Human CD56+CD39+ dNK cells support fetal survival through controlling trophoblastic cell fate: immune mechanisms of recurrent early pregnancy loss.

Decidual natural killer (dNK) cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy in both mice and humans, and emerging single-cell transcriptomic studies have uncovered various human dNK subsets that are disrupted in patients experiencing recurrent early pregnancy loss (RPL) at early gestational stage, suggesting a connection between abnormal proportions or characteristics of dNK subsets and RPL pathogenesis. However, the functional mechanisms underlying this association remain unclear. Here, we established a mouse model by adoptively transferring human dNK cells into pregnant NOG (NOD/Shi-scid/IL-2Rγnull) mice, where human dNK cells predominantly homed into the uteri of recipients. Using this model, we observed a strong correlation between the properties of human dNK cells and pregnancy outcome. The transfer of dNK cells from RPL patients (dNK-RPL) remarkably worsened early pregnancy loss and impaired placental trophoblast cell differentiation in the recipients. These adverse effects were effectively reversed by transferring CD56+CD39+ dNK cells. Mechanistic studies revealed that CD56+CD39+ dNK subset facilitates early differentiation of mouse trophoblast stem cells (mTSCs) towards both invasive and syncytial pathways through secreting macrophage colony-stimulating factor (M-CSF). Administration of recombinant M-CSF to NOG mice transferred with dNK-RPL efficiently rescued the exacerbated pregnancy outcomes and fetal/placental development. Collectively, this study established a novel humanized mouse model featuring functional human dNK cells homing into the uteri of recipients and uncovered the pivotal role of M-CSF in fetal-supporting function of CD56+CD39+ dNK cells during early pregnancy, highlighting that M-CSF may be a previously unappreciated therapeutic target for intervening RPL.

Immune interactions and regulation with CD39+ extracellular vesicles from platelet concentrates.

Introduction: CD39 plays an important role in the immunoregulation and inhibition of effector cells. It is expressed on immune cells, including Tregs, and on extracellular vesicles (EVs) budding from the plasma membrane. Platelet transfusion may induce alloimmunization against HLA-I antigens, leading to refractoriness to platelet transfusion with severe consequences for patients. Tregs may play a key role in determining whether alloimmunization occurs in patients with hematologic disorders. We hypothesized that CD39+ EVs might play an immunoregulatory role, particularly in the context of platelet transfusions in patients with hematologic disorders. Such alloimmunization leads to the production of alloantibodies and is sensitive to the regulatory action of CD39. Methods: We characterized CD39+ EVs in platelet concentrates by flow cytometry. The absolute numbers and cellular origins of CD39+ EVs were evaluated. We also performed functional tests to evaluate interactions with immune cells and their functions. Results: We found that CD39+ EVs from platelet concentrates had an inhibitory phenotype that could be transferred to the immune cells with which they interacted: CD4+ and CD8+ T lymphocytes (TLs), dendritic cells, monocytes, and B lymphocytes (BLs). Moreover, the concentration of CD39+ EVs in platelet concentrates varied and was very high in 10% of concentrates. The number of these EVs present was determinant for EV-cell interactions. Finally, functional interactions were observed with BLs, CD4+ TLs and CD39+ EVs for immunoglobulin production and lymphoproliferation, with potential implications for the immunological management of patients.

ENTPD1 (CD39) and NT5E (CD73) expression in human medulloblastoma: an in silico analysis.

Medulloblastoma is the most common malignant tumor in the pediatric population. Its classification has incorporated key molecular variations alongside histological characterization. CD39 (also known as ENTPD1) and CD73 (also known as NT5E), enzymes of the purinergic signaling pathway, act in synergy to generate extracellular adenosine, creating an immunosuppressive tumor microenvironment. Our study examined the expression of mRNA of these genes in previously described transcriptome data sets of medulloblastoma patient samples from the Cavalli Cohort (n = 763). Survival distribution was estimated according to the Kaplan-Meier method using a median cut-off and log-rank statistics (p ≤ 0.05). In non-WNT and non-SHH medulloblastoma Group 4 (n = 264), the high expression of ENTPD1 and NT5E was significantly related to a lower overall survival (p = 2.7e-04; p = 2.6e-03). In the SHH-activated group (n = 172), the high expression of ENTPD1 was significantly related to lower overall survival (p = 7.8e-03), while the high expression of NT5E was significantly related to greater overall survival (p = 0.017). In the WNT group (n = 63), the expressions of ENTPD1 and NT5E were not significantly correlated with overall survival (p = 0.212; p = 0.101). In non-WNT and non-SHH medulloblastoma Group 3 (n = 113), the high expression of ENTPD1 was significantly related to greater survival (p = 0.034), while expression of NT5E was not significantly related to survival of patients (p = 0.124). This in silico analysis indicates that ENTPD1 (CD39) and NT5E (CD73) can be seen as potential prognostic markers and therapeutic targets for primary medulloblastomas in non-WNT and non-SHH Group 4.

CD39 is induced by nutrient deprivation in colorectal cancer cells and contributes to the tolerance to hypoxia.

Rapid tumor growth and insufficient blood supply leads to the development of a hypoxic and nutrient deprived microenvironment. To survive, tumor cells need to tolerate these adverse conditions. Here we found the expression of CD39 was enhanced in necrotic regions distant from blood vessels. We speculate that this is a strategy for tumor cells to actively adapt to the hostile environment. Further studies showed that CD39 was induced by nutrient deprivation through the AMPK signalling pathway. We next explored the significance of CD39 for tumor cells. Our results showed that CD39 reduced cellular oxygen consumption, which could be significant for tumor cells if the available oxygen is limited. Metabolomics analysis showed that overexpression of CD39 significantly altered cellular metabolism, and tricarboxylic acid (TCA) cycle was identified as the most impacted metabolic pathway. In order to explore the molecular mechanism, we performed RNA-seq analysis. The results showed that CD39 significantly up-regulated the expression of pyruvate dehydrogenase kinase isozyme 2 (PDK2), thus inhibiting the activity of pyruvate dehydrogenase (PDH) and TCA cycle. Finally, CD39 was shown to protect tumor cells from hypoxia-induced cell death and reduce intratumoral hypoxia levels. CD39 has attracted a great deal of attention as a newly discovered immune checkpoint molecule in recent years. Our results indicate that CD39 not only plays a role in immune regulation, but also enables tumor cells to tolerate hypoxia by inhibiting TCA cycle and reducing cellular oxygen consumption. This study provides evidence that targeting CD39 may be a novel strategy to prevent adaptation of tumor cells in stressed conditions.

CD39 transforming cancer therapy by modulating tumor microenvironment.

CD39 is a pivotal enzyme in cancer, regulating immune response and tumor progression via extracellular ATP and adenosine in the tumor microenvironment (TME). Beyond its established immunoregulatory function, CD39 influences cancer cell angiogenesis and metabolism, opening new frontiers for therapeutic interventions. Current research faces gaps in understanding CD39's full impact across cancer types, with ongoing debates about its potential beyond modulating immune evasion. This review distills CD39's multifaceted roles, examining its dual actions and implications for cancer prognosis and treatment. We analyze the latest therapeutic strategies, highlighting the need for an integrated approach that combines molecular insights with TME dynamics to innovate cancer care. This synthesis underscores CD39's integral role, charting a course for precision oncology that seeks to unravel controversies and harness CD39's therapeutic promise for improved cancer outcomes.

Synergistic upregulation of PD­L1 in tumor cells and CD39 in tumor­infiltrating CD8+ T cells leads to poor prognosis in patients with hepatocellular carcinoma.

The immune escape of tumor cells and functional status of tumor-infiltrating T cells may serve pivotal roles in the tumor immune microenvironment and progression of hepatocellular carcinoma (HCC). The present study enrolled 91 patients with HCC and examined programmed cell death ligand 1 (PD-L1) expression in tumor cells and CD39 expression in tumor-infiltrating CD8+ T cells in patient samples using multiplex immunofluorescence assays. The impact of PD-L1 and CD39 expression levels on the prognosis of patients with HCC was investigated utilizing Kaplan-Meier analyses. The individual upregulation of PD-L1 in tumor cells, as well as the individual upregulation of CD39 expression in tumor-infiltrating CD8+ T cells did not significantly affect the prognosis of patients with HCC. However, the simultaneous upregulation of both PD-L1 in tumor cells and CD39 in tumor-infiltrating CD8+ T cells was associated with reduced overall survival in patients with HCC. Therefore, the results of the present study suggested that the interplay between tumor cell immune escape and tumor-infiltrating immune cell functional status within the tumor immune microenvironment may have had a substantial impact on the prognosis of patients with HCC. Mechanistically, increased expression levels of PD-L1 in tumor cells may improve the immune escape capacity of tumors, whilst upregulation of CD39 in tumor-infiltrating T cells may be associated with T cell exhaustion. Therefore, the upregulation of PD-L1 expression in tumor cells, in conjunction with the exhaustion of tumor-infiltrating CD8+ T cells, could serve as a future potential prognostic indicator of patients with HCC.

The significance of CD8+ tumor-infiltrating lymphocytes exhaustion heterogeneity and its underlying mechanism in diffuse large B-cell lymphoma.

CD8+ tumor-infiltrating lymphocytes (TILs) exhaustion is a major barrier to effective tumor control in diffuse large B-cell lymphoma (DLBCL) and may consist of heterogeneous populations with different functional states. We profiled the CD8+TILs exhaustion heterogeneity and explored its clinical significance as well as the underlying mechanism through single-cell RNA sequencing (n = 7), bulk RNA sequencing (n = 3300), immunohistochemistry (n = 116), and reverse transcription-quantitative polymerase chain reaction (n = 95), and somatic mutation data (n = 48). Our results demonstrated that exhausted CD8+TILs in DLBCL were composed of progenitor and terminal states characterized by CCL5 and TUBA1B, respectively. High terminally exhausted CD8+TILs indicated an immunosuppressive tumor microenvironment, activated B-cell-like subtype, inferior prognosis, and poor response to immune checkpoint blockade therapy in DLBCL. Our study further demonstrated that the CD39/A2AR-related signaling may be the potential pathway that promoted the transition of progenitor toward terminally exhausted CD8+TILs in DLBCL. Furthermore, the CD39/A2AR-related pathway in DLBCL may be regulated by BATF and STAT3 in exhausted CD8+TILs, and MYD88 mutation in tumor cells. Our study highlights CD8+TILs exhaustion heterogeneity and its possible regulatory mechanism provides a novel prognostic indicator and can facilitate the optimization of individualized immunotherapy.

IFN-γ-Preconditioned Human Gingival-Derived Mesenchymal Stromal Cells Inhibit Plasmacytoid Dendritic Cells via Adenosine.

Plasmacytoid dendritic cells (pDCs) are vital players in antiviral immune responses because of their high levels of IFN-α secretion. However, this attribute has also implicated them as critical factors behind the immunopathogenesis of inflammatory diseases, and no currently available therapy can efficiently inhibit pDCs' aberrant activation. Mesenchymal stromal cells (MSCs) possess stromal immunomodulatory functionality, regulating immune cell activation through several mechanisms, including the adenosinergic (CD39/CD73/adenosine) pathway. The IFN-γ preconditioning of bone marrow MSCs improves their inhibitory properties for therapy applications; however, isolating human gingival tissue-derived MSCs (hGMSCs) is more accessible. These cells have shown better immunomodulatory effects, yet the outcome of IFN-γ preconditioning and its impact on the adenosinergic pathway has not been evaluated. This study first validated the immunoregulatory properties of primary-cultured hGMSCs, and the results showed that IFN-γ preconditioning strengthens CD39/CD73 coexpression, adenosine production, and the regulatory properties of hGMSC, which were confirmed by describing for the first time their ability to reduce pDC activation and their IFN-α secretion and to increase the frequency of CD73+ pDC. In addition, when CD73's enzymatic activity was neutralized in hGMSCs, adenosine production and the IFN-γ preconditioning effect were restrained. This evidence might be applied to design hGMSCs- and adenosine-based immunotherapeutic strategies for treating inflammatory disorders that are associated with pDC overactivation.

ADA/CD26 axis increases intra-tumor PD-1+CD28+CD8+ T-cell fitness and affects NSCLC prognosis and response to ICB.

Improving cancer immunotherapy efficacy hinges on identifying key T-cell populations critical for tumor control and response to Immune Checkpoint Blockade (ICB). We have recently reported that while the co-expression of PD-1 and CD28 is associated with impaired functionality in peripheral blood, it significantly enhances T-cell fitness in the tumor site of non-small cell lung cancer (NSCLC) patients. To uncover the underlying mechanisms, we explored the role of CD26, a key player in T-cell activation through its interaction with adenosine deaminase (ADA), a crucial intra/extracellular enzyme able to neutralize local adenosine (ADO). We found that an autocrine ADA/CD26 axis enhances CD8+PD-1+CD28+ T-cell function, particularly within an immunosuppressive environment marked by CD39 expression. Then, we interrogated the TCGA and OAK datasets to gain insight into the prognostic/predictive potential of our findings. We identified a signature predicting overall survival (OS) in LUAD patients and response to atezolizumab in advanced LUAD cases. These findings suggest promising avenues for therapeutic intervention targeting the ADA/CD26 axis.

Moderate expression of CD39 in GPC3-CAR-T cells shows high efficacy against hepatocellular carcinoma.

CD39 serves as a crucial biomarker for neoantigen-specific CD8+ T cells and is associated with antitumor activity and exhaustion. However, the relationship between CD39 expression levels and the function of chimeric antigen receptor T (CAR-T) cells remains controversial. This study aimed to investigate the role of CD39 in the functional performance of CAR-T cells against hepatocellular carcinoma (HCC) and explore the therapeutic potential of CD39 modulators, such as mitochondrial division inhibitor-1 (mdivi-1), or knockdown CD39 through short hairpin RNA. Our findings demonstrated that glypican-3-CAR-T cells with moderate CD39 expression exhibited a strong antitumor activity, while high and low levels of CD39 led to an impaired cellular function. Methods modulating the proportion of CD39 intermediate (CD39int)-phenotype CAR-T cells such as mdivi-1 and CD39 knockdown enhanced and impaired T cell function, respectively. The combination of mdivi-1 and CD39 knockdown in CAR-T cells yielded the highest proportion of infiltrated CD39int CAR-T cells and demonstrated a robust antitumor activity in vivo. In conclusion, this study revealed the crucial role of CD39 in CAR-T cell function, demonstrated the potential therapeutic efficacy of combining mdivi-1 with CD39 knockdown in HCC, and provided a novel treatment strategy for HCC patients in the field of cellular immunotherapy.

Foxp3+CD39+CD73+ regulatory T-cells are decreased in the peripheral blood of women with deep infiltrating endometriosis.

Endometriosis's pathophysiology remains incompletely understood, with evidence pointing towards a dysregulated immune response. Regulatory T (Treg) cells, pivotal in maintaining self-tolerance, may facilitate the survival of ectopic endometrial cells within the abdominal cavity, thereby contributing to endometriosis development. This study aimed to assess the prevalence of CD39+CD73+ suppressor Treg cell subsets in the peripheral blood of endometriosis patients. This research focuses on the pivotal role of regulatory T-cells (Tregs), which are essential for maintaining immune tolerance and preventing autoimmune diseases. A case-control study was conducted, including 32 women diagnosed with endometriosis and 22 control subjects. The frequency of peripheral blood CD39+CD73+ suppressor Treg cells was quantified using flow cytometry. No significant differences were observed in the frequency of CD3+CD4+CD25High cells (Median [M]: 10.1; Interquartile Range [IQR]: 6.32‒18.3 vs. M: 9.72; IQR: 6.22-19.8) or CD3+CD4+CD25HighCD39+Foxp3+ cells (M: 31.1; IQR: 19.7-44.0 vs. M: 30.55; IQR: 18.5-45.5) between controls and patients. However, a significantly lower frequency of CD3+CD4+CD25HighCD39+CD73+ cells was observed in the endometriosis group compared to controls (M: 1.98; IQR: 0.0377-3.17 vs. M: 2.25; IQR: 0.50-4.08; p = 0.0483), suggesting a reduction in systemic immune tolerance among these patients. This finding highlights the potential role of CD39 and CD73 expression on Treg cells as biomarkers for assessing disease severity and progression. Furthermore, elucidating the mechanisms driving these alterations may unveil new therapeutic strategies to restore immune equilibrium and mitigate endometriosis symptoms.

The presence of cytotoxic CD4 and exhausted-like CD8+ T-cells is a signature of active tuberculosis.

Chronic infections induce CD4+ T-cells with cytotoxic functions (CD4 CTLs); at present, it is still unknown whether latent tuberculosis (LTB) and active tuberculosis (ATB) induce CD4 CTLs. Plasma and cells from four patient groups-uninfected contact (UC), LTB, and ATB (divided as sensitive [DS-TB]- or resistant [DR-TB]-drug)-were evaluated by flow cytometry, q-PCR, and proteomics. The data showed that ATB patients had an increased frequency of CD4+ T-cells and a decreased frequency of CD8+ T-cells. The latter displays an exhausted-like profile characterized by CD39, CD279, and TIM-3 expression. ATB had a high frequency of CD4 + perforin+ cells, suggesting a CD4 CTL profile. The expression (at the transcriptional level) of granzyme A, granzyme B, granulysin, and perforin, as well as the genes T-bet (Tbx21) and NKG2D (Klrk1), in enriched CD4+ T-cells, confirmed the cytotoxic signature of CD4+ T-cells during ATB (which was stronger in DS-TB than in DR-TB). Moreover, proteomic analysis revealed the presence of HSP70 (in DS-TB) and annexin A5 (in DR-TB), which are molecules that have been associated with favoring the CD4 CTL profile. Finally, we found that lipids from Mycobacterium tuberculosis increased the presence of CD4 CTLs in DR-TB patients. Our data suggest that ATB is characterized by exhausted-like CD8+ T-cells, which, together with a specific microenvironment, favor the presence of CD4 CTLs.

Development of endothelial-targeted CD39 as a therapy for ischemic stroke.

BACKGROUND: Ischemic stroke is characterized by a necrotic lesion in the brain surrounded by an area of dying cells termed the penumbra. Salvaging the penumbra either with thrombolysis or mechanical retrieval is the cornerstone of stroke management. At-risk neuronal cells release extracellular adenosine triphosphate, triggering microglial activation and causing a thromboinflammatory response, culminating in endothelial activation and vascular disruption. This is further aggravated by ischemia-reperfusion injury that follows all reperfusion therapies. The ecto-enzyme CD39 regulates extracellular adenosine triphosphate by hydrolyzing it to adenosine, which has antithrombotic and anti-inflammatory properties and reverses ischemia-reperfusion injury. OBJECTIVES: The objective off the study was to determine the efficacy of our therapeutic, anti-VCAM-CD39 in ischaemic stroke. METHODS: We developed anti-VCAM-CD39 that targets the antithrombotic and anti-inflammatory properties of recombinant CD39 to the activated endothelium of the penumbra by binding to vascular cell adhesion molecule (VCAM)-1. Mice were subjected to 30 minutes of middle cerebral artery occlusion and analyzed at 24 hours. Anti-VCAM-CD39 or control agents (saline, nontargeted CD39, or anti-VCAM-inactive CD39) were given at 3 hours after middle cerebral artery occlusion. RESULTS: Anti-VCAM-CD39 treatment reduced neurologic deficit; magnetic resonance imaging confirmed significantly smaller infarcts together with an increase in cerebrovascular perfusion. Anti-VCAM-CD39 also restored blood-brain barrier integrity and reduced microglial activation. Coadministration of anti-VCAM-CD39 with thrombolytics (tissue plasminogen activator [tPA]) further reduced infarct volumes and attenuated blood-brain barrier permeability with no associated increase in intracranial hemorrhage. CONCLUSION: Anti-VCAM-CD39, uniquely targeted to endothelial cells, could be a new stroke therapy even when administered 3 hours postischemia and may further synergize with thrombolytic therapy to improve stroke outcomes.

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction.

Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

The roles of adenosine signaling in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with multiple etiological factors. Immune disorder contributes to SLE development and is an important clinical manifestation of SLE patients. Immune dysfunction is characterized by abnormal of B cells, T cells, monocyte-macrophages and dendritic cells (DCs), in both quantity and quality. Adenosine is a critical factor for human immune homeostasis, which acts as an immunosuppressive signal and can prevent the hyperactivity of human immune system. Adenosine levels are significant decreased in serum from SLE patients. Adenosine level is regulated by the CD39, CD73 and adenosine deaminase (ADA). CD39/CD73/ADA catalyzed the cascade enzymatic reaction, which contained the adenosine generation and degradation. Adenosine affects the function of various immune cells via bind to the adenosine receptors, which are expressed on the cell surface. This review aims to export the changes of immune cells and adenosine signal pathway in SLE, as well as the effect of adenosine signal pathway in SLE development.

Genetically predicted blood metabolites mediate the association between circulating immune cells and pancreatic cancer: A Mendelian randomization study.

BACKGROUND: Pancreatic cancer is characterized by metabolic dysregulation and unique immunological profiles. Nevertheless, the comprehensive understanding of immune and metabolic dysregulation of pancreatic cancer remains unclear. In the present study, we aimed to investigate the causal relationship of circulating immune cells and pancreatic cancer and identify the blood metabolites as potential mediators. METHODS: The exposure and outcome genome-wide association studies (GWAS) data used in the present study were obtained from the GWAS open-access database (https://gwas.mrcieu.ac.uk). The study used 731 circulating immune cell features, 1400 types of blood metabolites and pancreatic cancer from GWAS. We then performed bidirectional Mendelian randomization (MR) analyses to explore the causal relationships between the circulating immune cells and pancreatic cancer, and two-step MR to discover potential mediating blood metabolites in this process. All statistical analyses were performed in R software. The STROBE-MR (i.e. Strengthening the Reporting of Observational Studies in Epidemiology using Mendelian Randomization) checklist for the reporting of MR studies was also used. RESULTS: MR analysis identified seven types of circulating immune cells causally associated with pancreatic cancer. Furthermore, there was no strong evidence that genetically predicted pancreatic cancer had an effect on these seven types of circulating immune cells. Further two-step MR analysis found 10 types of blood metabolites were causally associated with pancreatic cancer and the associations between circulating CD39+CD8+ T cells and pancreatic cancer were mediated by blood orotates with proportions of 5.18% (p = 0.016). CONCLUSIONS: The present study provides evidence supporting the causal relationships between various circulating immune cells, especially CD39+CD8+ T cells, and pancreatic cancer, with a potential effect mediated by blood orotates. Further research is needed on additional risk factors as potential mediators and establish a comprehensive immunity-metabolism network in pancreatic cancer.

STAT activation in regulatory CD4+ T cells of patients with primary sclerosing cholangitis.

INTRODUCTION: Regulatory CD4+ T cells (Tregs) are pivotal for inhibition of autoimmunity. Primary sclerosing cholangitis (PSC) is an autoimmune cholestatic liver disease of unknown etiology where contribution of Tregs is still unclear. Activation of the JAK-STAT pathway critically modifies functions of Tregs. In PSC, we studied activation of STAT proteins and Treg functions in response to cytokines. METHODS: In 51 patients with PSC, 10 disease controls (chronic replicative hepatitis C), and 36 healthy controls we analyzed frequencies of Foxp3+CD25+CD127lowCD4+ Tregs, their expression of ectonucleotidase CD39, and cytokine-induced phosphorylation of STAT1, 3, 5, and 6 using phospho-flow cytometry. In parallel, we measured cytokines IFN-gamma, interleukin (IL)-6, IL-2, and IL-4 in serum via bead-based immunoassays. RESULTS: In patients with PSC, ex vivo frequencies of peripheral Tregs and their expression of CD39 were significantly reduced (p < .05 each). Furthermore, serum levels of IFN-gamma, IL-6, IL-2, and IL-4 were markedly higher in PSC (p < .05 each). Unlike activation of STAT1, STAT5, and STAT6, IL-6 induced increased phosphorylation of STAT3 in Tregs of PSC-patients (p = .0434). Finally, STAT3 activation in Tregs correlated with leukocyte counts. CONCLUSIONS: In PSC, we observed enhanced STAT3 responsiveness of CD4+ Tregs together with reduced CD39 expression probably reflecting inflammatory activity of the disease.

Generation and characterization of antagonistic anti-human CD39 nanobodies.

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti-CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39. From cDNA-immunized alpacas we selected 16 clones from seven nanobody families that bind to two distinct epitopes of human CD39. Among these, clone SB24 inhibited the enzymatic activity of CD39. Of note, SB24 blocked ATP degradation by both soluble and cell surface CD39 as a 15kD monomeric nanobody. Dimerization via fusion to an immunoglobulin Fc portion further increased the blocking potency of SB24 on CD39-transfected HEK cells. Finally, we confirmed the CD39 blocking properties of SB24 on human PBMCs. In summary, SB24 provides a new small biological antagonist of human CD39 with potential application in cancer therapy.

The Scutellaria-Coptis herb couple and its active small-molecule ingredient wogonoside alleviate cytokine storm by regulating the CD39/NLRP3/GSDMD signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: A drug pair is a fundamental aspect of traditional Chinese medicine prescriptions. Scutellaria baicalensis Georgi and Coptis chinensis Franch, commonly used as an herb couple (SBCC), are representative heat-clearing and dampness-drying drugs. They possess functions such as clearing heat, drying dampness, purging fire, and detoxifying. These herbs are used in both traditional and modern medicine for treating inflammation. AIM OF THE STUDY: This study investigated the effects of SBCC on cytokine storm syndrome (CSS) and explored its potential regulatory mechanism. MATERIALS AND METHODS: We assessed the impact of SBCC in a sepsis-induced acute lung injury mouse model by administering an intraperitoneal injection of LPS (15 mg/kg). The cytokine levels in the serum and lungs, the wet-to-dry ratio of the lungs, and lung histopathological changes were evaluated. The macrophages in the lung tissue were examined through transmission electron microscopy. Western blot was used to measure the levels of the CD39/NLRP3/GSDMD pathway-related proteins. Immunofluorescence imaging was used to assess the activation of pro-caspase-1 and ASC and their interaction. AMP-Glo™ assay was used to screen for active ingredients in SBCC targeting CD39. One of the ingredients was selected, and its effect on cell viability was assessed. We induced inflammation in macrophages using LPS + ATP and detected the levels of proinflammatory factors. The images of cell membrane large pores were captured using scanning electron microscopy, the interaction between NLRP3 and ASC was detected using immunofluorescence imaging, and the levels of CD39/NLRP3/GSDMD pathway-related proteins were assessed using Western blot. RESULTS: SBCC administration effectively mitigated LPS-induced cytokine storm, pulmonary edema and lung injury. Furthermore, it repressed the programmed death of lung tissue macrophages by inhibiting the NLRP3/GSDMD pyroptosis pathway and regulating the CD39 purinergic pathway. Based on the results of the AMP-Glo™ assay, we selected wogonoside for further valuation. Wogonoside alleviated LPS + ATP-induced inflammatory damage by regulating the inhibiting the NLRP3/GSDMD pyroptosis pathway and regulating the CD39 purinergic pathway. However, its effect on NLRP3 is not mediated though CD39. CONCLUSION: SBCC and its active small-molecule ingredient, wogonoside, improved CSS by regulating the NLRP3/GSDMD pyroptosis pathway and its upstream CD39 purinergic pathway. It is essential to note that the regulatory effect of wogonoside on NLRP3 is not mediated by CD39.

Regulatory role of CD39 and CD73 in tumor immunity.

CD39 is the rate-limiting enzyme for the molecular signal cascade leading to the generation of ADP and adenosine monophosphate (AMP). In conjunction with CD73, CD39 converts adenosine triphosphate (ATP) to ADP and AMP, which leads to the accumulation of immunosuppressive adenosine in the tumor microenvironment. This review focuses on the role of CD39 and CD73 in immune response and malignant progression, including the expression of CD39 within the tumor microenvironment and its relationship to immune effector cells, and its role in antigen presentation. The role of CD39- and CD73-targeting therapeutics and cancer-directed clinical trials investigating CD39 modulation are also explored.

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