Immune related endonucleases and GTPases are not associated with tumor response in patients with advanced non-small cell lung cancer treated with checkpoint inhibitors.

Immune related endonucleases have recently been described as potential therapeutic targets and predictors of response to treatment with immune checkpoint inhibitors (ICI). The aim is to evaluate the association between the expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d and Cytohesin-3) in parallel with the more common ICI-predictive markers, PD-L1 expression and Tumor Mutation Burden (TMB) with response to ICI therapy in an advanced non-small cell lung cancer (NSCLC) cohort. METHODS: Patients with advanced NSCLC treated with ICI single agent were divided into responders and non-responders according to RECIST v1.1 and duration of response (DOR) criteria. Immunohistochemistry was performed on pretreatment tumor tissue samples for PD-L1, CD73, CD39, VISTA, Arl4d, and Cytohesin-3 expression. TMB was estimated with NEOplus v2 RUO (NEO New Oncology GmbH) hybrid capture next generation sequencing assay. Resistance mutations in STK11/KEAP1 and positive predictive mutations in ARID1A/POLE were also evaluated. RESULTS: Included were 56 patients who were treated with ICI single agent. The median progression-free and overall survival for the whole cohort was 3.0 (95% CI, 2.4-3.6) and 15 (95% CI, 9.7-20.2) months, respectively. The distribution of CD73 in tumor cells and CD39, VISTA, Arl4d and Cytohesin-3 expression in immune cells were not different between responders and non-responders. Also, PD-L1 and TMB were not predictive for response. The frequency of STK11, KEAP1 and ARID1A mutations was low and only observed in the non-responder group. CONCLUSION: Separate and combined expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d, and Cytohesin-3) was not associated with response in our cohort of advanced NSCLC patients receiving single agent ICI. To confirm our findings the analysis of independent larger cohorts is warranted.

Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens.

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.

Flow cytometric expression of CD71, CD81, CD44 and CD39 in B cell lymphoma.

Flow cytometry is a useful ancillary tool for the diagnosis of nodal B cell lymphomas. Well-established antigens have diagnostic limitations. This study aimed to assess the expression of CD71, CD81, CD44 and CD39 by flow cytometry in B cell lymphomas. Expression of these 4 antigens was queried in 185 samples with a diagnosis of a B cell lymphoma according to a histological examination of the lymph node and the World Health Organization (WHO) classification (follicular lymphoma [FL, n = 96], diffuse large B cell lymphoma/High grade B cell lymphoma [DLBCL/HGBH, n = 48], marginal zone lymphoma/lymphoplasmacytic lymphoma [MZL/LPL, n = 14], chronic lymphocytic leukemia/small lymphocytic lymphoma [CLL, n = 10], mantle cell lymphoma [MCL, n = 11], Burkitt lymphoma [BL, n = 4] and other [n = 2]). CD81 was bright and CD44 was dim in germinal center-derived malignancies, particularly aggressive lymphomas (BL and CD10-positive DLBCL/HGBL). CD81 was very dim in CLL. CD71 was bright in aggressive lymphomas (DLBCL/HGBL and BL). CD39 was bright in CD10-negative DLBCL. CD71 appeared valuable in the differential diagnosis between indolent and aggressive lymphomas, CD39 between CD10-negative DLBCL and MZL/LPL and CD81 between MCL and CLL. To conclude, we report the expression of CD71, CD81, CD44 and CD39 by FC in B cell lymphomas. Further studies will have to determine the value they add to specific FC panels.

ENTPD1 (CD39) Expression Inhibits UVR-Induced DNA Damage Repair through Purinergic Signaling and Is Associated with Metastasis in Human Cutaneous Squamous Cell Carcinoma.

UVR and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells promote cSCC carcinogenesis, and in other solid tumors, infiltrating regulatory T cells and CD8+ T cells express ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39), an ectoenzyme that catalyzes the rate-limiting step in converting extracellular adenosine triphosphate (ATP) to extracellular adenosine (ADO). We previously showed that extracellular purine nucleotides influence DNA damage repair. In this study, we investigate whether DNA damage repair is modulated through purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared with the expression on T cells from blood or nonlesional skin, and accordingly, concentrations of derivative extracellular adenosine diphosphate (ADP), adenosine monophosphate (AMP), and ADO are increased in tumors compared with those in normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize than in those that are nonmetastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL-27-dependent manner. Finally, increased extracellular ADO is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DNA damage repair secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DNA damage repair through purinergic signaling to promote skin carcinogenesis and metastasis.

Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer.

Adoptive T cell therapy (ACT) using ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate complete regression of certain human cancers. The impact of TIL phenotypes on clinical success of TIL-ACT is currently unclear. Using high-dimensional analysis of human ACT products, we identified a memory-progenitor CD39-negative stem-like phenotype (CD39-CD69-) associated with complete cancer regression and TIL persistence and a terminally differentiated CD39-positive state (CD39+CD69+) associated with poor TIL persistence. Most antitumor neoantigen-reactive TILs were found in the differentiated CD39+ state. However, ACT responders retained a pool of CD39- stem-like neoantigen-specific TILs that was lacking in ACT nonresponders. Tumor-reactive stem-like TILs were capable of self-renewal, expansion, persistence, and superior antitumor response in vivo. These data suggest that TIL subsets mediating ACT response are distinct from TIL subsets enriched for antitumor reactivity.

Human platelets express functional ectonucleotidases that restrict platelet activation signaling.

Platelets play central role in thrombosis and haemostasis. Platelets store adenine nucleotides in their dense granules, which are released upon agonist-stimulation. Level of these nucleotides in extracellular fluid is regulated by activities of ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73) expressed on platelet surface. Here we demonstrate that, expression of surface-bound ectonucleotidases rose significantly in platelets, concomitant with upregulation of their enzymatic activities, when cells were stimulated with thrombin. Interestingly, inhibition of CD73 in thrombin-treated platelets led to enhanced tyrosine phosphorylation of proteins and rise in intracellular free calcium, [Ca2+]i, thus signifying the inhibitory role of the ectonucleotidase on agonist-mediated platelet signaling.

Sergentomyia schwetzi: Salivary gland transcriptome, proteome and enzymatic activities in two lineages adapted to different blood sources.

During the blood feeding, sand fly females inject saliva containing immunomodulatory and anti-haemostatic molecules into their vertebrate hosts. The saliva composition is species-specific, likely due to an adaptation to particular haemostatic pathways of their preferred host. Research on sand fly saliva is limited to the representatives of two best-studied genera, Phlebotomus and Lutzomyia. Although the members of the genus Sergentomyia are highly abundant in many areas in the Old World, their role in human disease transmission remains uncertain. Most Sergentomyia spp. preferentially attack various species of reptiles, but feeding on warm-blooded vertebrates, including humans and domestic animals, has been repeatedly described, especially for Sergentomyia schwetzi, of which salivary gland transcriptome and proteome is analyzed in the current study. Illumina RNA sequencing and de novo assembly of the reads and their annotation revealed 17,293 sequences homologous to other arthropods' proteins. In the sialome, all proteins typical for sand fly saliva were identified-antigen 5-related, lufaxin, yellow-related, PpSP15-like, D7-related, ParSP25-like, and silk proteins, as well as less frequent salivary proteins included 71kDa-like, ParSP80-like, SP16-like, and ParSP17-like proteins. Salivary enzymes include apyrase, hyaluronidase, endonuclease, amylase, lipase A2, adenosine deaminase, pyrophosphatase, 5'nucleotidase, and ribonuclease. Proteomics analysis of salivary glands identified 631 proteins, 81 of which are likely secreted into the saliva. We also compared two S. schwetzi lineages derived from the same origin. These lineages were adapted for over 40 generations for blood feeding either on mice (S-M) or geckos (S-G), two vertebrate hosts with different haemostatic mechanisms. Altogether, 20 and 40 annotated salivary transcripts were up-regulated in the S-M and S-G lineage, respectively. Proteomic comparison revealed ten salivary proteins more abundant in the lineage S-M, whereas 66 salivary proteins were enriched in the lineage S-G. No difference between lineages was found for apyrase activity; contrarily the hyaluronidase activity was significantly higher in the lineage feeding on mice.

Enhanced Phenotype Definition for Precision Isolation of Precursor Exhausted Tumor-Infiltrating CD8 T Cells.

In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo. PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve in vivo persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in Slamf6 and lack Entpd1 and Havcr2 expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39- Tim3- Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39- Tim3- Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and in vivo persistence of transferred cells in the context of ACT.

Deficient Resident Memory T Cell and CD8 T Cell Response to Commensals in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The intestinal microbiota is closely associated with resident memory lymphocytes in mucosal tissue. We sought to understand how acquired cellular and humoral immunity to the microbiota differ in health versus inflammatory bowel disease [IBD]. METHODS: Resident memory T cells [Trm] in colonic biopsies and local antibody responses to intraepithelial microbes were analysed. Systemic antigen-specific immune T and B cell memory to a panel of commensal microbes was assessed. RESULTS: Systemically, healthy blood showed CD4 and occasional CD8 memory T cell responses to selected intestinal bacteria, but few memory B cell responses. In IBD, CD8 memory T cell responses decreased although B cell responses and circulating plasmablasts increased. Possibly secondary to loss of systemic CD8 T cell responses in IBD, dramatically reduced numbers of mucosal CD8+ Trm and γδ T cells were observed. IgA responses to intraepithelial bacteria were increased. Colonic Trm expressed CD39 and CD73 ectonucleotidases, characteristic of regulatory T cells. Cytokines/factors required for Trm differentiation were identified, and in vitro-generated Trm expressed regulatory T cell function via CD39. Cognate interaction between T cells and dendritic cells induced T-bet expression in dendritic cells, a key mechanism in regulating cell-mediated mucosal responses. CONCLUSIONS: A previously unrecognised imbalance exists between cellular and humoral immunity to the microbiota in IBD, with loss of mucosal T cell-mediated barrier immunity and uncontrolled antibody responses. Regulatory function of Trm may explain their association with intestinal health. Promoting Trm and their interaction with dendritic cells, rather than immunosuppression, may reinforce tissue immunity, improve barrier function, and prevent B cell dysfunction in microbiota-associated disease and IBD aetiology.

Development of a selective and highly sensitive fluorescence assay for nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39).

Ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5'-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z'-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 µM, and a kcat value of 119 × 10-3 s-1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 µM and 82.5 × 10-3 s-1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.

Regulatory T cells and asthma.

Asthma is a chronic disease of airway inflammation due to excessive T helper cell type 2 (Th2) response. Present treatment based on inhalation of synthetic glucocorticoids can only control Th2-driven chronic eosinophilic inflammation, but cannot change the immune tolerance of the body to external allergens. Regulatory T cells (Tregs) are the main negative regulatory cells of the immune response. Tregs play a great role in regulating allergic, autoimmune, graft-versus-host responses, and other immune responses. In this review, we will discuss the classification and biological characteristics, the established immunomodulatory mechanisms, and the characteristics of induced differentiation of Tregs. We will also discuss the progress of Tregs in the field of asthma. We believe that further studies on the regulatory mechanisms of Tregs will provide better treatments and control strategies for asthma.

Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates.

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types1-4. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients5,6. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control2,4,7-10, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8+ TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein-Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8+ TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8+ TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39- CD8+ TILs. Furthermore, frequencies of CD39 expression among CD8+ TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells.

Functional heterogeneity of circulating T regulatory cell subsets in breast cancer patients.

BACKGROUND: Regulatory T cells (Tregs) play a major role in tumor escape from immunosurveillance by suppressing effector cells. The number of Tregs is increased in tumor sites and peripheral blood of breast cancer patients. However, the data regarding phenotypic and functional heterogeneity of Treg subpopulations in breast cancer are limited. The present study aimed to investigate the number and suppressive potential of Tregs that possess natural naïve-(N nTregs), effector/memory-like (EM nTregs), and Tr1-like phenotypes in breast cancer patients and healthy women. METHODS: The study included 10 HW and 17 primary breast cancer patients. Numbers of CD4+CD25+FoxP3+CD45RA+ N nTregs, CD4+CD25+FoxP3+CD45RA- EM nTregs, and CD4+IL-4-IL-10+ Tr1 subsets and the expression of CTLA-4, CD39, GITR, LAP, and IL-35 by these Treg subsets were measured in freshly obtained peripheral blood by flow cytometry. RESULTS: Herein, we demonstrate that the percentages of N nTregs, EM nTregs, CD25+ and FoxP3+ Tr1 cells are elevated in the peripheral blood of breast cancer patients, but do not correlate with cancer stages. Nevertheless, the frequency of CD25+ Tr1 cells was associated with nodal involvement, while the number of EM nTregs correlated with clinical outcome. The expression of CTLA-4 and IL-35 by all assessed Treg subsets was increased throughout all tumor stages (I-III). CONCLUSIONS: Collectively, the current study shows phenotypic alterations in suppressive receptors of Treg subsets, suggesting that breast cancer patients have increased activity of N nTregs, EM nTregs and Tr1 cells; and EM nTregs and CD25+ Tr1 cells represent prospective markers for assessing disease prognosis.

Simultaneous accurate quantification of HO-1, CD39, and CD73 in human calcified aortic valves using multiple enzyme digestion - filter aided sample pretreatment (MED-FASP) method and targeted proteomics.

Several proteins such as membrane-associated ectonucleotidases: ecto-5'-nucleotidase (E5NT/CD73) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), and intracellular heme oxygenase-1 (HO-1) may contribute to protection from inflammation-related diseases such as calcific aortic valve stenosis (CAS). Accurate quantification of these proteins could contribute to better understanding of the disease mechanisms and identification of biomarkers. This report presents development and validation of quantification method for E5NT/CD73, ENTPD1/CD39 and HO-1. The multiplexed targeted proteomic assay involved antibody-free, multiple-enzyme digestion, filter-assisted sample preparation (MED-FASP) strategy and a nanoflow liquid chromatography/mass spectrometry under multiple reaction monitoring mode (LC-MRM/MS). The method developed presented high sensitivity (LLOQ of 5 pg/mL for each of the analytes) and accuracy that ranged from 92.0% to 107.0%, and was successfully applied for the absolute quantification of HO-1, CD39 and CD73 proteins in homogenates of human calcified and non-calcified valves. The absolute CD39 and CD73 concentrations were lower in calcified aortic valves (as compared to non-stenotic ones) and were found to be: 1.16 ± 0.39 vs. 3.15 ± 0.37 pmol/mg protein and 1.94 ± 0.21 vs. 2.39 ± 0.39 pmol/mg protein, respectively, while the quantity of HO-1 was elevated in calcified valves (10.72 ± 1.18 vs. 4.28 ± 0.42 amol/mg protein). These results were consistent but more reproducible as compared to immunoassays. In conclusion, multiplexed quantification of HO-1, CD39 and CD73 proteins by LC-MRM/MS works well in challenging human tissues such as aortic valves. This analysis confirmed the relevance of these proteins in pathogenesis of CAS and could be extended to other biomedical investigations.

Changes in CD73, CD39 and CD26 expression on T-lymphocytes of ANCA-associated vasculitis patients suggest impairment in adenosine generation and turn-over.

Extracellular adenosine, generated via the concerted action of CD39 and CD73, contributes to T-cell differentiation and function. Adenosine concentrations are furthermore influenced by adenosine deaminase binding protein CD26. Because aberrant T-cell phenotypes had been reported in anti-neutrophil cytoplasmic auto-antibody (ANCA)-associated vasculitis (AAV) patients, an impaired expression of these molecules on T-cells of AAV patients was hypothesized in the present study. While in AAV patients (n = 29) CD26 was increased on CD4+ lymphocytes, CD39 and CD73 were generally reduced on patients' T-cells. In CD4+ cells significant differences in CD73 expression were confined to memory CD45RA- cells, while in CD4- lymphocytes differences were significant in both naïve CD45RA+ and memory CD45RA- cells. The percentage of CD4-CD73+ cells correlated with micro-RNA (miR)-31 expression, a putative regulator of factor inhibiting hypoxia-inducible factor 1 alpha (FIH-1), inversely with serum C-reactive protein (CRP) and positively with estimated glomerular filtration rate (eGFR). No correlation with disease activity, duration, and ANCA profile was found. It remains to be assessed if a decreased CD73 and CD39 expression underlies functional impairment of lymphocytes in AAV patients. Likewise, the relations between frequencies of CD4-CD73+ cells and serum CRP or eGFR require further functional elucidation.

A self-assembled amphiphilic imidazolium-based ATP probe.

A novel amphiphilic imidazolium-based probe containing a dansyl fluorophore and a long cetyl chain has been developed for ATP recognition. The probe forms self-assembled micelle-like aggregates at low concentration in its aqueous solution and can selectively recognize ATP among other bioactive anions with a significant enhancement in fluorescence emission.

Characteristics of peripheral blood CD4+CD25+ regulatory T cells and related cytokines in severe atopic dermatitis.

Regulatory T cells (Tregs) have been suggested to play a role in the pathogenesis of atopic dermatitis (AD). However, alterations in the ability of Tregs remain to be determined. To investigate the expression of various surface receptors on CD4(+)CD25(high) regulatory T cells and to investigate their capacity for inhibiting the proliferation of CD4(+) CD25(-) effector T cells (Teffs). Peripheral blood samples were obtained from 15 patients with severe atopic dermatitis (AD) and 20 control subjects. FACs was then carried out to analyze the expression levels of FoxP3, CD152 (CTLA-4), CD39, CD73, CD223 (LAG-3), CCR4, CCR5, and CCR10 on Tregs. The proliferative responses of Teffs were assessed in the absence or presence of autologous Tregs and the TGF-ß1 and IL-10 levels in the culture supernatant and sera were detected by enzyme-linked immunosorbent assay (ELISA). The CD152, CD39, CD73, CCR4, and CCR5 expression levels on Tregs were higher in patients with severe AD than in the controls. Tregs showed an attenuated suppressive function of the proliferation of autologous Teffs in severe AD. The concentrations of IL-10 and TGF-ß in the culture supernatants of Tregs were lower in the AD group than in the control. The attenuated ability of Tregs to suppress Teff proliferation may be responsible for the autoimmune reaction of severe AD.

A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39(+)Foxp3(+) T cells and Treg function.

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (Tregs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3(+) Tregs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3(+) Tregs, PSA enriched Foxp3 expression, enhanced the frequency of CD39(+)HLA-DR(+) cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4(+)Foxp3(+) T cells and enhanced suppressive function of circulating Foxp3(+) Tregs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.

High circulating CD39(+) regulatory T cells predict poor survival for sepsis patients.

BACKGROUND: Sepsis encompasses two phases, the 'hyper'-reactive phase and the 'hypo'-reactive phase. The initial inflammatory stage is quickly counterbalanced by an anti-inflammatory response, which compromises the immune system, leading to immune suppression. Regulatory T cells (Tregs) have been implicated in the pathogenesis of sepsis by inducing immunosuppression; however, the role of CD39(+) Tregs in the process of sepsis is uncertain. This study investigated the dynamic levels of CD39(+) Tregs and their phenotypic change in sepsis. METHODS: Fourteen patients with systemic inflammatory response syndrome (SIRS), 42 patients with sepsis, and 14 healthy controls were enrolled. Sequential blood samples were used to analyze the numbers of CD39(+) Tregs and their phenotypic changes. Survival at 28 days was used to evaluate the capacity of CD39(+) Treg levels to predict mortality in sepsis patients. RESULTS: Sepsis patients displayed a high percentage (3.13%, 1.46%, and 0.35%, respectively) and mean fluorescence intensity (MFI) (59.65, 29.7, and 24.3, respectively) of CD39(+) Tregs compared with SIRS patients and healthy subjects. High-level expression of CD39(+) Tregs was correlated with the severity of sepsis, which was reflected by the sepsis-related organ failure assessment score (r=0.322 and r=0.31, respectively). In addition, the expression of CD39(+) Tregs was associated with survival of sepsis patients (p<0.01). By receiver-operating characteristic (ROC) curve analysis, the percentage and MFI of CD39(+) Tregs showed similar sensitivities and specificities to predict mortality (74.2% and 85.1%, and 73.9% and 84.1%, respectively). Using Kaplan-Meier curves to assess the impact of CD39(+) Tregs percentage and MFI on overall survival, we found that a high CD39(+) Tregs percentage (p<0.001; >4.1%) and MFI (p<0.001; >49.2) were significantly associated with mortality. Phenotypically, CD39(+) Tregs from sepsis patients showed high expression of CD38 and PD-1 (p<0.01 and p<0.01 respectively). CONCLUSIONS: Increased expression of CD39(+) Tregs was associated with a poor prognosis for sepsis patients, which suggests that CD39(+) Treg levels could be used as a biomarker to predict the outcome of sepsis patients.

Overexpression of CD39 and high tumoral CD39⁺/CD8⁺ ratio are associated with adverse prognosis in resectable gastric cancer.

CD39/ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) is a cell surface-located, rate-limiting enzyme in the generation of adenosine, and plays a crucial role in tumor development. We examined co-expression of CD39 and CD8in gastric cancer (GC) and showed that the expression of CD39 and CD8 increased significantly in tumor tissues compared to paired peritumor tissues. The expression of tumoral CD39 (tCD39), but not tumoral CD8 (tCD8), was related to overall survival. Furthermore, the CD39(+)/CD8(+) ratio was associated with poor prognosis in resected GC patients. Taken together, our data indicate that highCD39 expression and high tCD39(+)/CD8(+) ratio in GC is a predictor of poor prognosis for GC patients after radical resection. Moreover, CD39 could serve as a potential target for cancer immunotherapy.