generated and used for transfection and heterologous expression in immortalized monkey COS-7 kidney cells that lack native NT5E protein. Enzyme histochemistry and Malachite green assays were performed to assess the biochemical activities of wild type and mutant fusion NT5E proteins. Subcellular trafficking of fusion NT5E proteins was monitored by confocal microscopy and western blot analysis of fractionated cell constituents. All 3 F1, F2, and F3 mutations result in a protein with significantly reduced trafficking to the plasma membrane and reduced ER retention as compared to wild type protein. Confocal immunofluorescence demonstrates vesicles containing DsRed-tagged NT5E proteins (F1, F2 and F3) in the cell synthetic apparatus. All 3 mutations resulted in absent NT5E enzymatic activity at the cell surface. In conclusion, three familial NT5E mutations (F1, F2, F3) result in novel trafficking defects associated with human disease. These novel genetic causes of human disease suggest that the syndrome of premature arterial calcification due to NT5E mutations may also involve a novel "trafficking-opathy".

Emerging connections between GPI-anchored proteins and their extracellular carriers in colorectal cancer

Article

Full-text available

May 2023

Although extracellular vesicles (EVs) were discovered over 40 years ago, there has been a resurgence of interest in secreted vesicles and their attendant cargo as novel modes of intracellular communication. In addition to vesicles, two amembranous nanoparticles, exomeres and supermeres, have been isolated and characterized recently. In this rapidly expanding field, it has been challenging to assign cargo and specific functions to a particular carrier. Refinement of isolation methods, well-controlled studies, and guidelines detailed by Minimal Information for Studies of Extracellular Vesicles (MISEV) are being employed to “bring order to chaos.” In this review, we will briefly summarize three types of extracellular carriers - small EVs (sEVs), exomeres, and supermeres - in the context of colorectal cancer (CRC). We found that a number of GPI-anchored proteins (GPI-APs) are overexpressed in CRC, are enriched in exosomes (a distinct subset of sEVs), and can be detected in exomeres and supermeres. This affords the opportunity to elaborate on GPI-AP biogenesis, modifications, and trafficking using DPEP1, a GPI-AP upregulated in CRC, as a prime example. We have cataloged the GPI-anchored proteins secreted in CRC and will highlight features of select CRC-associated GPI-anchored proteins we have detected. Finally, we will discuss the remaining challenges and future opportunities in studying these secreted GPI-APs in CRC.

CD73 as a potential opportunity for cancer immunotherapy

Article

Dec 2018

Introduction: Cancer cells apply various mechanisms to induce and enhance immune escape. The complex network of immune-response modulating factors in the tumor microenvironment is a reason for the difficulties encountered when attempting to treat many cancers. Adenosine is a potent immune-modulating factor that can be generated through the degradation of ATP by cooperative action of NTPDase1 (CD39) and ecto-5ʹ-nucleotidase (CD73) molecules. Overexpression of CD73 on tumor and immune cells leads to the presence of a high concentration of this factor in the tumor region. Upregulation of CD73 is associated with the overproduction of adenosine; it suppresses anti-tumor immune responses and helps proliferation, angiogenesis and metastasis. Areas covered: We attempt to clarify the immunobiology of CD73 in association with its role in cancer development, angiogenesis and metastasis. Moreover, we have reviewed CD73-targeting studies and highlighted CD73 as a potent target for cancer immunotherapy. Expert opinion: It seems that blockade of CD73 in combination with immune checkpoint inhibitors such as anti-PD-L1 and anti-CTLA-4, can be a novel promising therapeutic strategy that can be evaluated in the future trials.

Integrin affinity modulation and survival signalling

Article

Jan 2008

Paul Anthony Elliott

Integrins are heterodimeric transmembrane proteins that provide a bi-directional link between the cell’s internal biological mechanisms and the extracellular environment. During inside-out signalling, intracellular messages converge on the integrin cytoplasmic domain to induce a conformational change. This is transmitted to the extracellular domain where it results in an alteration in affinity for integrin ligands such as fibronectin and laminin. In this way the cell has developed the ability to modulate the critical functions of adhesion and cell movement. In outside-in signalling, the integrin performs a more complex function than simple adhesion; upon binding to ligand, the integrin extracellular domain undergoes a conformational change which is transmitted to the cytoplasmic domain. This alters the integrin’s cytoplasmic domain affinity for intracellular signalling proteins and results in the activation of intracellular second messenger pathways. In this way, the extracellular milieu is able to influence intracellular signalling including those involved in apoptosis. This thesis demonstrates data which provide original insights into bi-directional integrin signalling: Inside-out signalling: Constitutively active Notch1 increases β3-integrin affinity and abrogates Hras-mediated integrin suppression without increasing expression of β3- integrin. Dominant-Negative Rras blocks Notch-mediated integrin activation and Notch1-mediated reversal of Hras and Raf-mediated integrin suppression and this is independent of erk phosphorylation. Notch1 induces Rras activation. Functional adhesion assays confirm that Notch1IC increases K562 adhesion in a β1-integrin dependent manner and this is abrogated by Dominant-Negative Rras. This data supports a mechanism in which Notch1 increases integrin affinity via activation of Rras. Outside-in signalling: Evidence is presented demonstrating that extracellular matrix proteins, laminin and fibronectin, activate β1-integrins to protect SCLC cells against the apoptotic effects of etoposide and ionizing radiation via PI3Kinase activation. This occurs in two ways: 1) PI3Kinase-dependent β1-integrin signalling resulting in phosphorylation of Bad and reduced caspase-9 cleavage and 2) a β1-integrinmediated over-riding of etoposide and radiotherapy-induced cell cycle S phase delay and G2/M arrest. β1-integrin-mediated outside-in survival signalling was investigated further in the in vivo setting; MatrigelTM, a basement membrane product rich in extracellular matrix proteins, promoted SCLC xenograft survival and growth in a β1-integrin and tyrosine kinase-dependent manner. This data provides novel insights into the critical functions that integrins play in adhesion and survival signalling.

Drugs targeting adenosine signaling pathways: A current view

Article

Jul 2023

Adenosine is an endogenous nucleoside that regulates many physiological and pathological processes. It is derived from either the intracellular or extracellular dephosphorylation of adenosine triphosphate and interacts with cell-surface G-protein-coupled receptors. Adenosine plays a substantial role in protecting against cell damage in areas of increased tissue metabolism and preventing organ dysfunction in pathological states. Targeting adenosine metabolism and receptor signaling may be an effective therapeutic approach for human diseases, including cardiovascular and central nervous system disorders, rheumatoid arthritis, asthma, renal diseases, and cancer. Several lines of evidence have shown that many drugs exert their beneficial effects by modulating adenosine signaling pathways but this knowledge urgently needs to be summarized, and most importantly, actualized. The present review collects pharmaceuticals and pharmacological or diagnostic tools that target adenosine signaling in their primary or secondary mode of action. We overviewed FDA-approved drugs as well as those currently being studied in clinical trials. Among them are already used in clinic A2A adenosine receptor modulators like istradefylline or regadenoson, but also plenty of anti-platelet, anti-inflammatory, or immunosuppressive, and anti-cancer drugs. On the other hand, we investigated dozens of specific adenosine pathway regulators that are tested in clinical trials to treat human infectious and noninfectious diseases. In conclusion, targeting purinergic signaling represents a great therapeutic challenge. The actual knowledge of the involvement of adenosinergic signaling as part of the mechanism of action of old drugs has open a path not only for drug-repurposing but also for new therapeutic strategies.

Anti-CD73 antibody activates human B cells, enhances humoral responses and induces redistribution of B cells in patients with cancer

Article

Full-text available

Dec 2022

Background CD73 is widely expressed on immune cells playing a critical role in immunomodulatory functions including cell adhesion and migration, as a costimulatory molecule for T cells and in production of adenosine. The function of CD73 expressed on B cells has not been fully characterized. Mupadolimab is an anti-human CD73 antibody that activates B cells. We evaluated the characteristics of this antibody and its effects on immune cells in vitro and in vivo. Methods Mupadolimab binding to CD73, inhibition of CD73 enzymatic activity, and effects on lymphocyte activation were evaluated in vitro by measuring changes in immunophenotype by flow cytometry. Cryogenic-transmission electron microscopy was used to determine epitope binding. Effects on human B cells in vivo were evaluated in immunodeficient NSG-SGM3 mice immunized with SARS-CoV-2 and influenza viral antigens. Safety and immune effects were evaluated in the completed dose escalation portion of a phase 1 trial conducted in patients with cancer. Results Mupadolimab binds to a unique epitope on CD73 POS B cells resulting in their activation and differentiation through B cell receptor signaling pathways. Mupadolimab induces expression of CD69, CD83, CD86 and MHC class II on B cells along with morphological transformation into plasmablasts and expression of CD27, CD38 and CD138. These effects are independent of adenosine. Mupadolimab binds to the N-terminal of CD73 in the closed position and competitively inhibits substrate binding. Mupadolimab enhanced antigen specific antibody response to SARS-CoV-2 spike protein and influenza hemagglutinin in humanized mouse models. Mupadolimab was evaluated as a monotherapy in a phase 1 trial ( NCT03454451 ) in 34 patients with advanced cancer and demonstrated binding to CD73 POS circulating cells and transient reduction in the number of B cells, with return of CD73 NEG B cells with memory phenotype. No dose-limiting toxicities or changes in serum immunoglobulins were seen. Conclusions Mupadolimab activates B cells and stimulates the production of antigen specific antibodies. The effects in patients with cancer suggest that activated, CD69 POS B cells redistribute to lymphoid tissues. Minor tumor regression was observed in several patients. These results support further investigation of mupadolimab as an immunotherapy for cancer and its potential use as a vaccine adjuvant. Trial registration number NCT03454451 .

Targeting two distinct epitopes on human CD73 with a bispecific antibody improves anticancer activity

Article

Full-text available

Sep 2022

Background Immunosuppressive extracellular adenosine is generated by the enzymatic activity of CD73. In preclinical models, antibodies (Abs) targeting different epitopes on CD73 exert anticancer activity through distinct mechanisms such as inhibition of enzymatic activity, engagement of Fc receptors, and spatial redistribution of CD73. Methods Using controlled Fab arm exchange, we generated biparatopic bispecific antibodies (bsAbs) from parental anti-CD73 Abs with distinct anticancer activities. The resulting anticancer activity was evaluated using in vitro and in vivo models. Results We demonstrate that different anticancer activities can be combined in a biparatopic bsAb. Remarkably, the bsAb significantly improved the enzyme inhibitory activity compared with the parental Abs, which led to neutralization of adenosine-mediated T-cell suppression as demonstrated by proliferation and interferon gamma (IFN-γ) production and prolonged survival of tumor-bearing mice. Additionally, the bsAb caused more efficient internalization of cell surface CD73 and stimulated potent Fc-mediated engagement of human immune effector cells in vitro and in vivo. Conclusions Our data collectively demonstrate that complementary anticancer mechanisms of action of distinct anti-CD73 Abs can be combined and enhanced in a biparatopic bsAb. The multiple mechanisms of action and superior activity compared with the monospecific parental Abs make the bsAb a promising candidate for therapeutic targeting of CD73 in cancer. This concept may greatly improve future Ab design.

The Multifaceted Actions of CD73 During Development and Suppressive Actions of Regulatory T Cells

Article

Full-text available

May 2022

Adenosine (Ado) has been shown to have immunosuppressive effects in a variety of diseases. It can either be released directly into the extracellular environment by cells, or it can be produced by degradation of ATP within the extracellular spaces. This extracellular pathway is facilitated by the concerted actions of the ectoenzymes CD39 and CD73. In a first step CD39 dephosphorylates ATP to ADP and AMP, respectively, and in a second step CD73 converts AMP to Ado. Thus, activity of CD73 on the cell surface of cells is the rate limiting step in the generation of extracellular Ado. Among T cells, CD73 is most abundantly expressed by regulatory T cells (Tregs) and is even upregulated after their activation. Functionally, the generation of Ado by CD73 ⁺ Tregs has been shown to play a role in immune suppression of dendritic cells, monocytes and T cells, and the defined expression of CD73 by Tregs in immunosuppressive environments, such as tumors, made CD73 a novel checkpoint inhibitor. Therefore, therapeutical intervention by anti-CD73 antibodies or by chemical inhibitors of the enzymatic function is currently under investigation in some preclinical animal models. In the following we summarize the expression pattern and the possible functions of CD73 in T cells and Tregs, and exemplify novel ways to manipulate CD73 functions in Tregs to stimulate anti-tumor immunity.

Leukocyte Membrane Enzymes Play the Cell Adhesion Game

Article

Full-text available

Nov 2021

For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.

Rôle des 5'-nucléotidases dans la biologie des cellules cancéreuses pulmonaires humaines et leurs réponses à des stress extérieurs

Thesis

Full-text available

Apr 2020

Muhammad Zawwad Raza

5’-nucleotidases are enzymes that are primarily involved in nucleotide metabolism by dephosphorylating both intracellular and extracellular nucleotides. So far, eight different 5’-nucleotidases have been characterized and are named based on their cellular localization and substrate specificity. Among these, cytosolic 5’-nucleotidase (cN-II) and ecto 5’-nucleotidase (eN, CD73) have shown significant roles in cancer. For example, cN-II mutant cells have shown an increased resistance to cytotoxic drugs in relapsed childhood acute lymphoid leukemia, and there is an increased importance of CD73 as a novel checkpoint for cancer immunotherapy due to its role in the production of immunosuppressive adenosine. However, the detailed understanding of the biological nature of their roles in cancer progression and resistance towards treatment is still a matter to be studied. During my PhD, we have studied the biological roles of these enzymes in cancer cell proliferation, migration and response towards stress conditions (nucleotide stress, cytotoxic drugs and hypoxia). To do so, we used human lung adenocarcinoma (NCI-H292) and breast cancer (MDA-MB-231) cell lines as well as mouse breast cancer cell line (4T1) and prepared for each cell line four distinct phenotypes expressing or not cN-II and/or CD73. Our results showed that knocking out cN-II and CD73 is associated with an increased cell migration and involves TIMP-2, MMP-2 and MMP-9 via activation of COX-2/PGE2/Akt pathway. We also showed that both cN-II and CD73 have a role to play in cells response to different stress conditions as well as sensitivity towards certain cytotoxic drugs. Indeed, CD73-deficiency was associated with increased sensitivity towards nucleotide stress and vincristine whereas decreased sensitivity towards mitomycin C

Antenatal Dexamethasone Treatment Induces Sex-dependent Upregulation of NTPDase1/CD39 and Ecto-5'-nucleotidase/CD73 in the Rat Fetal Brain

Article

Full-text available

Aug 2022
CELL MOL NEUROBIOL

Dexamethasone (DEX) is frequently used to treat women at risk of preterm delivery, but although indispensable for the completion of organ maturation in the fetus, antenatal DEX treatment may exert adverse sex-dimorphic neurodevelopmental effects. Literature findings implicated oxidative stress in adverse effects of DEX treatment. Purinergic signaling is involved in neurodevelopment and controlled by ectonucleotidases, among which in the brain the most abundant are ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5ʹ-nucleotidase (e5ʹNT/CD73), which jointly dephosphorylate ATP to adenosine. They are also involved in cell adhesion and migration, processes integral to brain development. Upregulation of CD39 and CD73 after DEX treatment was reported in adult rat hippocampus. We investigated the effects of maternal DEX treatment on CD39 and CD73 expression and enzymatic activity in the rat fetal brain of both sexes, in the context of oxidative status of the brain tissue. Fetuses were obtained at embryonic day (ED) 21, from Wistar rat dams treated with 0.5 mg DEX/kg/day, at ED 16, 17, and 18, and brains were processed and used for further analysis. Sex-specific increase in CD39 and CD73 expression and in the corresponding enzyme activities was induced in the brain of antenatally DEX-treated fetuses, more prominently in males. The oxidative stress induction after antenatal DEX treatment was confirmed in both sexes, although showing a slight bias in males. Due to the involvement of purinergic system in crucial neurodevelopmental processes, future investigations are needed to determine the role of these observed changes in the adverse effects of antenatal DEX treatment.

Antenatal Dexamethasone Treatment Induces Sex-dependent Up-regulation of NTPDase1/CD39 and Ecto-5ʹ-nucleotidase/CD73 in the Rat Fetal Brain

Preprint

Full-text available

Feb 2021

Dexamethasone (DEX) is frequently used to treat women at risk of preterm delivery, but although indispensable for completion of organ maturation in fetus, antenatal DEX treatment may exert adverse sex-dimorphic neurodevelopmental effects. Literature findings implicated oxidative stress in adverse effects of DEX treatment.. Purinergic signalling is involved in neurodevelopment, and controled by ectonucleotidases, among which in the brain are most abundant ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5ʹ-nucleotidase (e5ʹNT/CD73), which jointly dephosphorylate ATP to adenosine. They are also involved in cell adhesion and migration, critical for brain development. Up-regulation of CD39 and CD73 after DEX treatment was reported in adult rat hippocampus. We investigated the effects of maternal DEX treatment on CD39 and CD73 expression and enzymatic activity in the rat fetal brain of both sexes, in the context of oxidative status of the brain tissue. Fetuses were obtained at embryonic day (ED) 21, from Wistar rat dams treated with 0.5 mg DEX/kg/day, at ED 16, 17, and 18, and brains were processed and used for further analysis. Sex-specific increase in CD39 and CD73 expression and in the corresponding enzyme activities was induced in the brain of antenatally DEX-treated fetuses, more prominently in males. The oxidative stress induction after antenatal DEX treatment, was confirmed in both sexes, altghough showing a slight bias in males. Due to involvement of purinergic system in crucial neurodevelopmental processes, future investigations are needed to determine the role of observed changes in the adverse effects of antenatal DEX treatment.

Characterization and Phase 1 Trial of a B Cell Activating Anti-CD73 Antibody for the Immunotherapy of COVID-19

Preprint

Full-text available

Sep 2020

COVID-19 is a global pandemic that has resulted in over 800,000 deaths. Robust humoral anti-viral immune responses have the potential to generate a diverse set of neutralizing antibodies to eliminate viruses and protect against re-infection, transmission, and the evolution of mutations that escape targeted therapeutics. CD73 is present on the majority of human B cells and a subset of T cells where it plays a role in lymphocyte activation and migration. CD73 also functions as an ectoenzyme that converts AMP into adenosine, which can be immunosuppressive. Here we report on CPI-006, a humanized FcγR binding-deficient IgG1 anti-CD73 antibody that blocks CD73 enzymatic activity and directly activates CD73+ B cells, inducing differentiation into plasmablasts, immunoglobulin class switching, and antibody secretion independent of adenosine. Immunophenotypic analysis of peripheral blood from advanced cancer patients receiving CPI-006 revealed evidence of B cell activation, clonal expansion, and development of memory B cells. These immune effects suggested that CPI-006 may be effective at enhancing the magnitude, diversity, and duration of humoral and cellular responses to viruses such as SARS-CoV-2. We have therefore initiated a Phase 1, single-dose, dose-escalation trial in hospitalized patients with mild to moderate COVID-19. The objectives of this trial are to evaluate the safety of CPI-006 in COVID-19 patients and to determine effects of CPI-006 on anti-SARS-CoV-2 antibody responses and the development of memory B cell and T cells. Ten patients have been enrolled in the trial receiving doses of 0.3 mg/kg or 1.0 mg/kg. All evaluable patients had low pre-treatment serum levels of anti-viral antibodies to the SARS-CoV-2 trimeric spike protein and its receptor binding domain, independent of the duration of their COVID-19 related symptoms prior to enrollment. Anti-viral antibody responses were induced 7 days after CPI-006 treatment and titers continued to rise past Day 56. Increases in the frequency of memory B cells and effector/memory T cells were observed 28 days after treatment. These preliminary results suggest that CPI-006 activates B cells and may enhance and prolong anti-SARS-CoV-2 antibody responses in patients with COVID-19. This approach may be useful for treating COVID-19 or as an adjuvant to enhance the efficacy of vaccines.

Novel multiparameter correlates of Coxiella burnetii infection and vaccination identified by longitudinal deep immune profiling

Article

Full-text available

Aug 2020

Q-fever is a flu-like illness caused by Coxiella burnetii (Cb), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb, consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb.

Therapeutic Value of CD73 as a Biomarker in Human Cancer

Article

Full-text available

Jan 2018

Context: Over the past several years, biomarkers have emerged as a diagnostic and therapeutic tool for cancer therapy. CD73 (ecto-5'-nucleotidase) which is a cell surface ectonucleotidase, mediates the conversion of extracellular Adenosine monophosphate (AMP) into adenosine through the purinergic signaling pathway. In this study the role of CD73 in different human cancers was investigated. Evidence Acquisition: The present study reviewed the articles related to the importance of CD73 as a therapeutic tool in human cancers which were published from 1990 to 2019. The publications were found by searching the valid databases for instance PubMed, Google Scholar, ProQuest, Scopus and Science Direct using keywords CD73, ectonucleotidase, therapy, cancer, etc. During the review process 90 articles were selected. Results: Different studies about the effect of CD73 on human malignancies show that CD73 is overexpressed in various types of cancer. Recent findings demonstrate that the extracellular adenosine can promote tumor growth and invasion. The significant regulatory role of CD73 has made it a suitable biomarker for cancer treatment. Conclusion: This study outlines the impact of CD73 on tumor growth, metastasis and angiogenesis. The findings are promising and can highlight the efficacy of this protein as a therapeutic tool in the context of anti-CD73 cancer therapy.

Unveiling the Role of Ecto-5′-Nucleotidase/CD73 in Astrocyte Migration by Using Pharmacological Tools

Article

Full-text available

Mar 2018

CD73 is a bifunctional glycosylphosphatidylinositol (GPI)-anchored membrane protein which functions as ecto-5′-nucleotidase and a membrane receptor for extracellular matrix protein (ECM). A large body of evidence demonstrates a critical involvement of altered purine metabolism and particularly, increased expression of CD73 in a number of human disorders, including cancer and immunodeficiency. Massive up-regulation of CD73 was also found in reactive astrocytes in several experimental models of human neuropathologies. In all the pathological contexts studied so far, the increased expression of CD73 has been associated with the altered ability of cells to adhere and/or migrate. Thus, we hypothesized that increased expression of CD73 in reactive astrocytes has a role in the process of astrocyte adhesion and migration. In the present study, the involvement of CD73 in astrocyte migration was investigated in the scratch wound assay (SW), using primary astrocyte culture prepared from neonatal rat cortex. The cultures were treated with one of the following pharmacological inhibitors which preferentially target individual functions of CD73: (a) α,β-methylene ADP (APCP), which inhibits the catalytic activity of CD73 (b) polyclonal anti-CD73 antibodies, which bind to the internal epitope of CD73 molecule and mask their surface exposure and (c) small interfering CD73-RNA (siCD73), which silences the expression of CD73 gene. It was concluded that approaches that reduce surface expression of CD73 increase migration velocity and promote wound closure in the scratch wound assay, while inhibition of the enzyme activity by APCP induces redistribution of CD73 molecules at the cell surface, thus indirectly affecting cell adhesion and migration. Application of anti-CD73 antibodies induces a decrease in CD73 activity and membrane expression, through CD73 molecules shedding and their release to the culture media. In addition, all applied pharmacological inhibitors differentially affect other aspects of astrocyte function in vitro, including reduced cell proliferation, altered expression of adenosine receptors and increased expression of ERK1/2. Altogether these data imply that CD73 participates in cell adhesion/migration and transmits extracellular signals through interactions with ECM.

Targeting immunosuppressive adenosine in cancer

Article

Full-text available

Nov 2017
NAT REV CANCER

Targeting immunosuppressive adenosine in cancer

Article

Full-text available

Oct 2017

Despite the success of anti-programmed cell death protein 1 (PD1), anti-PD1 ligand 1 (PDL1) and anti-cytotoxic T lymphocyte antigen 4 (CTLA4) therapies in advanced cancer, a considerable proportion of patients remain unresponsive to these treatments (known as innate resistance). In addition, one-third of patients relapse after initial response (known as adaptive resistance), which suggests that multiple non-redundant immunosuppressive mechanisms coexist within the tumour microenvironment. A major immunosuppressive mechanism is the adenosinergic pathway, which now represents an attractive new therapeutic target for cancer therapy. Activation of this pathway occurs within hypoxic tumours, where extracellular adenosine exerts local suppression through tumour-intrinsic and host-mediated mechanisms. Preclinical studies in mice with adenosine receptor antagonists and antibodies have reported favourable antitumour immune responses with some definition of the mechanism of action. Currently, agents targeting the adenosinergic pathway are undergoing first-in-human clinical trials as single agents and in combination with anti-PD1 or anti-PDL1 therapies. In this Review, we describe the complex interplay of adenosine and adenosine receptors in the development of primary tumours and metastases and discuss the merits of targeting one or more components that compose the adenosinergic pathway. We also review the early clinical data relating to therapeutic agents inhibiting the adenosinergic pathway.

Immunosuppressive enzymes in the tumor microenvironment

Article

Aug 2017

Antigen encounter by T lymphocytes induces important metabolic changes. Anti-tumor T lymphocytes enter in a metabolic competition with tumors, which divert feedback mechanisms of the immune response. Immunosuppressive enzymes, modifying the nutrient availability and leading to the production of toxic catabolites, represent one of these mechanisms, contributing to the metabolic halo in which T lymphocytes evolve during immune responses. Two classes of immunosuppressive enzymes, expressed by the tumor cells or by cells of the microenvironment, have been described: those catabolizing essential or semi-essential amino acids, tryptophan, arginine and phenylalanine and the ectoenzymes, which degrade the ATP to produce adenosine. These enzymes are described, as well as some of the ongoing clinical trials aiming to block them in cancer treatment. This article is protected by copyright. All rights reserved.

CD73 expression identifies a subset of IgM+ antigen-experienced cells with memory attributes that is T cell and CD40 signaling dependent

Article

Jul 2017

B cell memory was long characterized as isotype-switched, somatically mutated and germinal center (GC)-derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype-switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T-dependent versus T-independent responses. We report that CD73-expression identifies a subset of antigen-experienced IgM+ cells that share attributes of functional B cell memory. This subset is reduced in the spleens of T cell-deficient and CD40-deficient mice and in mixed marrow chimeras made with mutant and wild-type marrow, the proportion of CD73+ IgM memory is restored in the T cell-deficient donor compartment but not in the CD40-deficient donor compartment, indicating that CD40-ligation is involved in its generation. We also report that CD40-signaling supports optimal expression of CD73 on splenic T cells and age-associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B-1 B cells and regulatory T- and B- cells. Our data indicate that in addition to promoting GC-associated memory generation during B cell differentiation, CD40-signaling can influence the composition of the unswitched memory B cell pool. They also raise the possibility that a fraction of ABCs may represent T-dependent IgM memory. This article is protected by copyright. All rights reserved.

The ectonucleotidases CD39 and CD73: Novel checkpoint inhibitor targets

Article

Full-text available

Mar 2017

Cancers are able to grow by subverting immune suppressive pathways, to prevent the malignant cells as being recognized as dangerous or foreign. This mechanism prevents the cancer from being eliminated by the immune system and allows disease to progress from a very early stage to a lethal state. Immunotherapies are newly developing interventions that modify the patient's immune system to fight cancer, by either directly stimulating rejection-type processes or blocking suppressive pathways. Extracellular adenosine generated by the ectonucleotidases CD39 and CD73 is a newly recognized “immune checkpoint mediator” that interferes with anti-tumor immune responses. In this review, we focus on CD39 and CD73 ectoenzymes and encompass aspects of the biochemistry of these molecules as well as detailing the distribution and function on immune cells. Effects of CD39 and CD73 inhibition in preclinical and clinical studies are discussed. Finally, we provide insights into potential clinical application of adenosinergic and other purinergic-targeting therapies and forecast how these might develop in combination with other anti-cancer modalities.

CD73 on B16F10 melanoma cells in CD73-deficient mice promotes tumor growth, angiogenesis, neovascularization, macrophage infiltration and metastasis

Article

Dec 2015

Ecto-5’-nucleotidase (CD73), an enzyme providing interstitial adenosine, mediates diverse physiological and pathological responses. In tumor progression, it has primarily an immunosuppressive role but is also thought to regulate neovascularization. However, the latter role is still in debate. When B16F10 melanoma was subcutaneously injected into CD73 knockout mice, changes in the tumor vasculature were not always observed. However, we demonstrated earlier that the growth and vascularization of B16F10 melanoma in CD73 knockout mice depend on the low presence of CD73 on tumor cells. To further analyze the role of CD73 on tumor growth and vascularization, we compared the changes in B16F10 melanoma subcutaneously injected into right flank of wild-type mice, CD73 knockout mice lacking host CD73 only, and CD73 knockout mice with tumor cell CD73 either inhibited with AOPCP (adenosine α,β-methylene 5’′-diphosphate) or permanently knocked down through genetic modification. We report here that both inhibition and knockdown of tumor CD73 further inhibited tumor growth compared to host CD73 knockout alone. MAP-kinase signaling pathway activation also decreased more strongly in the stable knockdown. There was a significant reduction in the angiogenic activation of blood microvessels as observed by decreased anti-VEGFR2 staining. Stable CD73 knockdown also reduced endothelial cell proliferation as measured by anti-CD105 staining. However, only chemical inhibition with AOPCP significantly augmented the reduction in intratumoral microvessel density induced by host CD73 knockout. Such reduction was not observed when tumor CD73 was knocked down due to the much slower tumor growth and decreased oxygen demand as indicated by the low expression of Bad, a hypoxia marker. Decreased CD73 activity also led to the decreased expression of angiogenic factors, including VEGF and bFGF that was only partially reversed by hypoxia in tumors treated with AOPCP. Both inhibition and knockdown of tumor CD73 significantly decreased tumor macrophage infiltration and induced microenvironment changes, thereby influencing MI or MII macrophage polarization. Additionally, tumor cell CD73 is important in metastasis formation through adenosine-independent attachment to endothelium. We conclude that even low tumor cell CD73 expression has an undeniable role in melanoma progression, including the regulation of many aspects of angiogenesis. CD73 is thus a viable target in anti-angiogenic melanoma therapy. Full text available for the next 50 days (up to 10th December) from link: http://authors.elsevier.com/a/1RvsS4qcwY8qlq

Role of adenosine during the embryogenesis of vertebrates

Article

Oct 2014

Alice Tocco

Extracellular adenosine belongs to the purinergic signalling pathway and regulatesvarious physiological processes through activation of specific receptors named adora. Theextracellular concentration of adenosine is regulated by several ecto-enzymes involved eitherin its generation or in its degradation but also by nucleoside transporters enabling its exitoutside or entry inside the cell. In adults, the functions of adenosine are quite well known,however, the its involvement during embryogenesis remains poorly studied. An excess ofadenosine in early phases of development is lethal in mouse and sea urchins, demonstratingthe importance of the extracellular adenosine level regulation during embryogenesis. The aimof my phD is to understand the role of adenosine during embryogenesis using Xenopus as avertebrate model. Indeed, the first in vivo evidence of the implication of the purinergic signallingpathway during vertebrate development, and in particular of ADP during eye formation hasbeen demonstrated using this model. The first part of this project was to characterize all theadenosine signalling pathway actors in Xenopus in order to generate the first comprehensiveand comparative embryonic expression map of these genes. This work allowed me to selectthe alkaline phosphatase alpl for functional studies based on its specific expression profile, inthe retina and kidney. These functional studies, mostly carried out by knockdown experiments,constituted the second part of this phD and showed the implication of this enzyme during theeye and kidney development.

CD73 and CD39 ectonucleotidases in T cell differentiation: Beyond immunosuppression

Article

Full-text available

Jul 2015

Extracellular ATP is a danger signal released by dying and damaged cells, and it functions as an immunostimulatory signal that promotes inflammation. However, extracellular adenosine acts as an immunoregulatory signal that modulates the function of several cellular components of the adaptive and innate immune response. Consequently, the balance between ATP and adenosine concentration is crucial in immune homeostasis. CD39 and CD73 are two ectonucleotidases that cooperate in the generation of extracellular adenosine through ATP hydrolysis, thus tilting the balance towards immunosuppressive microenvironments. Extracellular adenosine can prevent activation, proliferation, cytokine production and cytotoxicity in T cells through the stimulation of the A2A receptor; however, recent evidence has shown that adenosine may also affect other processes in T-cell biology. In this review, we discuss evidence that supports a role of CD73 and CD39 ectonucleotidases in controlling naive T-cell homeostasis and memory cell survival through adenosine production. Finally, we propose a novel hypothesis of a possible role of these ectonucleotidases and autocrine adenosine signaling in controlling T-cell differentiation. Copyright © 2015. Published by Elsevier B.V.

The role of ecto-5′-nucleotidase in endothelial dysfunction and vascular pathologies

Article

May 2015

Ecto-5'-nucleotidase (e5NT, CD73) is an enzyme that is highly expressed in endothelium and is involved in the extracellular nucleotide catabolism. CD73 converts AMP to adenosine that via specific subtypes of P1 receptor mediates cytoprotection involving diverse mechanisms such as vasodilatation, suppression of inflammation, inhibition of thrombosis and anti-adrenergic effect. Physiological intravascular concentration of adenosine is in nanomolar range, but could become micromolar in response to various forms of stress. Endothelium is a major site for both CD73 mediated production of adenosine and its cytoprotective effect. Nucleotides (predominantly ATP or ADP) that could be released from different cells via controlled specific of unspecific mechanisms constitute a major source of substrate for adenosine production via CD73. Direct effects of extracellular nucleotides (mediated by P2 receptors) are typically opposite to adenosine P1 mediated activities. Retention of nucleotides and decreased adenosine production due to loss of CD73 function may have negative implications and could be important cause of various pathologies. Protective role of CD73 was indicated in ectopic calcification, atherosclerosis, rejection after xenotransplantation and thrombosis. Reduced activity of CD73 due to lymphocyte contact with endothelium increases its permeability that leads to enhanced leukocyte transmigration. Upregulation of endothelial CD73 may therefore be protective in a number of cardiovascular pathologies. Such effect has been confirmed for some common drugs such as statins and it could be part of its pleiotropic portfolio. Activation of CD73 could be a new target for specific treatment strategy that in particular will enhance endothelial protection. Copyright © 2015. Published by Elsevier Urban & Partner Sp. z o.o.

Targeting Cancer-Derived Adenosine: New Therapeutic Approaches

Article

Full-text available

Jul 2014

Unlabelled: CD73 generation of immunosuppressive adenosine within the hypoxic tumor microenvironment causes dysregulation of immune cell infiltrates, resulting in tumor progression, metastases, and poor disease outcomes. Therapies targeted toward the adenosinergic pathway, such as antibodies targeting CD73 and CD39, have proven efficacy in mouse tumor models; however, humanized versions are only in preliminary development. In contrast, A(2A) adenosine receptor antagonists have progressed to late-stage clinical trials in Parkinson disease, yet evidence of their role in oncology is limited. This review will compare the merits and challenges of these therapeutic approaches, identifying tumor indications and combinations that may be fruitful as they progress to the clinic. Significance: High concentrations of immunosuppressive adenosine have been reported in cancers, and adenosine is implicated in the growth of tumors. This brief review delineates the current treatment strategies and tumor subtypes that will benefit from targeting adenosinergic pathways, alone or in combination with contemporary approaches to cancer treatment.

A 2A blockade enhances anti-metastatic immune responses

Article

Full-text available

Dec 2013

The specific targeting of tumor-elicited immunosuppression is a promising strategy for the treatment of cancer. We have recently demonstrated that targeting the immunosuppressive pathway mediated by CD73-derived adenosine through the blockade of A2A/A2B adenosine receptors significantly reduced the metastatic potential of CD73(+) breast carcinomas and melanomas via both immunological and non-immunological mechanisms.

HIV "Elite Controllers" Are Characterized by a High Frequency of Memory CD8+CD73+ T Cells Involved in the Antigen-Specific CD8+ T-cell Response

Article

Full-text available

Dec 2013
J INFECT DIS

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by chronic immune activation and suppressed T-lymphocyte functions. Here we report that CD73, both a coactivator molecule of T cells and an immunosuppressive ecto-enzyme through adenosine production, is only weakly expressed by CD8+ T cells of HIV-infected patients and only partially restored after successful antiviral treatment. CD73 expression on CD8+ T cells correlates inversely with cell activation both ex vivo and in vitro. However, CD8+ T cells from HIV controllers (HICs), which spontaneously control HIV replication, express CD73 strongly, despite residual immune activation. Finally, we demonstrate that CD73 is involved in the HIV-specific CD8+ T-cell expansion. Thus, we show that CD73 is central to the functionality of HIV-specific CD8+ T cells and that the preservation of HIV-specific CD73+CD8+ T cells is a characteristic of HICs. These observations reveal a novel mechanism involved in the control of viral replication.

Blockade of A 2A receptors potently suppresses the metastasis of CD73 + tumors

Article

Full-text available

Aug 2013
P NATL ACAD SCI USA

CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A2A/A2B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.2 breast tumors) and when CD73 was ectopically expressed (B16F10 melanoma). A2A(-/-) mice were strongly protected against tumor metastasis, indicating that host A2A receptors enhanced tumor metastasis. A2A blockade enhanced natural killer (NK) cell maturation and cytotoxic function in vitro, reduced metastasis in a perforin-dependent manner, and enhanced NK cell expression of granzyme B in vivo, strongly suggesting that the antimetastatic effect of A2A blockade was due to enhanced NK cell function. Interestingly, A2B blockade had no effect on NK cell cytotoxicity, indicating that an NK cell-independent mechanism also contributed to the increased metastasis of CD73(+) tumors. Our results thus revealed that CD73 promotes tumor metastasis through multiple mechanisms, including suppression of NK cell function. Furthermore, our data strongly suggest that A2A or A2B antagonists may be useful for the treatment of metastatic disease. Overall, our study has potential therapeutic implications given that A2A/A2B receptor antagonists have already entered clinical trials in other therapeutic settings.

Graft-versus-Host Disease Is Enhanced by Selective CD73 Blockade in Mice

Article

Full-text available

Mar 2013
PLOS ONE

CD73 functions as an ecto-5'-nucleotidase to produce extracellular adenosine that has anti-inflammatory and immunosuppressive activity. We here demonstrate that CD73 helps control graft-versus-host disease (GVHD) in mouse models. Survival of wild-type (WT) recipients of either allogeneic donor naïve CD73 knock-out (KO) or WT T cells was similar suggesting that donor naïve T cell CD73 did not contribute to GVHD. By contrast, donor CD73 KO CD4(+)CD25(+) regulatory T cells (Treg) had significantly impaired ability to mitigate GVHD mortality compared to WT Treg, suggesting that CD73 on Treg is critical for GVHD protection. However, compared to donor CD73, recipient CD73 is more effective in limiting GVHD. Pharmacological blockade of A2A receptor exacerbated GVHD in WT recipients, but not in CD73 KO recipients, suggesting that A2 receptor signaling is primarily implicated in CD73-mediated GVHD protection. Moreover, pharmacological blockade of CD73 enzymatic activity induced stronger alloreactive T cell activity, worsened GVHD and enhanced the graft-versus-leukemia (GVL) effect. These findings suggest that both donor and recipient CD73 protects against GVHD but also limits GVL effects. Thus, either enhancing or blocking CD73 activity has great potential clinical application in allogeneic bone marrow transplants.

Dual, enzymatic and non-enzymatic, function of ecto-5'-nucleotidase (eN, CD73) in migration and invasion of A375 melanoma cells

Article

Nov 2012
ACTA BIOCHIM POL

Ecto-5'-nucleotidase (eN, CD73) mediates extracellular adenosine production from 5'-AMP. Non-enzymatic functions of eN have also been reported. The aim of the study was to investigate the role of ecto-5'-nucleotidase in aggressive melanoma behaviour. Analysis of the involvement of eN in adhesion, migration and invasion revealed eN functions unknown to date. We found that following eN blockade by concanavalin A, the strength of adhesion to different ECM proteins was not altered, but at the same time the invasion ability of the cells was decreased. On the other hand, knocking down eN in melanoma cells did not influence cell invasion but abolished their migration on tenascin C (TnC). Ecto-5'-nucleotidase seems to fulfil a more distinct role as a receptor than as an enzyme in the cell interaction and mobility on TnC. Ecto-5'-nucleotidase activates also focal adhesion kinase and enhances the formation of complexes upon cell adhesion to TnC. All these observations prove that an eN-TnC complex is involved in cell migration and invasion and thus in the regulation of melanoma progression.

CD73-Generated Adenosine: Orchestrating the Tumor-Stroma Interplay to Promote Cancer Growth

Article

Full-text available

Oct 2012
J BIOMED BIOTECHNOL

Despite the coming of age of cancer immunotherapy, clinical benefits are still modest. An important barrier to successful cancer immunotherapy is that tumors employ a number of mechanisms to facilitate immune escape, including the production of anti-inflammatory cytokines, the recruitment of regulatory immune subsets, and the production of immunosuppressive metabolites. Significant therapeutic opportunity exists in targeting these immunosuppressive pathways. One such immunosuppressive pathway is the production of extracellular adenosine by CD73, an ectonucleotidase overexpressed in various types of cancer. We hereafter review the biology of CD73 and its role in cancer progression and metastasis. We describe the role of extracellular adenosine in promoting tumor growth through paracrine and autocrine action on tumor cells, endothelial cells, and immune cells.

Altered Fate of Tendon-Derived Stem Cells Isolated from a Failed Tendon-Healing Animal Model of Tendinopathy

Article

Full-text available

Oct 2012
STEM CELLS DEV

We hypothesized that altered fate of tendon-derived stem cells (TDSCs) might contribute to chondro-ossification and failed healing in the collagenase-induced (CI) tendon injury model. This study aimed to compare the yield, proliferative capacity, immunophenotypes, senescence and differentiation potential of TDSCs isolated from healthy (HT) and CI tendons. TDSCs were isolated from CI and healthy SD rat patellar tendons. The yield, proliferative capacity, immunophenotypes and senescence of TDSCs (CI) and TDSCs (HT) were compared by colony-forming unit assay, BrdU assay, flow cytometry and β-galactosidase activity assay, respectively. Their osteogenic and chondrogenic differentiation potentials and mRNA expression of tendon-related markers were compared using standard assays. More TDSCs which showed lower proliferative potential and higher cellular senescence were present in the CI patellar tendons compared to HT tendons. There was higher ALP activity and mineralization in TDSCs (CI) in both basal and osteogenic media. More chondrocyte-like cells and higher proteoglycan deposition, Sox9 and collagen type II expression were observed in TDSCs (CI) pellets upon chondrogenic induction. There was higher protein expression of Sox9 but lower mRNA expression of Col1a1, Scx and Tnmd in TDSCs (CI) in basal medium. In conclusion, TDSCs (CI) showed altered fate, higher cellular senescence but lower proliferative capacity compared to TDSCs (HT), which might contribute to pathological chondro-ossification and failed tendon healing in this animal model.

The evaluation of CD39, CD73, and HIF-1 α expression besides their related miRNAs in PBMCs of women with Recurrent Pregnancy Loss

Article

Mar 2023
J REPROD IMMUNOL

The molecular mechanisms involved in the pathogenesis of recurrent pregnancy loss (RPL) are not completely recognized. The present study aimed to assess the molecules associated with ATP catabolism and hypoxia besides their related miRNAs in patients with RPL. The frequency of Th17 and Treg cells in PBMCs of RPL women and healthy pregnant women were evaluated with Flow cytometry. The expression levels of CD39, CD73, and Hypoxia-inducible factor-alpha (HIF-1α), miR-18a, miR-30a, and miR-206 in PBMCs of two groups were measured with real-time PCR and western blotting. Then, serum levels of IGF-1, TGF-β, and HIF-1α were measured by ELISA. Our results indicated a higher (p = 0.0002) and lower (p < 0.0001) frequency of Th17 and Treg lymphocytes in RPL women, respectively. The expression level of CD39 decreased in PBMCs of RPL women whereas the level of CD73 and HIF-α increased (p = 0.0010, 0.0023, 0.0006 respectively). The results of CD39 and CD37 were also confirmed by protein analysis (p = 0.0047, 0.0364 respectively). Almost, the same results for CD39 and CD73 expression at mRNA and protein levels were observed in isolated Treg cells. Moreover, we found the higher expression of miR-206 and miRNA-30a (p = 0.0038, 0.0123), but the lower expression of miRNA-18a (p = 0.0101) in RPL. The concentration level of IGF-1, and TGF-β reduced (p = 0.0017, 0.0065 respectively) while the level of HIF-α elevated (p = 0.0235) in serum samples of RPL. In conclusion, we observed the dysregulation of molecules that are involved in ATP catabolism and hypoxia, including CD39, CD73, and HIF-1a which is related to miR-18a, miR-30a, and miR-206 change in RPL women. It may be potentially used for RPL prognosis by more comprehensive future studies.

The adenosine pathway in immuno-oncology

Article

Jun 2020

Cancer immunotherapy based on immune-checkpoint inhibition or adoptive cell therapy has revolutionized cancer care. Nevertheless, a large proportion of patients do not benefit from such treatments. Over the past decade, remarkable progress has been made in the development of ‘next-generation’ therapeutics in immuno-oncology, with inhibitors of extracellular adenosine (eADO) signalling constituting an expanding class of agents. Induced by tissue hypoxia, inflammation, tissue repair and specific oncogenic pathways, the adenosinergic axis is a broadly immunosuppressive pathway that regulates both innate and adaptive immune responses. Inhibition of eADO-generating enzymes and/or eADO receptors can promote antitumour immunity through multiple mechanisms, including enhancement of T cell and natural killer cell function, suppression of the pro-tumourigenic effects of myeloid cells and other immunoregulatory cells, and promotion of antigen presentation. With several clinical trials currently evaluating inhibitors of the eADO pathway in patients with cancer, we herein review the pathophysiological function of eADO with a focus on effects on antitumour immunity. We also discuss the treatment opportunities, potential limitations and biomarker-based strategies related to adenosine-targeted therapy in oncology.

Cell type- And tissue-specific functions of ecto-5=-nucleotidase (CD73)

Article

Aug 2019

CD73 is a ubiquitously-expressed glycosylphosphatidylinositol (GPI)-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate (AMP) to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, CD73 has many protective physiological functions that need to be taken into account for new targeted therapies. This review covers CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene ( NT5E) cause the rare disease 'arterial calcifications due to deficiency of CD73' (ACDC). Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. For example, in the context of the central nervous system (CNS), CD73 is anti-nociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, but this is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia/reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury are less clear. For example, CD73 has a promoting role in liver fibrosis, but protects in the case of lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.

Complex regulation of ecto-5′-nucleotidase/CD73 and A2AR-MEdiated adenosine signaling at neurovascular unit: A link between acute and chronic neuroinflammation

Article

Apr 2019

Nadezda Nedeljkovic

The review summarizes available data regarding the complex regulation of CD73 at the neurovascular unit (NVU) during neuroinflammation. Based on available data we propose the biphasic pattern of CD73 regulation at NVU, with an early attenuation and a postponed up-regulation of CD73 activity. Transient attenuation of CD73 activity on leukocyte/vascular endothelium and leukocyte/ astrocyte surface, required for the initiation of a neuroinflammatory response, may be effectuated either by catalytic inhibition of CD73 and/or by shedding of the CD73 molecule from the cell surface, while postponed induction of CD73 is effectuated by transcriptional upregulation of Nt5e and posttranslational modifications. Neuroinflammatory conditions are also associated with significant enhancement and gain-of-function of A2AR-mediated adenosine signaling. However, in contrast to the temporary prevalence of A2AR over A1R signaling during an acute inflammatory response, prolonged induction of A2AR and resulting perpetual CD73/A2AR coupling may be a contributing factors in the transition between acute and chronic neuroinflammation. Thus, pharmacological targeting of the CD73/A2AR axis may attenuate inflammatory response and ameliorate neurological deficits in chronic neuroinflammatory conditions.

Immunomodulation of Tumor Vessels: It Takes Two to Tango

Article

Aug 2018
TRENDS IMMUNOL

The density of intratumoral CD8+ T cells predicts patient survival and responsiveness to immunotherapy. Effector T cell infiltration in turn is controlled by the tumor vasculature which co-evolves together with an immune-suppressive environment. At the T cell-vascular interface, endothelial cells actively suppress T cell trafficking and function. Conversely, forced activation, normalization, and differentiation of tumor vessels into high endothelial venule entrance portals for lymphocytes can facilitate T cell extravasation. Emerging evidence demonstrates that this process is not exclusively controlled by the endothelium. Indeed, tumor vasculature and CD4+ and/or CD8+ T cells may regulate each other: increasing local effector T cell numbers or re-invigorating pre-existing T cells via immune checkpoint blockade can directly affect the vasculature. A deeper understanding of the orchestration and duration of this reciprocal relationship may help shape the design of future immunotherapies.

Brain cortical injury induces changes in peripheral lymphocyte ectonucleotidase activities

Article

Jan 2013

Injury and other pathological conditions induce a massive release of ATP and ADP that initiate an immune response. Extracellular nucleotides are degraded by ectonucleotidases: enzymes from E-NTPDase and E-NPP families sequentially hydrolyze ATP and ADP to AMP, which is further hydrolyzed by ecto-5'-nucleotidase to adenosine that exerts suppressive effects on immune cells. We investigated the ectonucleotidase activities of peripheral lymphocytes at different post-injury times after an unilateral brain injury in the rat. Significant and dynamic changes in the lymphocytic ecto-nucleotidase activities were obtained. ATP-and ADP-hydrolysis changes, together with their calculated ratios, indicate the major contribution of E-NTPDase 1 and its comparable upregulation between sham operation and injury. AMP hydrolysis changes were more brain-injury specific, with a longer-lasting lymphocytic response induced by cortical stab injury (CSI). In summary, CSI and sham operation induce the upregulation of the whole enzyme chain for adenine nucleotide hydroly-sis in lymphocytes, suggesting an important roles of ectonucleotidases in the course of recovery after brain injury.

Targeting CD73 and downstream adenosine receptor signaling in triple-negative breast cancer

Article

Full-text available

May 2014
EXPERT OPIN THER TAR

Introduction: Despite significant improvements in diagnosis and therapy over the past 20 years, breast cancer remains a worldwide public health issue. In particular, triple negative breast cancer (TNBC), a subset of very aggressive breast tumors, is associated with a poor prognosis and has very few efficient therapeutic options. The ectonucleotidase CD73 has recently emerged as a promising new target for TNBC in preclinical models. Pharmacological targeting of CD73 and downstream adenosine A2A/A2B receptor signaling is currently an active field of research that could lead to the development of new cancer therapeutics, including options against TNBC. Areas covered: This article reviews the basic structural and molecular features of CD73 and its role in the development of cancer, with a particular focus on CD73's role in the biology of TNBC. Expert opinion: It was recently demonstrated that CD73 expression in TNBC is associated with worse clinical outcomes and increased resistance to anthracycline chemotherapy. Targeted blockade of the CD73/A2A axis has been shown to impair various aspects of tumorigenesis and displays synergism with other anti-cancer treatments in preclinical studies. Hence, we strongly argue for the development of CD73 inhibitors and for the repositioning of A2A antagonists in cancer.

Ectoenzymes in leukocyte migration and their therapeutic potential

Article

Mar 2014
SEMIN IMMUNOPATHOL

Inflammation causes or accompanies a huge variety of diseases. Migration of leukocytes from the blood into the tissues, in the tissues, and from the tissues to lymphatic vasculature is crucial in the formation and resolution of inflammatory infiltrates. In addition to classical adhesion and activation molecules, several other molecules are known to contribute to the leukocyte traffic. Several of them belong to ectoenzymes, which are cell surface molecules having catalytically active sites outside the cell. We will review here how several ectoenzymes present on leukocytes or endothelial cell surface function as adhesins and/or modulate the extravasation cascade through their enzymatic activities. Moreover, their therapeutic potential as immune modulators in different experimental inflammation models and in clinical trials will be discussed.

Immunity, inflammation and cancer: A leading role for adenosine

Article

Full-text available

Nov 2013

Cancer is a complex disease that is dictated by both cancer cell-intrinsic and cell-extrinsic processes. Adenosine is an ancient extracellular signalling molecule that can regulate almost all aspects of tissue function. As such, several studies have recently highlighted a crucial role for adenosine signalling in regulating the various aspects of cell-intrinsic and cell-extrinsic processes of cancer development. This Review critically discusses the role of adenosine and its receptors in regulating the complex interplay among immune, inflammatory, endothelial and cancer cells during the course of neoplastic disease.

Nucleotidases and Nucleoside Analog Cytotoxicity

Article

Jan 2006

Nucleoside analogs are an important part of therapeutic strategies in a broad range of diseases, especially cancer and viral infections. Most nucleoside analogs need to be phosphorylated to attain full clinical potency; thus, knowledge of the metabolism of this class of drugs is required to improve their clinical use. The 5′-nucleotidases are a family of enzymes that catalyze the final dephosphorylation step of nucleotides in the cell and, by opposing the activation step catalyzed by nucleoside kinases, initiate subsequent purine and pyrimidine catabolism. They also catalyze a critical step in nucleotide analog degradation; therefore, their expression and regulation in various tissues will likely have an impact on a nucleoside drug’s half-life in the human body. Numerous studies in vitro and in vivo indicate that increased expression of 5′_nucleotidase may decrease nucleoside analog activation and thereby contribute to drug resistance. Because cloned 5′_nucleotidases have been described in human tissues, it is not always possible to assess which particular 5′-nucleotidase is important in nucleoside drug catabolism. In this chapter, we review the properties of all cloned 5′-nucleotidases and the important role of these enzymes in nucleoside analog metabolism and clinical resistance.

Opportunities and challenges for anti-CD73 cancer therapy

Article

Sep 2012
IMMUNOTHERAPY-UK

Bin Zhang

Applicability of microplate assay coupled to Fiske–Subbarow reducer for the determination of phosphorous produced by in vivo human lymphocytes: PKC is probably cross talking with ecto 5′-nucleotidase

Article

Aug 2005
MICROCHEM J

In this research, the phorbol ester, phorbol 12-myristate 13-acetate (PMA), was used to assess the effect of protein kinase C (PKC) activation on the specific activity of ecto-5′-nucleotidase (eNT) in human lymphocytes. PMA mimics the effects of diacylglycerol, a natural compound released by the hydrolysis of the glycosilphosphatidilinositol (GPI) moiety, in activating PKC. In order to evaluate the activity of eNT in living lymphocytes, a micro-assay method was established with a low detection limit for inorganic phosphate (Pi) of 0.94 nmol Pi assay−1. The dephosphorylation of Adenosine monophosphate (AMP) by functional lymphocytes was evaluated and the contribution of the eNT activity was calculated by its inhibition with the specific eNT inhibitor α,β-methylene ADP (MADP) and the use of the broad spectrum phosphatases inhibitor (but not eNT), levamisole. Under the conditions tested, we obtained an AMPase value of 8.05±4.4 nmol Pi million cells−1 h−1. The addition of MADP to the incubation media decreased the AMPase activity to 2.43±0.9 nmol Pi million cells−1 h−1 (p<0.05). On the other hand, when lymphocytes were incubated with PMA, an increase of 182% in the AMPase activity was observed. However, the addition of levamisole inhibited the AMPase activity by about 17%, while the co-incubation of cells with PMA and levamisole reduced only an 8% of the total PMA-increased AMPase activity. These results show that (1) a non-radioactive micro-method can be used to assess the Pi production in living cells; (2) the obtained data strongly suggest that eNT is the main ecto-enzyme present on the surface of circulating lymphocytes responsible for the hydrolysis of extracellular AMP; and (3) that PKC is cross talking with eNT.

Cellular function and molecular structure of ecto-nucleotidases

Article

Full-text available

May 2012
PURINERG SIGNAL

Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ecto-nucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.

Airas L, Niemela J, Salmi M, Puurunen T, Smith DJ, Jalkanen S.. Differential regulation and function of CD73, a glycosyl-phosphatidylinositol-linked 70-kD adhesion molecule, on lymphocytes and endothelial cells. J Cell Biol 136: 421-431

Article

Full-text available

Jan 1997
J CELL BIOL

CD73, otherwise known as ecto-5-nucleoti-dase, is a glycosyl-phosphatidylinositol–linked 70-kD molecule expressed on different cell types, including vascular endothelial cells (EC) and certain subtypes of lymphocytes. There is strong evidence for lymphocyte CD73 having a role in several immunological phenomena such as lymphocyte activation, proliferation, and adhesion to endothelium, but the physiological role of CD73 in other cell types is less clear. To compare the biological characteristics of CD73 in different cell types, we have studied the structure, function, and surface modulation of CD73 on lymphocytes and EC. CD73 molecules on lymphocytes are shed from the cell surface as a consequence of triggering with an anti-CD73 mAb, mimicking ligand binding. In contrast, triggering of endothelial CD73 does not have any effect on its expression. Lymphocyte CD73 is susceptible to phosphatidylinositol phospholipase, whereas only a small portion of CD73 on EC could be removed by this enzyme. Furthermore, CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon mAb triggering, whereas triggering of lymphocyte CD73 can induce tyrosine phosphorylation. Despite the functional differences, CD73 molecules on lymphocytes and EC were practically identical structurally, when studied at the protein, mRNA, and cDNA level. Thus, CD73 is an interesting example of a molecule which lacks structural variants but yet has a wide diversity of biological functions. We suggest that the ligand-induced shedding of lymphocyte CD73 represents an important and novel means of controlling lymphocyte– EC interactions.

A novel LFA-1 activation epitope maps to the I domain

Article

Full-text available

Mar 1993

Robert Clive Landis

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA-1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM-1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA-1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.

Interaction of leukocyte integrins with ligand is necessary but not sufficient for function

Article

Full-text available

Apr 1992

The leukocyte integrins (CD11/CD18 or beta 2-type integrins) are expressed exclusively on leukocytes and participate in many adhesion-dependent functions (Arnaout, M.A. 1990. Blood. 75:1037-1050; Springer, T. A. 1990. Nature. (Lond.) 346:425-434; Dustin, M. L., and T. S. Springer. 1991. Annu. Rev. Immunol. 9:27-66). The avidity of leukocyte integrin binding to their ligands or counter-receptors is dependent upon response to intracellular signals (Wright, S. D., and B. C. Meyer. 1986. J. Immunol. 136:1759-1764; Dustin, M. A., and T. S. Springer. 1989. Nature (Lond.). 341:619-624). We have investigated the effects of a novel mAb (mAb 24) which defines a leukocyte integrin alpha subunit epitope that is Mg(2+)-dependent and may be used as a "reporter" of the activation state of these receptors (Dransfield, I., and N. Hogg. 1989. EMBO (Eur. Mol. Biol. Organ) J. 8:3759-3765; Dransfield, I., A.-M. Buckle, and N. Hogg. 1990. Immunol. Rev. 114:29-44; Dransfield, I., C. Cabañas, A. Craig, and N. Hogg. 1992. J. Cell Biol.) Data is presented to show that this mAb inhibits monocyte-dependent, antigen-specific T cell proliferation and IL-2-activated natural killer cell assays which are both dependent on lymphocyte function-associated antigen-1 (LFA-1), and complement receptor type 3 (CR3)-mediated neutrophil chemotaxis to f-Met-Leu-Phe. This inhibitory effect is not caused by the prevention of receptor/ligand binding because LFA-1/ICAM-1, LFA-1/ICAM-2,3 and CR3/iC3b interactions are, under activating conditions, promoted rather than blocked by mAb 24. As it does not interfere with mitogen-stimulated T cell proliferation, it is unlikely that mAb 24 transduces a "negative" or antiproliferative signal to the T cells to which it is bound. Using a model system of transient activation of LFA-1, we have found that mAb 24 prevents "deadhesion" of receptor/ligand pairs, possibly locking leukocyte integrins in an "active" conformation. It is speculated that inhibition of leukocyte integrin function by this mAb reflects the necessity for dynamic leukocyte integrin/ligand interactions.

Koopman G, van Kooyk Y, de Graaff M, Meyer CJ, Figdor CG & Pals ST. Triggering of the CD44 antigen on T lymphocytes promotes T cell adhesion through the LFA-1 pathway. J Immunol145: 3589-3593

Article

Full-text available

Jan 1991

The CD44 molecule, a molecule which has been previously known as Hermes, Pgp-1, extracellular matrix receptor III, and In(Lu)-related p80, is currently thought to be involved in several steps of normal immune cell function, including lymphocyte adhesion to high endothelial venules and to the extracellular matrix and T cell activation. We now demonstrate that triggering of CD44 on T lymphocytes by anti-CD44 mAb promotes cell adhesion. The induced homotypic adhesion is mediated by lymphocyte function-associated antigen-1 (LFA-1), because it was inhibited by anti-LFA-1 antibodies and not by anti-LFA-3 antibodies. This notion is supported by the temperature and Mg2+ dependence which is characteristic of LFA-1-mediated adhesion. Moreover, the sensitivity of CD44-induced adhesion to AMG and H7, which both prevent the activation of protein kinase C, and to cytochalasin B, which inhibits microfilament formation, suggests that the activation of the LFA-1 pathway via CD44 involves protein kinase C activation and requires an intact cytoskeleton.

T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1

Article

Full-text available

Nov 1989

Effective interaction between T cells and their targets requires that recognition of specific antigen be coordinated with increased cell-cell adhesion. We show that antigen-receptor cross-linking increases the strength of the adhesion mechanism between lymphocyte function-associated molecule-1 (LFA-1) and intercellular adhesion molecules (ICAMs), with intracellular signals transmitted from the T-cell antigen receptor to the LFA-1 adhesion molecule. The increase in avidity is rapid and transient, providing a dynamic mechanism for antigen-specific regulation of lymphocyte adhesion and de-adhesion.

van Kooyk, Y., van de Wiel-van Kemenade, P., Weder, P., Kuijpers, T.W. & Figdor, C.G. Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes. Nature 342, 811-813

Article

Full-text available

Jan 1990

The lymphocyte function-associated molecule LFA-1 (CD11a/CD18) plays a key part in lymphocyte adhesion. Lymphocytes do not adhere spontaneously; activation of protein kinase C (PKC) by phorbol esters, however, gives rise to strong LFA-1-dependent adhesion, indicating that activation of LFA-1 is required to induce cell adhesion. We have now investigated whether the functionally important CD2 and CD3 surface structures on T lymphocytes are involved in the activation of LFA-1. The stimulation of these molecules, which causes activation of PKC, strongly promoted LFA-1-dependent adhesion. Furthermore, we demonstrate by using cells from an LFA-1-deficient patient that this enhanced lymphocyte adhesion is caused by activation of the LFA-1 molecule and not by activation of its ligands. LFA-1 was persistently activated by triggering through CD2 but only transiently by triggering through CD3. We postulate that CD2 and CD3 can differentially regulate the affinity of LFA-1 for its ligands by modulating its molecular conformation through PKC-dependent mechanisms.

Extracellular Ca2+ modulates leukocyte function-associated antigen-1 cell surface distribution on T lymphocytes and consequently affects cell adhesion

Article

Full-text available

Apr 1994

Transition of leukocyte function-associated antigen-1 (LFA-1), from an inactive into an activate state depends on the presence of extracellular Mg2+ and/or Ca2+ ions. Although Mg2+ is directly involved in ligand binding, the role of Ca2+ in LFA-1 mediated adhesion remained obscure. We now demonstrate that binding of Ca2+, but not Mg2+, directly correlates with clustering of LFA-1 molecules at the cell surface of T cells, thereby facilitating LFA-1-ligand interaction. Using a reporter antibody (NKI-L16) that recognizes a Ca(2+)-dependent epitope on LFA-1, we found that Ca2+ can be bound by LFA-1 with different strength. We noticed that weak binding of Ca2+ is associated with a dispersed LFA-1 surface distribution on T cells and with non-responsiveness of these cells to stimuli known to activate LFA-1. In contrast, stable binding of Ca2+ by LFA-1 correlates with a patch-like surface distribution and vivid ligand binding after activation of LFA-1. Mg(2+)-dependent ligand binding does not affect binding of Ca2+ by LFA-1 as measured by NKI-L16 expression, suggesting that Mg2+ binds to a distinct site, and that both cations are important to mediate adhesion. Only Sr2+ ions can replace Ca2+ to express the L16 epitope, and to induce clustering of LFA-1 at the cell surface. We conclude that Ca2+ is involved in avidity regulation of LFA-1 by clustering of LFA-1 molecules at the cell surface, whereas Mg2+ is important in regulation of the affinity of LFA-1 for its ligands.

Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes

Article

Full-text available

May 1995

The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.

CD73 is involved in lymphocyte binding to the endothelium: characterization of lymphocyte-vascular adhesion protein 2 identifies it as CD73

Article

Full-text available

Dec 1995

We have recently described a monoclonal antibody (mAb) 4G4 recognizing a 70-kD molecule constitutively expressed on human endothelial cells and on subpopulations of lymphocytes. We showed that this molecule, which we named lymphocyte-vascular adhesion protein 2 (L-VAP-2), mediates lymphocyte adhesion to cultured endothelial cells. Protein sequencing of tryptic peptides from immunoaffinity-purified L-VAP-2 revealed sequence identity between L-VAP-2 and CD73 (ecto-5'-nucleotidase, E.C.3.1.3.5), and COS cells transfected with a CD73 cDNA were positively stained with the mAb 4G4, which recognizes L-VAP-2. mAb 4G4 was also able to partially inhibit the ecto-5'-nucleotidase activity of peripheral blood lymphocytes. Moreover, cross-precipitation studies performed with mAb 4G4 and a CD73 workshop mAb 1E9 showed that these two antibodies recognize the same molecule. Since the tissue distribution and biochemical characteristics of the two molecules are also similar, we conclude that L-VAP-2 and CD73 are the same glycoprotein. Adhesion experiments showed significantly increased binding of freshly isolated lymphocytes to COS cells transfected with a CD73 cDNA, as compared to mock-transfected COS cells, and binding of lymphocytes to CD73-expressing COS cells was inhibited by the presence of mAb 4G4 in the adhesion assay. CD73 is a glycosyl phosphatidylinositol-linked molecule previously shown to have a cosignalling role in T lymphocyte proliferation. Our data suggest that it also has a function in mediating lymphocyte adhesion to the endothelium.

A novel LFA-1 activation epitope maps to the I domain

Article

Full-text available

Apr 1993

T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1

Article

Full-text available

Apr 1996

Many leukocyte integrins require activation before they can adhere to their ligands. For example, stimulation of T cells enables the integrin LFA-1 to bind to ligand. This study compares two well known protocols for inducing T cell LFA-1 mediated adhesion to intercellular adhesion molecule-1 (ICAM)-1. We how that treatment with high concentrations of the divalent cation Mg2+ induces a high affinity state of LFA-1, which is reflected in the binding of soluble ICAM-1 and correlates with the expression of the epitope recognized by mAb 24. The second stimulation protocol with the phorbol ester phorbol-12,13-dibutyrate (PDBu) does not induce a high affinity state of LFA-1, and in this situation, adhesion is dependent on cell spreading and intracellular events involving protein kinase C, [Ca2+]i, and actin polymerization. These low affinity LFA-1 receptors are responsible for the initial contact with immobilized ligand because, unlike the Mg2+-stimulated receptors, adhesion is not blocked by soluble ICAM-1. Finally, we used a third method of inducing LFA-1-mediated adhesion by stimulation of T cells through the TCR/CD3 complex. This procedure, which is considered to be a more physiologic trigger for LFA-1 activation, resembles the phorbol ester protocol in that high affinity LFA-1 receptors are not induced and cell adhesion depends on involvement of the cytoskeleton and cell spreading.

CD73 mediates adhesion of B cells to follicular dendritic cells

Article

Full-text available

Oct 1996

Lymphocyte-vascular adhesion protein-2 was recently identified as CD73. The CD73 molecule, otherwise known as ecto-5'-nucleotidase, is a lymphocyte maturation marker that is involved in intracellular signaling, and lymphocyte proliferation and activation. We now show that CD73, in addition to mediating lymphocyte binding to endothelial cells, also mediates adhesion between B cells and follicular dendritic cells (FDC), as a monoclonal antibody (MoAb) against CD73 inhibited the aggregation of isolated germinal center B cells and FDC in vitro. Cytocentrifuge preparations of isolated germinal center cells and two-color immunofluorescence stainings of different tonsillar B-cell populations show that CD73 is expressed on FDC and on small, recirculating IgD+ B cells, but only on a few B cells inside the germinal center. Thus, we propose that CD73 on FDC has an important role in controlling B cell-FDC interactions and B-cell maturation in germinal centers.

GlyCAM-1, a physiologic ligand for L-selectin, activates beta 2 integrins on naive peripheral lymphocytes

Article

Full-text available

Nov 1996

Naive T cells are selectively recruited from the blood into peripheral lymph nodes during lymphocyte recirculation. L-selectin, a lectin-like receptor, mediates the initial attachment of lymphocytes to high endothelial venules (HEV) in lymph nodes. A subsequent step involving the activation of beta 2 integrins has been proposed to facilitate firm adhesion, but the activating signals are poorly understood. We report here that either antibody-mediated cross-linking of L-selectin on human lymphocytes or treatment of the cells with GlyCAM-1, an HEV-derived, secreted ligand for L-selectin, stimulates their binding to ICAM-1 through the beta 2 integrin pathway. Furthermore, GlyCAM-1 causes the rapid expression of a neoepitope on beta 2 integrins associated with a high-avidity state. Naive (CD45RA+), but not memory (CD45R0+) lymphocytes, respond to L-selectin cross-linking or GlyCAM-1 treatment. Thus, the complexing of L-selectin by specific ligands may provide key signals to naive lymphocytes, contributing to their selective recruitment into peripheral lymphoid organs.

Differential Regulation and Function of CD73, a Glycosyl-Phosphatidylinositol–linked 70-kD Adhesion Molecule, on Lymphocytes and Endothelial Cells

Article

Full-text available

Feb 1997

CD73, otherwise known as ecto-5'-nucleotidase, is a glycosyl-phosphatidylinositol-linked 70-kD molecule expressed on different cell types, including vascular endothelial cells (EC) and certain subtypes of lymphocytes. There is strong evidence for lymphocyte CD73 having a role in several immunological phenomena such as lymphocyte activation, proliferation, and adhesion to endothelium, but the physiological role of CD73 in other cell types is less clear. To compare the biological characteristics of CD73 in different cell types, we have studied the structure, function, and surface modulation of CD73 on lymphocytes and EC. CD73 molecules on lymphocytes are shed from the cell surface as a consequence of triggering with an anti-CD73 mAb, mimicking ligand binding. In contrast, triggering of endothelial CD73 does not have any effect on its expression. Lymphocyte CD73 is susceptible to phosphatidylinositol phospholipase, whereas only a small portion of CD73 on EC could be removed by this enzyme. Furthermore, CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon mAb triggering, whereas triggering of lymphocyte CD73 can induce tyrosine phosphorylation. Despite the functional differences, CD73 molecules on lymphocytes and EC were practically identical structurally, when studied at the protein, mRNA, and cDNA level. Thus, CD73 is an interesting example of a molecule which lacks structural variants but yet has a wide diversity of biological functions. We suggest that the ligand-induced shedding of lymphocyte CD73 represents an important and novel means of controlling lymphocyte-EC interactions.

Functional rafts in cell membranes

Article

Full-text available

Jul 1997

A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. It is proposed that these rafts function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.

Ly-6C regulates endothelial adhesion and homing of CD8(+) T cells by activating integrin-dependent adhesion pathways

Article

Full-text available

Jul 1997

Ly-6C belongs to the Ly-6 family of glycosyl phosphatidylinositol-anchored surface glycoproteins and is expressed on a subset of mature CD8(+) T cells. Ly-6C ligation can mediate T cell activation and causes interleukin 2 secretion in cytolytic T cell clones. We characterize herein a new mAb 1G7.G10 against Ly-6C that recognizes an epitope involved in lymphocyte adhesion and in lymphocyte homing. Pretreatment of lymph node lymphocytes and of purified CD8(+) T cells (but not of lymphocytes depleted of CD8(+) T cells) with 1G7.G10 reduced their in vitro binding to lymph node high endothelial venules by 28% and 34%, respectively. This effect was bypassed by cross-linking Ly-6C molecules with 1G7.G10 and a second-step antibody. The in vivo homing of (donor) CD8(+) T lymphocytes to lymph nodes was reduced by Ly-6C blocking with 1G7. G10 (whole antibody) or with its fragments [F(ab) or F(ab)2] by 20% or by 32% and 48%, respectively. Cross-linking of Ly-6C in vitro induced very late antigen-4 and lymphocyte function-associated antigen 1-mediated aggregation of CD8(+) T cells, suggesting that ligand binding to Ly-6C leads to activation of integrins. This activation may facilitate homing of Ly-6C+ CD8(+) T cells in vivo.

Vascular Adhesion Protein 1 (VAP-1) Mediates Lymphocyte Subtype-specific, Selectin-independent Recognition of Vascular Endothelium in Human Lymph Nodes

Article

Full-text available

Aug 1997
J EXP MED

Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1-dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and alpha4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.

LFA-1–mediated Adhesion Is Regulated by Cytoskeletal Restraint and by a Ca-dependent Protease, Calpain

Article

Full-text available

Feb 1998
J CELL BIOL

The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca2+ mobilizers, ionomycin, 2, 5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)-CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.

Urokinase Receptor (CD87) Regulates Leukocyte Recruitment via 2 Integrins In Vivo

Article

Full-text available

Sep 1998
J EXP MED

The urokinase receptor (CD87; uPAR) is found in close association with beta 2 integrins on leukocytes. We studied the functional consequence of this association for leukocyte adhesion and migration. In vivo, the beta 2 integrin-dependent recruitment of leukocytes to the inflamed peritoneum of uPAR-deficient mice was significantly reduced as compared with wild-type animals. In vitro, beta 2 integrin-mediated adhesion of leukocytes to endothelium was lost upon removal of uPAR from the leukocyte surface by phosphatidyl-inositol-specific phospholipase C. Leukocyte adhesion was reconstituted when soluble intact uPAR, but not a truncated form lacking the uPA-binding domain, was allowed to reassociate with the cell surface. uPAR ligation with a monoclonal antibody induced adhesion of monocytic cells and neutrophils to vascular endothelium by six- to eightfold, whereas ligation with inactivated uPA significantly reduced cell-to-cell adhesion irrespective of the beta 2 integrin-stimulating pathway. These data indicate that beta 2 integrin-mediated leukocyte-endothelial cell interactions and recruitment to inflamed areas require the presence of uPAR and define a new phenotype for uPAR-deficient mice. Moreover, uPAR ligation differentially modulates leukocyte adhesion to endothelium and provides novel targets for therapeutic strategies in inflammation-related vascular pathologies.

Integrin Leukocyte Function-associated Antigen-1-mediated Cell Binding Can Be Activated by Clustering of Membrane Rafts

Article

Full-text available

Jan 2000

The leukocyte function-associated antigen-1 (LFA-1) integrin (CD11a/CD18) is an important adhesion molecule for lymphocyte migration and the initiation of an immune response. At the cell surface, LFA-1 activity can be regulated by divalent cations that enhance receptor affinity but also by membrane clustering induced by treatment of cells with substances such as phorbol esters. Membrane clustering leads to increased LFA-1 avidity. We report here that LFA-1-mediated binding of mouse thymocytes or activated T lymphocytes to intercellular adhesion molecule 1 can be rapidly induced by clustering of membrane rafts using antibodies to the glycosylphophatidylinositol-anchored molecule CD24 or cholera toxin (CTx). CD24 and CD18 were found to co-localize in rafts and cross-linking with CTx lead to enhanced LFA-1 clustering. We observed that disruption of raft integrity by lowering the membrane cholesterol content abolished the CTx and the phorbol 12-myristate 13-acetate-induced LFA-1 binding but left the ability to activate LFA-1 with Mg(2+)/EGTA unimpaired. In contrast to activation with Mg(2+)/EGTA, activation via raft clustering was dependent on PI3-kinase, required cytoskeletal mobility, and was accompanied by Tyr phosphorylation of a 18-kDa protein. Our results support the notion that rafts as preformed adhesion platforms could be important for the rapid regulation of lymphocyte adhesion.

Triggering of the CD44 antigen on T lymphocytes promotes T cell adhesion through the LFA-1 pathway.

Article

Dec 1990

CD73 mediates adhesion of B cells to follicular dendritic cells

Article

Sep 1996

Lymphocyte-vascular adhesion protein-2 was recently identified as CD73. The CD73 molecule, otherwise known as ecto-5′-nucleotidase, is a lymphocyte maturation marker that is involved in intracellular signaling, and lymphocyte proliferation and activation. We now show that CD73, in addition to mediating lymphocyte binding to endothelial cells, also mediates adhesion between B cells and follicular dendritic cells (FDC), as a monoclonal antibody (MoAb) against CD73 inhibited the aggregation of isolated germinal center B cells and FDC in vitro. Cytocentrifuge preparations of isolated germinal center cells and two- color immunofluorescence stainings of different tonsillar B-cell populations show that CD73 is expressed on FDC and on small, recirculating IgD+ B cells, but only on a few B cells inside the germinal center. Thus, we propose that CD73 on FDC has an important role in controlling B cell-FDC interactions and B-cell maturation in germinal centers.

Chemokines

Article

Aug 1997

Barrett Rollins

Extracellular Ca2+ modulates leukocyte function-associated antigen-1 cell surface distribution on T lymphocytes and consequently affects cell adhesion

Article

Mar 1994

Yvette van Kooyk

Interaction of leukocyte integrins with ligand is necessary but not sufficient for function

Article

Mar 1992

Ian Dransfield

GlyCAM-1, a physiologic ligand for L-selectin, activates beta 2 integrins on naive peripheral lymphocytes

Article

Oct 1996

S T Hwang

Calcium-activated neutral protease and its endogenous inhibitor. Activation at the cell membrane and biological function

Article

Sep 1987

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca2+ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.

Engagement of MHC class I molecules induces cell adhesion via both LFA-1-dependent and LFA-1-independent pathways

Article

May 1992

We here demonstrate that ligand binding to MHC class I molecules induces homotypic cell adhesion of lymphocytes and monocytes. mAb to beta 2-microglobulin caused sustained, largely LFA-1-independent adhesion whereas mAb to the MHC class I alpha H chain caused transient LFA-1-dependent adhesion. Both the protein kinase C inhibitor sphingosine and the tyrosine kinase inhibitor genistein abrogated MHC class I-mediated cellular adhesion. These results indicate that MHC class I molecules transduce signals that induce cell adhesion and suggest that interaction between MHC class I-restricted T cells and APC may result in reciprocal enhanced adhesiveness of these cells.

Zimmermann H5'-Nucleotidase: molecular structure and functional aspects. Biochem J 285(Pt 2): 345-365

Article

Aug 1992

H Zimmermann

5'-Nucleotidase activity in vertebrate tissues reflects the activity of four different forms of enzymes, a surface-located GPI-anchored ecto-form (e-N), a soluble form derived from it by cleavage of the membrane anchor (e-N(s)), and two cytosolic enzymes (c-N-I and c-N-II). Only the surface-located ecto-5'nucleotidase (e-N) has been characterized in molecular terms. It is expected that future sequence work will reveal that the closely related cytoplasmic forms c-N-I and c-N-II represent different genomic products. As yet the possibility of tissue-specific forms of 5'-nucleotidase as well as of splicing variants cannot be excluded. The e-N form reveals significant sequence identities with multispecific nucleotidases in bacteria, indicating common origin. Multispecific 5'-nucleotidases also occur in yeast but plant enzymes generally display marked substrate specificity. A characterization of the three-dimensional structure and of the active site would provide a basis for the interpretation of substrate specificities. The production of nucleosides is the general result of 5'nucleotidase activity. But the physiological implication of this catalytic process at thc cellular or tissue level varies. The intracellular enzymes c-N-I and c-N-II are involved in the catabolism of AMP and IMP and also of other mononucleotides and correspondingly produce the nucleoside. They are thought to be of particular importance for the hydrolysis of AMP and IMP in situations of low energy charge. The stimulatory effect of ATP and/or ADP on both affinity and rate seems to be of major physiological significance. A detailed analysis of the general biochemical properties with highly purified enzyme preparations is essential for setting the functional interpretations on a reliable basis. Furthermore, any general functional interpretation of the cytosolic enzymes requires both intimate knowledge of other cytosolic enzymes capable of hydrolysing 5'-mononucleotides as well as of the actual free concentrations of the metabolites concerned. Thc surface-located enzyme is involved in a number of functions which presumably do not mutually exclude each other. They rather supplement each other in their contribution to the physiology of cellular metabolism and tissue function. It is important to note that expression of ecto-5'-nucleotidase does not occur on all cells and can be developmentally regulated. Ecto-5'-nucleotidase is involved in the salvage of extracellular nucleotides. In addition, the enzyme has a major role in the control of tissue homeostasis. It effects the final step in the complete hydrolysis of thc extracellular messenger ATP. and, at the same time, produces adenosine as a further extracellular messenger. But this polysialyated GPI-anchored metalloprotein might not only function as an enzyme. The increased expression of the protein at the surface of activated cells and during certain stages of cell maturation, its capability to bind fibronectin as well as the presence of the HNK-I epitope in some tissues implies an involvement in cellular interactions such as cell recognition and cell adhesion or in cell matrix relations. This calls for a detailed analysis also of the carbohydrate chains, including possible developmental changes in the glycosylation pattern. Studies with lymphocytes even imply a function in signal transfer from the cell surface to the interior. Possibly a new mechanism for activation of intracellular messengers via GPI-anchored proteins will evolve. Finally, consensus should be sought on 5'-nucleotidase nomenclature. Since conventional enzyme nomenclature groups all forms described into one category the literature has become confusing. For example, at the bacterial level, periplasmic 5'nuclcotidase is multispecific and its relation to the vertebrate ecto-5'-nucleotidase can only be deduced from the primary structure. The cytolic 5'-nucleotidases have kinetic properties and physiological functions completely different from those of the ectoenzyme and the soluble form derived from it. It is suggested that current enzyme nomenclature is extended by introducing subclasses of EC 3.1.3.5: 1 for c-N-I, 2 for c-N-II, and 3 for e-N and e-N(s). 5'-Nucleotidases from bacteria and plants could then be grouped according to their sequence identities with one of the vertebrate enzymes.

Stimulation of B lymphocytes through surface Ig receptors induces LFA-1 and ICAM-1-dependent adhesion

Article

Jun 1991

Engagement of the surface Ig receptor with anti-IgM antibodies stimulates murine B lymphocytes to markedly increase their expression of the cell adhesion molecules ICAM-1 and LFA-1. Stimulated B cells display increased homotypic adhesiveness and form spontaneous heterotypic conjugates with T lymphocytes. This latter T-B cell interaction is further enhanced if T cells have been previously activated with phorbol esters. In all cases, the formation of cell-cell conjugates is dependent on LFA-1-ICAM-1-mediated interactions as assessed in mAb blocking experiments. B lymphocytes stimulated with anti-IgM display a marked increase in binding to ICAM-1-transfected L cells. This cell-cell interaction is inhibited by anti-LFA-1 mAb binding to the B lymphocyte. Together, these results demonstrate that there is an induction of both ICAM-1 and LFA-1 on stimulated B cells and a corresponding increase in the adhesiveness of these cells. These findings suggest that Ag binding to the surface Ig receptor could prepare a B lymphocyte for subsequent interaction with a T lymphocyte. This provides insight into how efficient T-B collaboration may occur between very infrequent Ag-specific lymphocytes.

Transmembrane signals generated through MHC class II, CD19, CD20, CD39, and CD40 antigens induce LFA-1-dependent and independent adhesion in human B cells through a tyrosine kinase-dependent pathway

Article

Jan 1992

Transmembrane signals generated following mAb binding to CD19, CD20, CD39, CD40, CD43, Leu-13 Ag, and HLA-D region gene products induced rapid and strong homotypic adhesion in a panel of human B cell lines. Lower levels of adhesion were also observed after engagement of CD21, CD22, and CD23. Adhesion induced by mAb binding to these Ag was identical with respect to the kinetics of adhesion and the morphology of the resulting cellular aggregates, and was distinct from PMA-induced adhesion in both of these properties. Adhesion was not observed in response to mAb binding to MHC class I, CD24, CD38, CD44, CD45RA, or CD72. In contrast to B cell lines, homotypic adhesion was not induced in two pre-B cell lines, in spite of their high level expression of CD19 and HLA-D. Adhesion induced by suboptimal stimulation through these surface Ag or by PMA was mediated primarily through LFA-1 and ICAM-1. However, optimal stimulation through CD19, CD20, CD39, CD40, and HLA-D induced strong homotypic adhesion that was not blocked by anti-LFA-1 mAb. This alternate pathway of adhesion was also observed in LFA-1-deficient cell lines and in the presence of EDTA, suggesting that adhesion was not mediated by integrins. Adhesion in response to engagement of cell-surface Ag was unaffected by H7 or genestein, but was significantly inhibited by staurosporine, and was completely ablated by sphingosine and herbimycin. These studies indicate that engagement of multiple B cell-surface molecules initiates a signal transduction cascade that involves tyrosine kinases but not protein kinase C, and which leads to homotypic adhesion. Furthermore, adhesion was mediated by at least two distinct cell-surface adhesion receptors: LFA-1/ICAM-1 and a heretofore unknown adhesion receptor.

Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity

Article

Jan 1992
CELL

Eugene C Butcher

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Human T cell activation: Synergy between CD73 (Ecto-5′-Nucleotidase) and signals delivered through CD3 and CD2 molecules

Article

Oct 1990

Interaction of the glycosyl phosphatidylinositol-linked differentiation Ag CD73 (ecto-5'-nucleotidase) with the CD73-specific mAb 1E9 generates agonistic signals that strongly synergize with T cell activation induced by CD3 and CD2 mAb. This synergy is observed only when 1E9 is immobilized on plastic and occurs in the absence of accessory cells or exogenous lymphokines. 1E9 induces a rapid (though transient) increase in [Ca2+]i in a minor proportion (20 to 30%) of unfractionated T lymphocytes (presumably CD73+ cells). However, this [Ca2+]i mobilization is not sufficient to fully activate CD73+ T cells, as shown by the requirement of additional signals such as CD3 or CD2 stimulation to initiate T cell proliferation. These signals cannot be substituted by the exogenous lymphokines, rIL-1, rIL-2, or rIL-4, or PMA (when T cells are rigorously depleted of monocytes). These data indicate that CD73 may behave as an accessory molecule regulating interactions between T cells and antigens or APC. A comparison was carried out with mAb 9.3 to the differentiation Ag CD28, another agonistic molecule with activating properties similar to CD73. Despite their lower percentage, the ability of CD73+ T cells to amplify the proliferation induced by CD3 or CD2 mAb was equivalent or even greater than that of CD28+ T cells. Once activated, CD73+ cells may recruit the remaining (CD73-) cells primed by CD3 or CD2 stimulation. Based on these data, we suggest that CD73+ T lymphocytes may be a specialized subset to amplify immune responses originated by the CD3 and CD2 activation pathways. Finally, the functional association between CD73 and integral membrane molecules like CD3 and CD2 suggests that GPI-anchored molecules may play a role in transmembrane signaling mediated by conventional second messenger systems.

Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5′-nucleotidase (CD73)

Article

Feb 1990

A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (ecto-5'-NT, CD73) was produced using highly purified human placental 5'-NT as immunogen. Antibodies 1E9.28.1 and 7G2.2.11 inhibit soluble placental 5'-NT activity and recognize lymphocyte CD73 in indirect immunofluorescence and immunoprecipitation assays. In addition, 1E9.28.1 induces vigorous T cell proliferation in the presence of submitogenic doses of phorbol myristate and F(ab')2 goat anti-mouse Ig. Both antibodies can be used to purify the three major forms of placental 5'-NT by affinity chromatography. By two-color immunofluorescence, CD73 was found to be expressed on 19 +/- 5% of CD3+, 11 +/- 4% of CD4+, 51 +/- 14% of CD8+, 25 +/- 8% of CD28+, 15 +/- 5% of CD29+, 27 +/- 7% of CD45RA+, and 70 +/- 6% of CD19+ lymphocytes. Within T cells, CD73 expression is restricted to the CD28+ subset. Thus, CD73 is found on subsets of both T and B lymphocytes, with the highest expression on B cells and CD8+ T cells. In sections of hyperplastic tonsil, CD73 expression is restricted to the small lymphocytes of the follicular mantle zone, a small subset of extrafollicular lymphocytes situated within the epithelium of the tonsillar crypt, and to follicular dendritic cells within the lower part of the "light-zone." CD73 is also detected on subsets of endothelial cells of capillaries and venules and the basal layer of non-keratinizing squamous epithelium and transitional cell type mucosa of many tissues. Given the tissue distribution of CD73, along with its glycosyl phosphatidylinositol membrane anchoring and the observation that some CD73 antibodies are mitogenic, we propose that this interesting antigen may play a role in cell activation, lymphocyte homing, and/or cell adhesion.

Regulated expression of Mg2+ binding epitope on leukocyte integrin α subunits

Article

Jan 1990

The leukocyte integrins LFA-1, CR3 and p150,95 are a family of heterodimeric receptors that mediate divalent cation-dependent cellular adhesion reactions. In this study we describe a novel antibody-defined epitope present on the leukocyte integrin alpha subunits indicating that the antibody recognizes a structural feature common to all three polypeptides. Antibody recognition further differs from that of previously described anti-leukocyte integrin antibodies in that it is strictly dependent upon the presence of Mg2+. This suggests that the epitope is located within, or in close proximity to, the three conserved cation binding domains and is therefore a measure of Mg2+ bound to the leukocyte integrins and thus reflects functionally active molecules. The epitope can be induced on polymorphonuclear leukocytes, a subset of T cells and on monocytes but is absent or much reduced at low temperature or in the presence of metabolic inhibitors. These observations have considerable implications for the regulation of leukocyte integrin function suggesting that Mg2+ binding to the extracellular domain(s) of the alpha subunits is controlled from within the cell. We suggest that one mechanism by which ligand binding by these molecules can be 'switched' on and off in response to external signals is by regulation of Mg2+ binding to these molecules, converting from the cation-free, 'inactive' to the Mg2+-bound, 'active' form.

Functional characterization of ecto-5′-nucleotidase-positive and -negative human T lymphocytes

Article

Apr 1989

Functional studies were performed on human peripheral blood T lymphocytes stained with goat anti-5'-nucleotidase antibodies and separated into ecto-5'-nucleotidase (ecto-5'-NT)-positive and -negative populations using the FACSTAR fluorescence-activated cell sorter. On the average, ecto-5'-NT+ T cells contained 34 +/- 13% CD4+ and 55 +/- 15% CD8+ cells, whereas ecto-5'-NT-T cells contained 65 +/- 12% CD4+ and 23 +/- 8% CD8+ cells. Staining with anti-5'-NT antibodies did not significantly alter the ability of unseparated T cells to proliferate in response to PHA or PMA, or in a MLR. However, prior incubation with anti-5'-NT antibodies did inhibit the ability of irradiated T cells to provide help for PWM-stimulated Ig synthesis by as much as 55%. In five separate experiments, ecto-5'-NT-T cells demonstrated an equal or better ability to incorporate [3H]TdR after PHA stimulation or in a MLR, as compared with ecto-5'-NT+ T cells. Similarly, ecto-5'-NT- T cells were not diminished in their ability to provide help for autologous B cells in a PWM-driven system. Clearly, the inability of ecto-5'-NT- T cells from patients with a variety of immunodeficiency diseases to function in these assays cannot be explained solely by their lack of ecto-5'-NT activity. In contrast, ecto-5'-NT-positive and -negative T cells showed markedly different dose-response curves for proliferation in response to PMA. Ecto-5'-NT+ T cells responded to lower doses of PMA (1.0 ng/ml) than did ecto-5'-NT- T cells and showed a two- to eight-fold greater rate of [3H]TdR incorporation at 3 to 10 ng of PMA per ml. Ecto-5'-NT+ T cells may have a protein kinase C that is more accessible or more easily activated or may utilize an alternate pathway of activation when stimulated with low concentrations of PMA.

Functional characterization of ecto-5'-nucleotidase (ecto-5'-NT) positive and negative human lymphocytes

Article

Feb 1989
ADV EXP MED BIOL

Permanent cell line expressing human factor VIII-related antigen established by hybridization

Article

Jul 1983

A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line.

Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm

Article

Feb 1994
CELL

Timothy A Springer

TABS, a T cell activation antigen that induces LFA-1-dependent aggregation

Article

Sep 1995

We describe here a mAb, DATK44, which induces homotypic aggregation of TK1 cells (a CD8 lymphoma). The glycoprotein recognized by DATK44 is of approximate m.w. 50 kDa and is expressed by monocytes, neutrophils, and subsets of lymphocytes, as well as on the high endothelial venule in peripheral and mesenteric lymph nodes. We named this Ag TABS (T cell activation B cell subset Ag), as TABS appears on T lymphocyte activation and is expressed at low and high levels by B cells. TABS is differentially regulated during T lymphocyte development, CD4+veCD8+ve thymocytes being TABShigh, while single positive CD4+ve and CD8+ve thymocytes are TABSdull CD4-veCD8-ve thymocytes are clearly split into dull and bright populations by the mAb. On exit from the thymus, T lymphocytes cease to express TABS, but T lymphocyte activation results in re-expression of TABS. TABS also shows tight coregulation with heat stable Ag on resting lymphocytes, but coexpression of these two molecules is lost upon lymphocyte activation. DATK44-induced aggregation of TK1 cells is temperature sensitive and blocked by pretreatment of the cells with metabolic inhibitors, genestein, dibutyl cAMP or cytochalasin B, while colchicine, staurosporin, sphingosine, okadaic acid, and W7 are without effect. DATK44-induced TK1 cell aggregation appears to be mediated by the LFA-1 pathway, as aggregation is blocked by anti-LFA-1 and anti-ICAM-1 mAbs but not by Abs capable of blocking CD44 and alpha 4 beta 7-mediated adhesion. Thus, TABS appears to be an adhesion inducer that selectively activates LFA-1-mediated lymphocyte aggregation.

The dynamic regulation of integrin adhesiveness

Article

Jul 1994
CURR BIOL

The integrins are a family of transmembrane heterodimeric adhesion molecules that play important roles in wound healing, immune system function and organ development. Recent studies indicate that adhesion of integrins to their ligands is not constitutive but is dynamically regulated by intracellular signal transduction pathways.

Calpain: New perspectives in molecular diversity and physiological-pathological involvement

Article

Sep 1994

Calpain, calcium-activated neutral protease, stands as a unique receptor for calcium signals in biological systems; its activation leads to irreversible proteolytic processing of substrate proteins, modifying cellular situations in a manner distinct from that of reversible processes including the phosphorylation-dephosphorylation reactions. Because the enzyme participates not only in normal intracellular signal transduction cascades but also in various pathological states including ischemia, calpain research has attracted tremendous interest in wide areas of life sciences in both basic and clinical terms. This review will address the new perspectives evoked by recent discoveries since 1990. Molecular biological studies have established that calpain in fact constitutes a large family of distinct isozymes differing in structure and distribution, whereas an increasing number of reports describe physiological-pathological involvement of calpain. Another major accomplishment is the technical breakthrough allowing spatial resolution of calpain action presenting a clearer in vivo picture of how calpain acts in cells and tissues.

New era of calpain research. Discovery of tissue-specific calpains

Article

May 1994

The recent discovery of several new calpain species other than the two species thus far studied reveals that calpain, especially the calpain large subunit, constitutes a family comprising at least six members that can be classified into ubiquitous (mu, m- and mu/m-types) and tissue-specific (p94 or nCL-1 specific for skeletal muscle, and nCL-2 and -2' specific for stomach) calpains. The newly identified tissue-specific calpains have various characteristics distinct from conventional calpains in structure, manner of expression, and enzyme activity. Unique features of tissue specific calpains are discussed together with the evolutionary view of the calpain large subunit.

Murine platelet endothelial cell adhesion molecule (PECAM-1)/CD31 modulates β2 integrins on lymphokine-activated killer cells

Article

Oct 1993

Lymphokine-activated killer (LAK) cells are able to colonize sites of tumor lesions in mouse and man. The molecular mechanisms of homing in on tumors are largely unknown. However, before LAK cells can reach the tumor, they must adhere to the vascular endothelial within the lesion and then extravasate. We developed a novel mAb, EA-3, which recognizes the murine homologue of the human adhesion molecule CD31. It is present on a subpopulation of murine LAK cells and all endothelial cells. CD31 was also involved in the adhesion of LAK cells to endothelium. Since CD31 can initiate integrin activation by inside-out signaling after binding to its ligand, EA-3 was used to minimic this in adhesion assays. It induces modifications in the beta 2 integrin LFA-1, leading to increased binding capacities of the cells to endothelium. In contrast, beta 1 integrins and RGD-binding integrins were not affected. These results suggest that expression of CD31 might confer adhesive advantages for LAK cells prone to tumor infiltration.

Lymphocyte-vascular adhesion protein-2 is a novel 70-kDa molecule involved in lymphocyte adhesion to vascular endothelium

Article

Nov 1993

Extravasation of leukocytes from the blood is essential for normal lymphocyte recirculation as well as in mounting an adequate inflammatory response in different tissues. Leukocyte migration from the blood is controlled by sophisticated interactions between surface receptors on leukocytes and their corresponding endothelial cell ligands. Here we describe a novel adhesion molecule, lymphocyte-vascular adhesion protein-2 (L-VAP-2), recognized by 4G4 mAb that was produced by immunizing mice with an enriched endothelial cell preparation isolated from inflamed human synovium. mAb 4G4 stains a subpopulation of venules in lymphoid and nonlymphoid tissues as well as a few high endothelial venules in lymphoid tissues. L-VAP-2 is constitutively expressed on human umbilical vein endothelial cells, and its expression cannot be up-regulated by cytokines and mitogens such as TNF-alpha, IL-1, and LPS. The Ag is expressed on approximately 20% of PBL, whereas granulocytes and monocytes are negative. On lymphocytes, L-VAP-2 is preferentially expressed on B cells and CD8+ T cells. The molecular mass of L-VAP-2 is approximately 70 kDa as determined from immunoprecipitated, 125I-labeled endothelial cell lysates. The involvement of L-VAP-2 in lymphocyte binding to endothelium was tested in vitro using human umbilical vein endothelial cells. Both the intact antibody and F(ab')2 fragments of it consistently inhibited lymphocyte binding to human umbilical vein endothelial cells by approximately 25%. On the basis of the molecular mass estimation and the staining of tissue sections, leukocyte populations, and ICAM-1 and ICAM-2 transfectants, L-VAP-2 appears to be a novel Ag involved in the lymphocyte-endothelial cell interaction.

Lymphocyte Homing and Homeostasis

Article

May 1996

The integration and control of systemic immune responses depends on the regulated trafficking of lymphocytes. This lymphocyte “homing” process disperses the immunologic repertoire, directs lymphocyte subsets to the specialized microenvironments that control their differentiation and regulate their survival, and targets immune effector cells to sites of antigenic or microbial invasion. Recent advances reveal that the exquisite specificity of lymphocyte homing is determined by combinatorial “decision processes” involving multistep sequential engagement of adhesion and signaling receptors. These homing-related interactions are seamlessly integrated into the overall interaction of the lymphocyte with its environment and participate directly in the control of lymphocyte function, life-span, and population dynamics. In this article a review of the molecular basis of lymphocyte homing is presented, and mechanisms by which homing physiology regulates the homeostasis of immunologic resources are proposed.

CD73 mediates lymphocyte binding to vascular endothelium in inflamed human skin

Article

Jan 1997

Inflammatory diseases of the skin are characterized by abundant lymphocytic infiltrates at the site of inflammation, which are critical for the perpetuation of chronic disease. Lymphocytes gain entry to the site of inflammation by the use of adhesion molecules, which recognize their counterparts on vascular endothelial cells. CD73 is a lymphocyte differentiation antigen, which has recently been shown to mediate lymphocyte binding to cultured endothelial cells. Here, we have examined its expression and function in inflammatory situations using inflammatory skin diseases as a model. In several idiopathic and allergic disorders of the skin, a vast majority of the skin-infiltrating lymphocytes were found to express CD73. However, on the circulating lymphocytes of these patients the expression of CD73 does not differ from that of healthy individuals. Of the peripheral blood lymphocytes (PBL) of patients, 13 % are CD73+; of these, 9 % express the cutaneous lymphocyte antigen (CLA), 32 % express CD45RO, and 86 % are L-selectin+. Only 1% of PBL express both CLA and CD73. In contrast, most skin-infiltrating lymphocytes express both molecules, which led us to investigate the role of CD73 in the skin-homing behavior of these cells. In the frozen-section adhesion assay, when PBL were treated with the anti-CD73 monoclonal antibody 4G4, their binding to the vascular endothelium in inflamed skin was inhibited by 70 %. Taken together, our results demonstrate that CD73+ lymphocytes preferentially accumulate into inflamed skin and, most importantly, that CD73 is involved in lymphocyte binding to vessels in inflamed skin. In the future, these findings may offer new means to treat inflammatory disorders of the skin.

Engagement of GPI-Linked CD48 Contributes to TCR Signals and Cytoskeletal Reorganization

Article

Jan 1999
IMMUNITY

GPI-linked proteins coassociate with intracellular tyrosine kinases in "lipid rafts" proposed to function as platforms for signal transduction and cytoskeletal reorganization. TCR activation requires both tyrosine kinase signals and cytoskeletal reorganization. How receptor engagement initiates cytoskeletal changes remains poorly understood. We investigated the consequences of recruiting GPI-linked CD48 and associated rafts to the site of T cell:APC contact by stimulating T cells with APCs that express the CD48 ligand CD2. We demonstrate that CD2:CD48 interactions enhance TCR-mediated functions. CD48/TCR coengagement qualitatively and quantitatively enhances lipid raft-dependent zeta association with the actin cytoskeleton and zeta tyrosine phosphorylation. This implicates lipid rafts as sites where receptor-induced signals and cytoskeletal reorganization are integrated and reveals a novel component of accessory molecule function.

Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes

Y Van Kooyk
P. Van De
P Wiel-Van Kemenade
T W Weder
C G Kuijpers
Figdor

van Kooyk, Y., P. van de. Wiel-van Kemenade, P. Weder, T. W. Kuijpers, and C. G. Figdor. 1989. Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes. Nature 342:811.

Jan 1997
909

B J Rollins

Rollins, B. J. 1997. Chemokines. Blood 90:909.

CD73 Engagement Promotes Lymphocyte Binding to Endothelial Cells Via a Lymphocyte Function-Associated Antigen-1-Dependent Mechanism

Abstract

No full-text available

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