CD39 delineates chimeric antigen receptor regulatory T cell subsets with distinct cytotoxic & regulatory functions against human islets.

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human ß cell line and human islet ß cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet ß cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, ß cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased ß cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet ß cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.

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.

IL-27 promotes pathogenic T cells in a mouse model of Sjögren's disease.

Sjögren's disease (SjD) is a chronic autoimmune disease characterized by focal lymphocytic inflammation in lacrimal and salivary glands. We recently identified IL-27 as a requisite signal for the spontaneous SjD-like manifestations in nonobese diabetic (NOD) mice. Here, we define T cell-intrinsic effects of IL-27 in lacrimal gland disease in NOD mice. IL-27 receptor was required by both CD4 T effector (Te) cells and CD8 T cells to mediate focal inflammation. Intrinsic IL-27 signaling was associated with PD-1 and ICOS expressing T follicular helper (Tfh)-like CD4 Te cells within lacrimal glands, including subsets defined by CD73 or CD39 expression. CD8 T cells capable of IL-27 signaling also expressed PD-1 with subsets expressing ICOS and CD73 demonstrating a T follicular cytotoxic (Tfc)-like cell phenotype and others expressing a CD39hi exhausted-like phenotype. These findings suggest IL-27 is a key early signal driving a follicular-type response in lacrimal gland inflammation in NOD mice.

CD200R activation on naïve T cells by B cells induces suppressive activity of T cells via IL-24.

CD200 is an anti-inflammatory protein that facilitates signal transduction through its receptor, CD200R, in cells, resulting in immune response suppression. This includes reducing M1-like macrophages, enhancing M2-like macrophages, inhibiting NK cell cytotoxicity, and downregulating CTL responses. Activation of CD200R has been found to modulate dendritic cells, leading to the induction or enhancement of Treg cells expressing Foxp3. However, the precise mechanisms behind this process are still unclear. Our previous study demonstrated that B cells in Peyer's patches can induce Treg cells, so-called Treg-of-B (P) cells, through STAT6 phosphorylation. This study aimed to investigate the role of CD200 in Treg-of-B (P) cell generation. To clarify the mechanisms, we used wild-type, STAT6 deficient, and IL-24 deficient T cells to generate Treg-of-B (P) cells, and antagonist antibodies (anti-CD200 and anti-IL-20RB), an agonist anti-CD200R antibody, CD39 inhibitors (ARL67156 and POM-1), a STAT6 inhibitor (AS1517499), and soluble IL-20RB were also applied. Our findings revealed that Peyer's patch B cells expressed CD200 to activate the CD200R on T cells and initiate the process of Treg-of-B (P) cells generation. CD200 and CD200R interaction triggers the phosphorylation of STAT6, which regulated the expression of CD200R, CD39, and IL-24 in T cells. CD39 regulated the expression of IL-24, which sustained the expression of CD223 and IL-10 and maintained the cell viability. In summary, the generation of Treg-of-B (P) cells by Peyer's patch B cells was through the CD200R-STAT6-CD39-IL-24 axis pathway.

CD39 expression by regulatory T cells participates in CD8+ T cell suppression during experimental Trypanosoma cruzi infection.

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection, specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein, we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model, we found that Treg cells play a role during the initial stages after T. cruzi infection, restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived, effector T cell subsets, without affecting memory precursor cell formation or the expression of activation, exhaustion and functional markers. In addition, Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially, the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses, preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model.

The adenosinergic signaling in the pathogenesis and treatment of multiple sclerosis.

Multiple sclerosis (MS) is a highly disabling, progressive neurodegenerative disease with no curative treatment available. Although significant progress has been made in understanding how MS develops, there remain aspects of disease pathogenesis that are yet to be fully elucidated. In this regard, studies have shown that dysfunctional adenosinergic signaling plays a pivotal role, as patients with MS have altered levels adenosine (ADO), adenosine receptors and proteins involved in the generation and termination of ADO signaling, such as CD39 and adenosine deaminase (ADA). We have therefore performed a literature review regarding the involvement of the adenosinergic system in the development of MS and propose mechanisms by which the modulation of this system can support drug development and repurposing.

Protein kinase inhibitor ceritinib blocks ectonucleotidase CD39 - a promising target for cancer immunotherapy.

BACKGROUND: An important mechanism, by which cancer cells achieve immune escape, is the release of extracellular adenosine into their microenvironment. Adenosine activates adenosine A2A and A2B receptors on immune cells constituting one of the strongest immunosuppressive mediators. In addition, extracellular adenosine promotes angiogenesis, tumor cell proliferation, and metastasis. Cancer cells upregulate ectonucleotidases, most importantly CD39 and CD73, which catalyze the hydrolysis of extracellular ATP to AMP (CD39) and further to adenosine (CD73). Inhibition of CD39 is thus expected to be an effective strategy for the (immuno)therapy of cancer. However, suitable small molecule inhibitors for CD39 are not available. Our aim was to identify drug-like CD39 inhibitors and evaluate them in vitro. METHODS: We pursued a repurposing approach by screening a self-compiled collection of approved, mostly ATP-competitive protein kinase inhibitors, on human CD39. The best hit compound was further characterized and evaluated in various orthogonal assays and enzyme preparations, and on human immune and cancer cells. RESULTS: The tyrosine kinase inhibitor ceritinib, a potent anticancer drug used for the treatment of anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer, was found to strongly inhibit CD39 showing selectivity versus other ectonucleotidases. The drug displays a non-competitive, allosteric mechanism of CD39 inhibition exhibiting potency in the low micromolar range, which is independent of substrate (ATP) concentration. We could show that ceritinib inhibits ATP dephosphorylation in peripheral blood mononuclear cells in a dose-dependent manner, resulting in a significant increase in ATP concentrations and preventing adenosine formation from ATP. Importantly, ceritinib (1-10 µM) substantially inhibited ATP hydrolysis in triple negative breast cancer and melanoma cells with high native expression of CD39. CONCLUSIONS: CD39 inhibition might contribute to the effects of the powerful anticancer drug ceritinib. Ceritinib is a novel CD39 inhibitor with high metabolic stability and optimized physicochemical properties; according to our knowledge, it is the first brain-permeant CD39 inhibitor. Our discovery will provide the basis (i) to develop more potent and balanced dual CD39/ALK inhibitors, and (ii) to optimize the ceritinib scaffold towards interaction with CD39 to obtain potent and selective drug-like CD39 inhibitors for future in vivo studies.

The percentage of CD39+ monocytes is higher in pregnant COVID-19+ patients than in nonpregnant COVID-19+ patients.

Current medical guidelines consider pregnant women with COVID-19 to be a high-risk group. Since physiological gestation downregulates the immunological response to maintain "maternal-fetal tolerance", SARS-CoV-2 infection may constitute a potentially threatening condition to both the mother and the fetus. To establish the immune profile in pregnant COVID-19+ patients, a cross-sectional study was conducted. Pregnant women with COVID-19 (P-COVID-19+; n = 15) were analyzed and compared with nonpregnant women with COVID-19 (NP-COVID-19+; n = 15) or those with physiological pregnancy (P-COVID-19-; n = 13). Serological cytokine and chemokine concentrations, leucocyte immunophenotypes, and mononuclear leucocyte responses to polyclonal stimuli were analyzed in all groups. Higher concentrations of serological TNF-α, IL-6, MIP1b and IL-4 were observed within the P-COVID-19+ group, while cytokines and chemokines secreted by peripheral leucocytes in response to LPS, IL-6 or PMA-ionomicin were similar among the groups. Immunophenotype analysis showed a lower percentage of HLA-DR+ monocytes in P-COVID-19+ than in P-COVID-19- and a higher percentage of CD39+ monocytes in P-COVID-19+ than in NP-COVID-19+. After whole blood polyclonal stimulation, similar percentages of T cells and TNF+ monocytes between groups were observed. Our results suggest that P-COVID-19+ elicits a strong inflammatory response similar to NP-COVID19+ but also displays an anti-inflammatory response that controls the ATP/adenosine balance and prevents hyperinflammatory damage in COVID-19.

Exhaustion of CD39-Expressing CD8+ T Cells in Crohn's Disease Is Linked to Clinical Outcome.

BACKGROUND & AIMS: Exhaustion of CD8 T cells has been suggested to inform different clinical outcomes in Crohn's disease, but detailed analyses are lacking. This study aimed to identify the role of exhaustion on a single-cell level and identify relevant CD8 T cell populations in Crohn's disease. METHODS: Blood and intestinal tissue from 58 patients with Crohn's disease (active disease or remission) were assessed for CD8 T cell expression of exhaustion markers and their cytokine profile by highly multiplexed flow and mass cytometry. Key disease-associated subsets were sorted and analyzed by RNA sequencing. CD39 inhibition assays were performed in vitro. RESULTS: Activated CD39+ and CD39+PD-1+ CD8 T cell subsets expressing multiple exhaustion markers were enriched at low frequency in active Crohn's disease. Their cytokine production capacity was inversely linked to the Harvey-Bradshaw Index. Subset-level protein and transcriptome profiling revealed co-existence of effector and exhaustion programs in CD39+ and CD39+ PD-1+CD8 T cells, with CD39+ cells likely originating from the intestine. CD39 enzymatic activity controlled T cell cytokine production. Importantly, transcriptional exhaustion signatures were enriched in remission in CD39-expressing subsets with up-regulation of TOX. Subset-level transcriptomics revealed a CD39-related gene module that is associated with the clinical course. CONCLUSIONS: These data showed a role for the exhaustion of peripheral CD39-expressing CD8 T cell subsets in Crohn's disease. Their low frequency illustrated the utility of single-cell cytometry methods for identification of relevant immune populations. Importantly, the link of their exhaustion status to the clinical activity and their specific gene signatures have implications for exhaustion-based personalized medicine approaches.

Higher proportions of CD39+ tumor-resident cytotoxic T cells predict recurrence-free survival in patients with stage III melanoma treated with adjuvant immunotherapy.

BACKGROUND: Adjuvant immune checkpoint inhibitor (ICI) immunotherapies have significantly reduced the recurrence rate in high-risk patients with stage III melanoma compared with surgery alone. However, 48% of anti-PD-1-treated patients will develop recurrent disease within 4 years. There is a need to identify biomarkers of recurrence after adjuvant ICI to enable identification of patients in need of alternative treatment strategies. As cytotoxic T cells are critical for the antitumor response to anti-PD-1, we sought to determine whether specific subsets were predictive of recurrence in anti-PD-1-treated high-risk patients with stage III melanoma. METHODS: Associations with recurrence in patients with stage III melanoma were sought by analyzing resection specimens (n=103) taken prior to adjuvant nivolumab/pembrolizumab±low-dose/low-interval ipilimumab. Multiplex immunohistochemistry was used to quantify intratumoral CD8+ T-cell populations using phenotypical markers CD39, CD103, and PD-1. RESULTS: With a median follow-up of 19.3 months, 37/103 (36%) of patients had a recurrence. Two CD8+ T-cell subpopulations were significantly associated with recurrence. First, CD39+ tumor-resident memory cells (CD39+CD103+PD-1+CD8+ (CD39+ Trm)) comprised a significantly higher proportion of CD8+ T cells in recurrence-free patients (p=0.0004). Conversely, bystander T cells (CD39-CD103-PD-1-CD8+) comprised a significantly greater proportion of T cells in patients who developed recurrence (p=0.0002). Spatial analysis identified that CD39+ Trms localized significantly closer to melanoma cells than bystander T cells. Multivariable analysis confirmed significantly improved recurrence-free survival (RFS) in patients with a high proportion of intratumoral CD39+ Trms (1-year RFS high 78.1% vs low 49.9%, HR 0.32, 95% CI 0.15 to 0.69), no complete lymph node dissection performed, and less advanced disease stage (HR 2.85, 95% CI 1.13 to 7.19, and HR 1.29, 95% CI 0.59 to 2.82). The final Cox regression model identified patients who developed recurrence with an area under the curve of 75.9% in the discovery cohort and 69.5% in a separate validation cohort (n=33) to predict recurrence status at 1 year. CONCLUSIONS: Adjuvant immunotherapy-treated patients with a high proportion of CD39+ Trms in their baseline melanoma resection have a significantly reduced risk of melanoma recurrence. This population of T cells may not only represent a biomarker of RFS following anti-PD-1 therapy, but may also be an avenue for therapeutic manipulation and enhancing outcomes for immunotherapy-treated patients with cancer.

Major depression favors the expansion of Th17-like cells and decrease the proportion of CD39+Treg cell subsets in response to myelin antigen in multiple sclerosis patients.

BACKGROUND: Mood disorders have been associated with risk of clinical relapses in multiple sclerosis (MS), a demyelinating disease mediated by myelin-specific T cells. OBJECTIVES: We aimed to investigate the impact of major depressive disorder (MDD) and cytokine profile of T-cells in relapsing remitting MS patients. METHODS: For our study, plasma and PBMC were obtained from 60 MS patients (30 with lifetime MDD) in remission phase. The PBMC cultures were stimulated with anti-CD3/anti-CD28 beads or myelin basic protein (MBP), and effector and regulatory T cell phenotypes were determined by flow cytometry. The cytokine levels, both in the plasma or in the supernatants collected from PBMC cultures, were quantified by Luminex. In some experiments, the effect of serotonin (5-HT) was investigated. RESULTS: Here, higher Th17-related cytokine levels in response to anti-CD3/anti-CD28 and MBP were quantified in the plasma and PBMC cultures of the MS/MDD group in comparison with MS patients. Further, elevated frequency of CD4+ and CD8+ T cells capable of producing IL-17, IL-22 and GM-CSF was observed in depressed patients. Interestingly, the percentage of myelin-specific IFN-γ+IL-17+ and IFN-γ+GM-CSF+ CD4+ T cells directly correlated with neurological disabilities. In contrast, the occurrence of MDD reduced the proportion of MBP-specific CD39+Tregs subsets. Notably, the severity of both neurological disorder and depressive symptoms inversely correlated with these Tregs. Finally, the addition of 5-HT downregulated the release of Th17-related cytokines in response to anti-CD3/anti-CD28 and myelin antigen. CONCLUSIONS: In summary, our findings suggested that recurrent major depression, by favoring imbalances of effector Th17 and Treg cell subsets, contributes to MS severity.

Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases.

COVID-19 is a disease characterized by acute respiratory failure and is a major health problem worldwide. Here, we aimed to investigate the role of CD39 expression in Treg cell subsets in COVID-19 immunopathogenesis and its relationship to disease severity. One hundred and ninety COVID-19 patients (juveniles, adults) and 43 volunteers as healthy controls were enrolled in our study. Flow cytometric analysis was performed using a 10-color monoclonal antibody panel from peripheral blood samples. In adult patients, CD39+ Tregs increased with disease severity. In contrast, CD39+ Tregs were decreased in juvenile patients in an age-dependent manner. Overall, our study reveals an interesting profile of CD39-expressing Tregs in adult and juvenile cases of COVID-19. Our results provide a better understanding of the possible role of Tregs in the mechanism of immune response in COVID-19 cases.

Systematic analysis of CD39, CD103, CD137, and PD-1 as biomarkers for naturally occurring tumor antigen-specific TILs.

The detection of tumor-specific T cells in solid tumors is integral to interrogate endogenous antitumor responses and to advance downstream therapeutic applications. Multiple biomarkers are reported to identify endogenous tumor-specific tumor-infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39; however, a direct comparison of these molecules has yet to be performed. We evaluated these biomarkers in primary human ovarian tumor samples using single-cell mass cytometry to compare their relative phenotypic profiles, and examined their response to autologous tumor cells ex vivo. PD-1+ , CD103+ , and CD39+ TILs all contain a CD137+ cell subset, while CD137+ TILs highly co-express the aforementioned markers. CD137+ TILs exhibit the highest expression of cytotoxic effector molecules compared to PD-1+ , CD103+ , or CD39+ TILs. Removal of CD137+ cells from PD-1+ , CD103+ , or CD39+ TILs diminish their IFN-γ secretion in response to autologous tumor cell stimulation, while CD137+ TILs maintain high HLA-dependent IFN-γ secretion. CD137+ TILs exhibited an exhausted phenotype but with CD28 co-expression, suggesting possible receptiveness to reinvigoration via immune checkpoint blockade. Together, our findings demonstrate that the antitumor abilities of PD-1+ , CD103+ , and CD39+ TILs are mainly derived from a subset of CD137-expressing TILs, implicating CD137 as a more selective biomarker for naturally occurring tumor-specific TILs.

Porcine-Stimulated Human Tr1 Cells Showed Enhanced Suppression in Xenoantigen Stimulation Response.

Type 1 regulatory T (Tr1) cells play a fundamental role in maintaining and inducing immune tolerance. Our preliminary study demonstrated that an interleukin- (IL-) 10-mediated pathway is a possible regulatory mechanism underlying the xenoantigen-specific human Treg enhanced suppressive capacity. Here, we developed a feasible protocol for expanding IL-10-induced xenoantigen-specific human Tr1 cells in vitro which would be more efficient in transplantation immunotherapy efficiency. In this study, xenoantigen-specific Tr1 cells are generated from human naive CD4+ T cells expanded for two subsequent xenoantigen-stimulation cycles with recombinant human IL-10. The phenotype and suppressive capacity of xenoantigen-stimulated Tr1 cells are assessed, and the mechanism of their suppression is studied. Tr1 cells can be induced by porcine xenoantigen stimulation combined with IL-10, IL-2, and IL-15, displaying an increased expression of CD49b, CTLA-4, and LAG-3 without expressing Foxp3 which also showed an effector memory Treg phenotype and expressed high levels of CD39. After xenoantigen stimulation, the IL-10 and IL-5 gene expression in Tr1 cells increased, secreting more IL-10, and xenoantigen-stimulated Tr1 cells changed their T cell receptor (TCR) Vß repertoire, increasing the expression of TCR Vß2, TCR Vß9, and TCR Vß13. In a pig to human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Tr1 cells displayed enhanced suppressive capacity via CD39 in a dose-dependent manner. Moreover, IL-5 could affect the proliferation of xenoantigen-specific Tr1 cells, but not their phenotypes' expression. This study provides a theory and feasible method for immune tolerance induction in clinical xenotransplantation.

Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Inhibitory Molecules PD-1, CD73 and CD39 Are Expressed by CD8+ T Cells in a Tissue-Dependent Manner and Can Inhibit T Cell Responses to Stimulation.

The salivary gland is an important tissue for persistence and transmission of multiple viruses. Previous work showed that salivary gland tissue-resident CD8+ T cells elicited by viruses were poorly functional ex vivo. Using a model of persistent murine cytomegalovirus (MCMV) infection, we now show that CD8+ T cells in the salivary gland and other non-lymphoid tissues of mice express multiple molecules associated with T cell exhaustion including PD-1, CD73 and CD39. Strikingly however, these molecules were expressed independently of virus or antigen. Rather, PD-1-expressing T cells remained PD-1+ after migration into tissues regardless of infection, while CD73 was activated on CD8+ T cells by TGF-ß signaling. Blockade of PD-L1, but not CD73, improved cytokine production by salivary gland T cells ex vivo and increased the expression of granzyme B after stimulation within the salivary gland. Nevertheless, salivary-gland localized CD8+ T cells could kill PD-L1-expressing targets in vivo, albeit with modest efficiency, and this was not improved by PD-L1 blockade. Moreover, the impact of PD-L1 blockade on granzyme B expression waned with time. In contrast, the function of kidney-localized T cells was improved by CD73 blockade, but was unaffected by PD-L1 blockade. These data show that tissue localization per se is associated with expression of inhibitory molecules that can impact T cell function, but that the functional impact of this expression is context- and tissue-dependent.

Ovarian Cancer-Associated Ascites Have High Proportions of Cytokine-Responsive CD56bright NK Cells.

Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients' blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.

CD39 Regulation and Functions in T Cells.

CD39 is an enzyme which is responsible, together with CD73, for a cascade converting adenosine triphosphate into adenosine diphosphate and cyclic adenosine monophosphate, ultimately leading to the release of an immunosuppressive form of adenosine in the tumor microenvironment. Here, we first review the environmental and genetic factors shaping CD39 expression. Second, we report CD39 functions in the T cell compartment, highlighting its role in regulatory T cells, conventional CD4+ T cells and CD8+ T cells. Finally, we compile a list of studies, from preclinical models to clinical trials, which have made essential contributions to the discovery of novel combinatorial approaches in the treatment of cancer.

Regulation of immune responses through CD39 and CD73 in cancer: Novel checkpoints.

The immunosuppressive tumor microenvironment has been implicated in attenuating anti-tumoral immune responses and tumor growth in various cancers. Inhibitory immune checkpoints have been introduced as the primary culprits for developing the immunosuppressive tumor microenvironment. Therefore, a better understanding of the cross-talk between inhibitory immune checkpoints in the tumor microenvironment can pave the way for introducing novel approaches for treating affected patients. Growing evidence indicates that CD39 and CD73, as novel checkpoints, can transform adenosine triphosphate (ATP)-mediated pro-inflammatory tumor microenvironment into an adenosine-mediated immunosuppressive one via the purinergic signaling pathway. Indeed, enzymatic processes of CD39 and CD73 have crucial roles in adjusting the extent, intensity, and chemical properties of purinergic signals. This study aims to review the biological function of CD39 and CD73 and shed light on their significance in regulating anti-tumoral immune responses in various cancers.