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Abstract The presence of Salmonella in dry fermented sausages is source of recalls and outbreaks. The genomic diversity of 173 Salmonella isolates from the dry fermented sausage production chains (pig carcasses, pork, and sausages) from France and Spain were investigated through their core phylogenomic relationships and accessory genome profiles. Ten different serovars and thirteen sequence type profiles were identified. The most frequent serovar from sausages was the monophasic variant of S. Typhimurium (1,4,[5],12:i:-, 72%) while S. Derby was in pig carcasses (51%). Phylogenomic clusters found in S. 1,4,[5],12:i:-, S. Derby, S. Rissen and S. Typhimurium serovars identified closely related isolates, with less than 10 alleles and 20 SNPs of difference, displaying Salmonella persistence along the pork production chain. Most of the S. 1,4,[5],12:i:- contained the Salmonella genomic island-4 (SGI-4), Tn21 and IncFIB plasmid. More than half of S. Derby strains contained the SGI-1 and Tn7. S. 1,4,[5],12:i:- genomes carried the most multidrug resistance genes (91% of the strains), whereas extended-spectrum β-lactamase genes were found in Typhimurium and Derby serovars. Salmonella monitoring and characterization in the pork production chains, specially S. 1,4,[5],12:i:- serovar, is of special importance due to its multidrug resistance capacity and persistence in dry fermented sausages.

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.

An increase in the extracellular concentration of ATP as a consequence of cellular stress or cell death results in the activation of immune cells. To prevent inflammation, extracellular ATP is rapidly metabolized to adenosine, which deploys an anti-inflammatory signaling cascade upon binding to P1 receptors on immune cells. The ectonucleotidases necessary for the degradation of ATP and generation of adenosine are present on the cell membrane of many immune cells, and their expression is tightly regulated under conditions of inflammation. The discovery that extracellular vesicles (EVs) carry purinergic enzyme activity has brought forward the concept of EVs as a new player in immune regulation. Adenosine-generating EVs derived from cancer cells suppress the anti-tumor response, while EVs derived from immune or mesenchymal stem cells contribute to the restoration of homeostasis after infection. Here we will review the existing knowledge on EVs containing purinergic enzymes and molecules, and discuss the relevance of these EVs in immune modulation and their potential for therapy.

Summary: Type I interferons (IFN-I) are important mediators of antiviral immunity and autoimmune diseases. Female plasmacytoid dendritic cells (pDCs) exert an elevated capacity to produce IFN-I upon toll-like receptor 7 (TLR7) activation compared to male pDCs, and both sex hormones and X-encoded genes have been implicated in these sex-specific differences. Using longitudinal samples from a trans men cohort receiving gender-affirming hormone therapy (GAHT), the impact of testosterone injections on TLR7-mediated IFN-I production by pDCs was assessed. Single-cell RNA analyses of pDCs showed downregulation of IFN-I-related gene expression signatures but also revealed transcriptional inter-donor heterogeneity. Longitudinal quantification showed continuous reduction of IFN-I protein production by pDCs and reduced expression of IFN-I-stimulated genes in peripheral blood mononuclear cells (PBMCs). These studies in trans men demonstrate that testosterone administration reduces IFN-I production by pDCs over time and provide insights into the immune-modulatory role of testosterone in sex-specific IFN-I-mediated immune responses.

Abstract Background Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel. Methods Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1β release after incubation with the P2X7-specific nbs. Results Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1β release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood–brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size. Conclusion Mechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage.

Ectonucleotidases associated to regulatory T cells are known modulators in the inflammatory environment. Here the authors describe CD8 T cell-derived extracellular vesicles bearing CD73 and suggest they function as an additional intrinsic modulator of immune responses.

Abstract Background: Hamburg is a city state of approximately 1.9 Mio inhabitants in Northern Germany. Currently, the COVID‐19 epidemic that had largely subsided during last summer is resurging in Hamburg and in other parts of the world, underlining the need for additional tools to monitor SARS‐CoV‐2 antibody responses. Aim: We aimed to develop and validate a simple, low‐cost assay for detecting antibodies against the native coronavirus 2 spike protein (CoV‐2 S) that does not require recombinant protein or virus. Method: We transiently co‐transfected HEK cells or CHO cells with expression vectors encoding CoV‐2 S and nuclear GFP. Spike protein‐specific antibodies in human serum samples bound to transfected cells were detected with fluorochrome conjugated secondary antibodies by flow cytometry orimmunofluorescence microscopy. We applied this assay to monitor antibody development in COVID‐19 patients, household contacts, and hospital personnel during the ongoing epidemic in the city state of Hamburg. Results: All recovered COVID‐19 patients showed high levels of CoV‐2 S‐specific antibodies. With one exception, all household members that did not develop symptoms also did not develop detectable antibodies. Similarly, lab personnel that worked during the epidemic and followed social distancing guidelines remained antibody‐negative. Conclusion: We conclude that high‐titer CoV‐2 S‐specific antibodies are found in most recovered COVID‐19 patients and in symptomatic contacts, but only rarely in asymptomatic contacts. The assay may help health care providers to monitor disease progression and antibody responses in vaccination trials, to identify health care personnel that likely are resistant to re‐infection, and recovered individuals with high antibody titers that may be suitable asplasma and/or antibody donors.

The search for new and effective treatment targets for cancer immunotherapy is an ongoing challenge. Alongside the more established inhibitory immune checkpoints, a novel potential target is CD73. As one of the key enzymes in the purinergic signalling pathway CD73 is responsible for the generation of immune suppressive adenosine. The expression of CD73 is higher in tumours than in the corresponding healthy tissues and associated with a poor prognosis. CD73, mainly by the production of adenosine, is critical in the suppression of an adequate anti-tumour immune response, but also in promoting cancer cell proliferation, tumour growth, angiogenesis, and metastasis. The upregulation of CD73 and generation of adenosine by tumour or tumour-associated immune cells is a common resistance mechanism to many cancer treatments such as chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Therefore, the inhibition of CD73 represents a new and promising approach to increase therapy efficacy. Several CD73 inhibitors have already been developed and successfully demonstrated anti-cancer activity in preclinical studies. Currently, clinical studies evaluate CD73 inhibitors in different therapy combinations and tumour entities. The initial results suggest that inhibiting CD73 could be an effective option to augment anti-cancer immunotherapeutic strategies. This review provides an overview of the rationale behind the CD73 inhibition in different treatment combinations and the role of CD73 as a prognostic marker.

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.

In multiple sclerosis (MS), relapse rate is decreased by 70-80% in the third trimester of pregnancy. However, the underlying mechanisms driving this effect are poorly understood. Evidence suggests that CD56bright NK cell frequencies increase during pregnancy. Here, we analyze pregnancy-related NK cell shifts in a large longitudinal cohort of pregnant women with and without MS, and provide in-depth phenotyping of NK cells. In healthy pregnancy and pregnancy in MS, peripheral blood NK cells showed significant frequency shifts, notably an increase of CD56bright NK cells and a decrease of CD56dim NK cells toward the third trimester, indicating a general rather than an MS-specific phenomenon of pregnancy. Additional follow-ups in women with MS showed a reversal of NK cell changes postpartum. Moreover, high-dimensional profiling revealed a specific CD56bright subset with receptor expression related to cytotoxicity and cell activity (e.g., CD16+ NKp46high NKG2Dhigh NKG2Ahigh phenotype) that may drive the expansion of CD56bright NK cells during pregnancy in MS. Our data confirm that pregnancy promotes pronounced shifts of NK cells toward the regulatory CD56bright population. Although exploratory results on in-depth CD56bright phenotype need to be confirmed in larger studies, our findings suggest an increased regulatory NK activity, thereby potentially contributing to disease amelioration of MS during pregnancy.

SARS-CoV-2 is still a major burden for global health despite effective vaccines. With the reduction of social distancing measures, infection rates are increasing in children, while data on the pediatric immune response to SARS-CoV-2 infection is still lacking. Although the typical disease course in children has been mild, emerging variants may present new challenges in this age group. Peripheral blood mononuclear cells (PBMC) from 51 convalescent children, 24 seronegative siblings from early 2020, and 51 unexposed controls were stimulated with SARS-CoV-2-derived peptide MegaPools from the ancestral and beta variants. Flow cytometric determination of activation-induced markers and secreted cytokines were used to quantify the CD4+ T cell response. The average time after infection was over 80 days. CD4+ T cell responses were detected in 61% of convalescent children and were markedly reduced in preschool children. Cross-reactive T cells for the SARS-CoV-2 beta variant were identified in 45% of cases after infection with an ancestral SARS-CoV-2 variant. The CD4+ T cell response was accompanied most predominantly by IFN-γ and Granzyme B secretion. An antiviral CD4+ T cell response was present in children after ancestral SARS-CoV-2 infection, which was reduced in the youngest age group. We detected significant cross-reactivity of CD4+ T cell responses to the more recently evolved immune-escaping beta variant. Our findings have epidemiologic relevance for children regarding novel viral variants of concern and vaccination efforts.

Summary: Extracellular ATP released to the ischemic brain parenchyma is quickly metabolized by ectonucleotidases. Among them, the ecto-5′-nucleotidase CD73 encoded by Nt5e generates immunosuppressive adenosine. Genetic deletion of Nt5e led to increased infarct size in the murine photothrombotic stroke model. We aimed at validating this result using the transient middle cerebral artery occlusion (tMCAO) stroke model that represents pathophysiological aspects of penumbra and reperfusion. Three days after tMACO, we did not detect a difference in stroke size between CD73-deficient (CD73−/−) and control mice. Consistent with this finding, CD73−/− and control mice showed comparable numbers and composition of brain-infiltrating leukocytes measured by flow cytometry. Using NanoString technology, we further demonstrated that CD73−/− and control mice do not differ regarding glia cell gene expression profiles. Our findings highlight the potential impact of stroke models on study outcome and the need for cross-validation of originally promising immunomodulatory candidates.

CD4+ T helper cells producing IL-17A (TH17 cells) can take on pathogenic or anti-inflammatory functions in context-specific manners. Here the authors show that the anti-inflammatory fate of TH17 cells contributes, via TGF-β signaling and induction of IL-10, to host immune tolerance, but also simultaneously dampens protective immunity against S. aureus.

CD8+ T cells are the most frequent T cell population in the immune cell compartment at the feto-maternal interface. Due to their cytotoxic potential, the presence of CD8+ T cells in the immune privileged pregnant uterus has raised considerable interest. Here, we review our current understanding of CD8+ T cell biology in the uterus of pregnant women and discuss this knowledge in relation to a recently published immune cell Atlas of human decidua. We describe how the expansion of CD8+ T cells with an effector memory phenotype often presenting markers of exhaustion is critical for a successful pregnancy, and host defense towards pathogens. Moreover, we review new evidence on the presence of long-lasting immunological memory to former pregnancies and discuss its impact on prospective pregnancy outcomes. The formation of fetal-specific memory CD8+ T cell subests in the uterus, in particular of tissue resident, and stem cell memory cells requires further investigation, but promises interesting results to come. Advancing the knowledge of CD8+ T cell biology in the pregnant uterus will be pivotal for understanding not only tissue-specific immune tolerance but also the etiology of complications during pregnancy, thus enabling preventive or therapeutic interventions in the future.

Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+ reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+ significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.

Type 1 diabetes (T1D) is a multifactorial disease of unknown aetiology. Studies focusing on environment-related prenatal changes, which might have an influence on the development of T1D, are still missing. Drugs, such as betamethasone, are used during this critical period without exploring possible effects later in life. Betamethasone can interact with the development and function of the two main players in T1D, the immune system and the pancreatic β-cells. Short-term or persistent changes in any of these two players may influence the initiation of the autoimmune reaction against β-cells. In this review, we focus on the ability of betamethasone to induce alterations in the immune system, impairing the recognition of autoantigens. At the same time, betamethasone affects β-cell gene expression and apoptosis rate, reducing the danger signals that will attract unwanted attention from the immune system. These effects may synergise to hinder the autoimmune attack. In this review, we compile scattered evidence to provide a better understanding of the basic relationship between betamethasone and T1D, laying the foundation for future studies on human cohorts that will help to fully grasp the role of betamethasone in the development of T1D.

Background: Paracetamol is the first choice for antipyretic or analgesic treatment throughout pregnancy. Products with Paracetamol are readily available over the counter and therefore easily accessible for self-medication. Epidemiological data on Paracetamol intake pattern during pregnancy and its potential immunological effects are sparse. We aimed to analyze a possible association between Paracetamol medication and numbers of hematopoietic stem cells (HSC) in cord blood. Methods: The objective was addressed in the PRINCE (PRENATAL DETERMINANTS OF CHILDREN'S HEALTH) study, a population-based prospective pregnancy cohort study initiated in 2011 at the University Medical Center in Hamburg, Germany. 518 healthy pregnant women with singleton pregnancies were recruited during the first trimester. Three examinations were scheduled at the end of the 1st (gestational week 12–14), the 2nd (gestational week 22–24) and the 3rd trimester (gestational week 34–36). For 146 of these women, cord blood flow cytometry data were available. Paracetamol intake was assessed for each trimester of pregnancy. Findings: Among the 518 enrolled women, 40% took Paracetamol as main analgesic treatment during pregnancy. The intake frequency and dosage of Paracetamol varied between the women and was overall low with a tendency towards higher frequencies and higher dosages in the third trimester. Paracetamol intake, particularly during the third trimester, resulted in decreased relative numbers of HSCs in cord blood, independent of maternal age, first-trimester BMI, parity, gestational age and birth weight (−0.286 (95% CI −0.592, 0.021), p = 0.068). Interpretation: Prenatal Paracetamol intake, especially during the third trimester, may be causally involved in decreasing HSCs in cord blood.

Summary: Identifying T cell clones associated with human autoimmunity has remained challenging. Intriguingly, many autoimmune diseases, including multiple sclerosis (MS), show strongly diminished activity during pregnancy, providing a unique research paradigm to explore dynamics of immune repertoire changes during active and inactive disease. Here, we characterize immunomodulation at the single-clone level by sequencing the T cell repertoire in healthy women and female MS patients over the course of pregnancy. Clonality is significantly reduced from the first to third trimester in MS patients, indicating that the T cell repertoire becomes less dominated by expanded clones. However, only a few T cell clones are substantially modulated during pregnancy in each patient. Moreover, relapse-associated T cell clones identified in an individual patient contract during pregnancy and expand during a postpartum relapse. Our data provide evidence that profiling the T cell repertoire during pregnancy could serve as a tool to discover and track “private” T cell clones associated with disease activity in autoimmunity. : Ramien et al. interrogate the immune repertoire in multiple sclerosis (MS) during pregnancy. They report a shift in T cell repertoire composition driven by a small number of “private” clones. This specific rather than global immunomodulation may help to explain the protective effect of pregnancy in human autoimmunity. Keywords: multiple sclerosis, pregnancy, T cell receptor α and β pairing, immunosequencing, repertoire sequencing, immune phenotyping, immune tolerance, human

Extracellular adenine nucleotides participate in cell-to-cell communication and modulate the immune response. The concerted action of ectonucleotidases CD39 and CD73 plays a major role in the local production of anti-inflammatory adenosine, but both ectonucleotidases are rarely co-expressed by human T cells. The expression of CD39 on T cells increases upon T cell activation and is high at sites of inflammation. CD73, in contrast, disappears from the cellular membrane after activation. The possibility that CD73 could act in trans would resolve the conundrum of both enzymes being co-expressed for the degradation of ATP and the generation of adenosine. An enzymatically active soluble form of CD73 has been reported, and AMPase activity has been detected in body fluids of patients with inflammation and cancer. It is not yet clear how CD73, a glycosylphosphatidylinositol (GPI)-anchored protein, is released from the cell membrane, but plausible mechanisms include cleavage by metalloproteinases and shedding mediated by cell-associated phospholipases. Importantly, like many other GPI-anchored proteins, CD73 at the cell membrane is preferentially localized in detergent-resistant domains or lipid rafts, which often contribute to extracellular vesicles (EVs). Indeed, CD73-containing vesicles of different size and origin and with immunomodulatory function have been found in the tumor microenvironment. The occurrence of CD73 as non-cell-bound molecule widens the range of action of this enzyme at sites of inflammation. In this review, we will discuss the generation of non-cell-bound CD73 and its physiological role in inflammation.

Background: Natural Killer T (NKT) cells are CD1d-restricted innate-like T cells that can rapidly release stored cytokines upon recognition of lipid antigens. In mice, type I NKT cells seem to promote liver inflammation, whereas type II NKT cells seem to restrict hepatitis. Here, we aimed at characterizing the role of human type I and type II NKT in patients with autoimmune hepatitis (AIH).Methods: NKT cells were analyzed by flow cytometry in peripheral blood and liver of AIH patients and control groups. α-galactosylceramide-loaded or sulfatide-loaded tetramers were used to detect type I or II NKT cells, respectively. Hepatic CD1d was stained by in situ-hybridization of liver biopsies.Results and Conclusions: Type II NKT cells were more prevalent in human peripheral blood and liver than type I NKT cells. In AIH patients, the frequency of sulfatide-reactive type II NKT cells was significantly increased in peripheral blood (0.11% of peripheral blood leukocytes) and liver (3.78% of intrahepatic leukocytes) compared to healthy individuals (0.05% and 1.82%) and patients with drug-induced liver injury (0.06% and 2.03%; p < 0.05). Intrahepatic type II NKT cells of AIH patients had a different cytokine profile than healthy subjects with an increased frequency of TNFα (77.8% vs. 59.1%, p < 0.05), decreased IFNγ (32.7% vs. 63.0%, p < 0.05) and a complete lack of IL-4 expressing cells (0% vs. 2.1%, p < 0.05). T cells in portal tracts expressed significantly more CD1d-RNA in AIH livers compared to controls. This study supports that in contrast to their assumed protective role in mice, human intrahepatic, sulfatide-reactive type II NKT cells displayed a proinflammatory cytokine profile in patients with AIH. Infiltrating T cells in portal areas of AIH patients overexpressed CD1d and could thereby activate type II NKT cells.

The ectoenzymes CD39 and CD73 degrade extracellular ATP to adenosine. ATP is released by stressed or damaged cells and provides pro-inflammatory signals to immune cells through P2 receptors. Adenosine, on the other hand, suppresses immune cells by stimulating P1 receptors. Thus, CD39 and CD73 can shape the quality of immune responses. Here we demonstrate that upregulation of CD39 is a consistent feature of activated conventional CD4+ and CD8+ T cells. Following stimulation in vitro, CD4+ and CD8+ T cells from human blood gained surface expression of CD39 but displayed only low levels of CD73. Activated human T cells from inflamed joints largely presented with a CD39+CD73- phenotype. In line, in spleens of mice with acute Listeria monocytogenes, listeria-specific CD4+ and CD8+ T cells acquired a CD39+CD73- phenotype. To test the function of CD39 in control of bacterial infection, CD39-deficient (CD39-/-) mice were infected with L. monocytogenes. CD39-/- mice showed better initial control of L. monocytogenes, which was associated with enhanced production of inflammatory cytokines. In the late stage of infection, CD39-/- mice accumulated more listeria-specific CD8+ T cells in the spleen than wildtype animals suggesting that CD39 attenuates the CD8+ T-cell response to infection. In conclusion, our results demonstrate that CD39 is upregulated on conventional CD4+ and CD8+ T cells at sites of acute infection and inflammation, and that CD39 dampens responses to bacterial infection.

Prenatal glucocorticoids are routinely administered to pregnant women at risk of preterm delivery in order to improve survival of the newborn. However, in half of the cases, birth occurs outside the beneficial period for lung development. Glucocorticoids are potent immune modulators and cause apoptotic death of immature T cells, and we have previously shown that prenatal betamethasone treatment at doses eliciting lung maturation induce profound thymocyte apoptosis in the offspring. Here, we asked if there are long-term consequences on the offspring’s immunity after this treatment. In the non-obese diabetic mouse model, prenatal betamethasone clearly decreased the frequency of pathogenic T cells and the incidence of type 1 diabetes (T1D). In contrast, in the lupus-prone MRL/lpr strain, prenatal glucocorticoids induced changes in the T cell repertoire that resulted in more autoreactive cells. Even though glucocorticoids transiently enhanced regulatory T cell (Treg) development, these cells did not have a protective effect in a model for multiple sclerosis which relies on a limited repertoire of pathogenic T cells for disease induction that were not affected by prenatal betamethasone. We conclude that prenatal steroid treatment, by inducing changes in the T cell receptor repertoire, has unforeseeable consequences on development of autoimmune disease. Our data should encourage further research to fully understand the consequences of this widely used treatment.

The recruitment and retention of Natural Killer (NK) cells in the liver are thought to play an important role during hepatotropic infections and liver cirrhosis. The aims of this study were to determine differences between liver-derived and peripheral blood-derived NK cells in the context of liver inflammation and cirrhosis. We conducted a prospective dual-center cross-sectional study in patients undergoing liver transplantation or tumor-free liver resections, in which both liver tissue and peripheral blood samples were obtained from each consenting study participants. Intrahepatic lymphocytes and PBMCs were stained, fixed and analyzed by flow cytometry. Our results showed that, within cirrhotic liver samples, intrahepatic NK cells were particularly enriched for CD49a+ NK cells when compared to tumor-free liver resection samples. CD49a+ liver-derived NK cells included populations of cells expressing CD25, CD34 and CXCR3. Moreover, CD49a+CD25+ liver-derived NK cells exhibited high proliferative capacity in vitro in response to low doses of IL-2. Our study identified a specific subset of CD49a+CD25+ NK cells in cirrhotic livers bearing functional features of proliferation.

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2(nd) SRCR domain with susceptibility to MS (P max(T) permutation = 1×10(-4)). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. - CD4(+) naïve cells, P = 0.0001; CD8(+) naïve cells, P

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-beta-lactamase hybrid protein and a fluorescent staining sensitive to beta-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-beta1A, and HeLa cells demonstrated that beta1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80(+), 11% of CD11c(+), 7% of CD49b(+), 5% of Gr1(+) cells, 2.3% of CD19(+), and 2.6% of CD3(+) cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19(+)CD21(+)CD23(+) follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-gammaR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-beta-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.