Essential role of smooth muscle Rac1 in severe asthma-associated airway remodelling.

BACKGROUND: Severe asthma is a chronic lung disease characterised by inflammation, airway hyperresponsiveness (AHR) and airway remodelling. The molecular mechanisms underlying uncontrolled airway smooth muscle cell (aSMC) proliferation involved in pulmonary remodelling are still largely unknown. Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 is activated in aSMC from allergic mice. The objective of this study was to assess the role of Rac1 in severe asthma-associated airway remodelling. METHODS AND RESULTS: Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from patients with severe asthma compared with control subjects. Inhibition of Rac1 by EHT1864 showed that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the signal transducer and activator of transcription 3 (STAT3) signalling pathway. In vivo, specific deletion of Rac1 in SMC or pharmacological inhibition of Rac1 by nebulisation of NSC23766 prevented AHR and aSMC hyperplasia in a mouse model of severe asthma. Moreover, the Rac1 inhibitor prevented goblet cell hyperplasia and epithelial cell hypertrophy whereas treatment with corticosteroids had less effect. Nebulisation of NSC23766 also decreased eosinophil accumulation in the bronchoalveolar lavage of asthmatic mice. CONCLUSION: This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation. It also provides evidence that Rac1 is causally involved in AHR and airway remodelling. Rac1 may represent as an interesting target for treating both AHR and airway remodelling of patients with severe asthma.

Blood CD9+ B cell, a biomarker of bronchiolitis obliterans syndrome after lung transplantation.

Bronchiolitis obliterans syndrome is the main limitation for long-term survival after lung transplantation. Some specific B cell populations are associated with long-term graft acceptance. We aimed to monitor the B cell profile during early development of bronchiolitis obliterans syndrome after lung transplantation. The B cell longitudinal profile was analyzed in peripheral blood mononuclear cells from patients with bronchiolitis obliterans syndrome and patients who remained stable over 3 years of follow-up. CD24hi CD38hi transitional B cells were increased in stable patients only, and reached a peak 24 months after transplantation, whereas they remained unchanged in patients who developed a bronchiolitis obliterans syndrome. These CD24hi CD38hi transitional B cells specifically secrete IL-10 and express CD9. Thus, patients with a total CD9+ B cell frequency below 6.6% displayed significantly higher incidence of bronchiolitis obliterans syndrome (AUC = 0.836, PPV = 0.75, NPV = 1). These data are the first to associate IL-10-secreting CD24hi CD38hi transitional B cells expressing CD9 with better allograft outcome in lung transplant recipients. CD9-expressing B cells appear as a contributor to a favorable environment essential for the maintenance of long-term stable graft function and as a new predictive biomarker of bronchiolitis obliterans syndrome-free survival.

Increased degradation of ATP is driven by memory regulatory T cells in kidney transplantation tolerance.

Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function.

RITA (Reactivating p53 and Inducing Tumor Apoptosis) is efficient against TP53abnormal myeloma cells independently of the p53 pathway.

BACKGROUND: The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells. METHODS: A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines. RESULTS: Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53(mutated) cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥ 1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤ 19%). CONCLUSION: These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.

Immunomodulation of B Lymphocytes by Prebiotics, Probiotics and Synbiotics: Application in Pathologies.

INTRODUCTION: Prebiotics, probiotics and synbiotics are known to have major beneficial effects on human health due to their ability to modify the composition and the function of the gut mucosa, the gut microbiota and the immune system. These components largely function in a healthy population throughout different periods of life to confer homeostasis. Indeed, they can modulate the composition of the gut microbiota by increasing bacteria strands that are beneficial for health, such as Firmicute and Bifidobacteria, and decreasing harmful bacteria, such as Enteroccocus. Their immunomodulation properties have been extensively studied in different innate cells (dendritic cells, macrophages, monocytes) and adaptive cells (Th, Treg, B cells). They can confer a protolerogenic environment but also modulate pro-inflammatory responses. Due to all these beneficial effects, these compounds have been investigated to prevent or to treat different diseases, such as cancer, diabetes, allergies, autoimmune diseases, etc. Regarding the literature, the effects of these components on dendritic cells, monocytes and T cells have been studied and presented in a number of reviews, but their impact on B-cell response has been less widely discussed. CONCLUSIONS: For the first time, we propose here a review of the literature on the immunomodulation of B-lymphocytes response by prebiotics, probiotics and synbiotics, both in healthy conditions and in pathologies. DISCUSSION: Promising studies have been performed in animal models, highlighting the potential of prebiotics, probiotics and synbiotics intake to treat or to prevent diseases associated with B-cell immunomodulation, but this needs to be validated in humans with a full characterization of B-cell subsets and not only the humoral response.

The immunomodulatory drug lenalidomide restores a vitamin D sensitive phenotype to the vitamin D resistant breast cancer cell line MDA-MB-231 through inhibition of BCL-2: potential for breast cancer therapeutics.

1α,25-Dihydroxyvitamin D3, (1,25-D3) the biologically active form of vitamin-D, is well established as a cancer cell growth inhibitor in addition to maintaining bone mineralization. In breast cancer cells, inhibitory effects on angiogenesis, and metastasis have been observed together with enhancement of apoptosis and induction of cell cycle arrest. There is a correlation between vitamin-D receptor expression on breast cancer cells and patient survival. However vitamin-D resistance and hypercalcaemia are key limiting factors in clinical use. The IMiD(®) immunomodulatory drug lenalidomide, (Revlimid(®), CC-5013) used in myeloma, can also modulate apoptotic and growth signalling. We studied whether lenalidomide treated breast cancer cells would acquire sensitivity to 1,25-D3 with resulting growth inhibition. The cell lines MCF-12A, MCF-7 and MDA-MB-231, representing non-tumorogenic, tumorogenic, and vitamin-D resistant lines respectively were treated with lenalidomide and/or 1,25-D3(at 100 nM). Whereas lenalidomide alone had no effect on cell growth, a 50% inhibition of cell growth by 1,25-D3 was achieved with additional 1 µM lenalidomide in resistant cells. This effect was through apoptosis measured by PARP cleavage and annexin-V expression. An apoptosis protein array showed that the 1,25-D3 and lenalidomide combination increased pro-apoptotic proteins (phosphorylated p53) and decreased BCL-2 expression. BCL-2 inhibition is proposed as a mechanism of action for the combined drugs in the MDA-MB-231 cell line. In vitamin D resistant cell lines MCF-7VDR and HBL-100 where the combination does not affect BCL-2-no inhibitory effect is observed. These results demonstrate the potential for the combinatorial use of lenalidomide and 1,25-D3 for vitamin D refractory tumours.

Oral exposure to bisphenol A exacerbates allergic inflammation in a mouse model of food allergy.

Allergic diseases are increasing worldwide, and their precise causes are not fully understood. However, this observation can be correlated with growing chemical pollution of the environment. Bisphenol A (BPA) alters the immune system, microbiota and barrier functions. Here, we studied the effect of oral BPA at levels equivalent to human exposure to understand the mechanisms of immunological, physiological and microbial action on food allergies. In a murine model of allergy, we evaluated the effect of direct oral exposure to BPA at 4 µg/kg bw/d corresponding to tolerable daily intake (TDI). We studied symptoms, intestinal physiology and humorall and cellular immune responses during food allergy. We explored the relationship between oral exposure to BPA and changes in the gut microenvironment. Markers of food allergy and intestinal permeability were increased following exposure to BPA. We also observed a modulated humorall and T-cell response with aggravation of food allergy inflammation. Moreover, BPA exposure induced gut dysbiosis and decreased microbial diversity induced by food allergy. Altogether, these results suggest that the 2015 European Food Safety Authority (EFSA) TDI should be reviewed to consider the immunotoxicity of BPA.

Maternal prebiotic supplementation impacts colitis development in offspring mice.

Background and aims: Maternal diet plays a key role in preventing or contributing to the development of chronic diseases, such as obesity, allergy, and brain disorders. Supplementation of maternal diet with prebiotics has been shown to reduce the risk of food allergies and affect the intestinal permeability in offspring later in life. However, its role in modulating the development of other intestinal disorders, such as colitis, remains unknown. Therefore, we investigated the effects of prebiotic supplementation in pregnant mice on the occurrence of colitis in their offspring. Materials and methods: Offspring from mothers, who were administered prebiotic galacto-oligosaccharides and inulin during gestation or fed a control diet, were subjected to three cycles of dextran sulphate sodium (DSS) treatment to induce chronic colitis, and their intestinal function and disease activity were evaluated. Colonic remodelling, gut microbiota composition, and lipidomic and transcriptomic profiles were also assessed. Results: DSS-treated offspring from prebiotic-fed mothers presented a higher disease score, increased weight loss, and increased faecal humidity than those from standard diet-fed mothers. DSS-treated offspring from prebiotic-fed mothers also showed increased number of colonic mucosal lymphocytes and macrophages than the control group, associated with the increased colonic concentrations of resolvin D5, protectin DX, and 14-hydroxydocosahexaenoic acid, and modulation of colonic gene expression. In addition, maternal prebiotic supplementation induced an overabundance of eight bacterial families and a decrease in the butyrate caecal concentration in DSS-treated offspring. Conclusion: Maternal prebiotic exposure modified the microbiota composition and function, lipid content, and transcriptome of the colon of the offspring. These modifications did not protect against colitis, but rather sensitised the mice to colitis development.

Human Milk Oligosaccharides: Their Effects on the Host and Their Potential as Therapeutic Agents.

Breastmilk is known to be very important for infants because it provides nutrients and immunological compounds. Among these compounds, human milk oligosaccharides (HMOs) represent the third most important component of breastmilk after lipids and lactose. Several experiments demonstrated the beneficial effects of these components on the microbiota, the immune system and epithelial barriers, which are three major biological systems. Indeed, HMOs induce bacterial colonization in the intestinal tract, which is beneficial for health. The gut bacteria can act directly and indirectly on the immune system by stimulating innate immunity and controlling inflammatory reactions and by inducing an adaptive immune response and a tolerogenic environment. In parallel, HMOs directly strengthen the intestinal epithelial barrier, protecting the host against pathogens. Here, we review the molecular mechanisms of HMOs in these different compartments and highlight their potential use as new therapeutic agents, especially in allergy prevention.

Prebiotic Supplementation During Pregnancy Modifies the Gut Microbiota and Increases Metabolites in Amniotic Fluid, Driving a Tolerogenic Environment In Utero.

The gut microbiota is influenced by environmental factors such as food. Maternal diet during pregnancy modifies the gut microbiota composition and function, leading to the production of specific compounds that are transferred to the fetus and enhance the ontogeny and maturation of the immune system. Prebiotics are fermented by gut bacteria, leading to the release of short-chain fatty acids that can specifically interact with the immune system, inducing a switch toward tolerogenic populations and therefore conferring health benefits. In this study, pregnant BALB/cJRj mice were fed either a control diet or a diet enriched in prebiotics (Galacto-oligosaccharides/Inulin). We hypothesized that galacto-oligosaccharides/inulin supplementation during gestation could modify the maternal microbiota, favoring healthy immune imprinting in the fetus. Galacto-oligosaccharides/inulin supplementation during gestation increases the abundance of Bacteroidetes and decreases that of Firmicutes in the gut microbiota, leading to increased production of fecal acetate, which was found for the first time in amniotic fluid. Prebiotic supplementation increased the abundance of regulatory B and T cells in gestational tissues and in the fetus. Interestingly, these regulatory cells remained later in life. In conclusion, prebiotic supplementation during pregnancy leads to the transmission of specific microbial and immune factors from mother to child, allowing the establishment of tolerogenic immune imprinting in the fetus that may be beneficial for infant health outcomes.

Prebiotic Supplementation During Gestation Induces a Tolerogenic Environment and a Protective Microbiota in Offspring Mitigating Food Allergy.

Food allergy is associated with alterations in the gut microbiota, epithelial barrier, and immune tolerance. These dysfunctions are observed within the first months of life, indicating that early intervention is crucial for disease prevention. Preventive nutritional strategies with prebiotics are an attractive option, as prebiotics such as galacto-oligosaccharides and inulin can promote tolerance, epithelial barrier reinforcement, and gut microbiota modulation. Nonetheless, the ideal period for intervention remains unknown. Here, we investigated whether galacto-oligosaccharide/inulin supplementation during gestation could protect offspring from wheat allergy development in BALB/cJRj mice. We demonstrated that gestational prebiotic supplementation promoted the presence of beneficial strains in the fecal microbiota of dams during gestation and partially during mid-lactation. This specific microbiota was transferred to their offspring and maintained to adulthood. The presence of B and T regulatory immune cell subsets was also increased in the lymph nodes of offspring born from supplemented mothers, suggestive of a more tolerogenic immune environment. Indeed, antenatal prebiotic supplementation reduced the development of wheat allergy symptoms in offspring. Our study thus demonstrates that prebiotic supplementation during pregnancy induces, in the offspring, a tolerogenic environment and a microbial imprint that mitigates food allergy development.

Der p 2.1 Peptide Abrogates House Dust Mites-Induced Asthma Features in Mice and Humanized Mice by Inhibiting DC-Mediated T Cell Polarization.

Asthma is a chronic airway disease often due to sensitization to aeroallergens, especially house dust mite allergens (HDMs). The Dermatophagoides pteronyssinus group 2 (Der p 2), is one of the most representative HDM allergens and is recognized by more than 90% of HDM-allergic patients. In mouse models, all asthma-related features can be prevented by prophylactic administration of Dermatophagoides pteronyssinus 2-derived peptide (Der p 2.1). However, it is unknown whether it is able to treat well-established asthma in mice and humans. We aimed here to evaluate the efficacy of Der p 2.1 immunotherapy in a mouse, humanized mouse, and asthmatic patients. Asthma related-features were analyzed through airway hyperresponsiveness (AHR), allergen-specific IgE, and lung histology in mice and humanized mice. Immune profile was analyzed using lung and blood from mice and severe asthmatic patients respectively. T cell and dendritic cell (DC) polarization was evaluated using co-culture of bone marrow derived cells (BMDCs) and naïve T cell from naïve mice. Mice and humanized mice both have a reduced AHR, lung tissue alteration, and HDM-specific IgE under Der p 2.1 treatment. Concerning the immune profile, T helper 2 cells (Th2) and T helper 17 cells (Th17) were significantly reduced in both mice and humanized mice lung and in peripheral blood mononuclear cells (PBMCs) from severe asthmatic patients after Der p 2.1 incubation. The downregulation of T cell polarization seems to be linked to an increase of IL-10-secreting DC under Der p 2.1 treatment in both mice and severe asthmatic patients. This study shows that allergen-derived peptide immunotherapy abrogates asthma-related features in mice and humanized mice by reducing Th2 and Th17 cells polarization via IL-10-secreting DC. These results suggest that Der p 2.1 peptide immunotherapy could be a promising approach to treat both Th2 and Th17 immunity in asthma.

Role of JAK inhibitors and immune cells in transplantation.

Immunosuppressive challenge after transplantation has dual objectives, namely, to efficiently inhibit immune populations involved in acute, chronic, humoral or cellular transplant rejection while minimizing the effect on immune integrity toward pathogens. The current immunosuppressive strategies show limited efficacy and remain associated with strong side effects, and thus, it is essential to develop new strategies. The use of Janus kinase (JAK) inhibitors is one of the new strategies focusing on cytokine pathways. Specifically, the first-generation JAK inhibitors (JAKis) showed low specificity toward the four known JAK molecules and did not exhibit better effects than calcineurin inhibitors, which constitute the standard treatment posttransplantation. However, because the new generation of JAKis present higher specificity, we are gaining further insights on the response of cells to these inhibitions. This review focuses on the impact of JAKis on different immune cell subsets, focusing on their role in transplantation.

Prebiotics: Mechanisms and Preventive Effects in Allergy.

Allergic diseases now affect over 30% of individuals in many communities, particularly young children, underscoring the need for effective prevention strategies in early life. These allergic conditions have been linked to environmental and lifestyle changes driving the dysfunction of three interdependent biological systems: microbiota, epithelial barrier and immune system. While this is multifactorial, dietary changes are of particular interest in the altered establishment and maturation of the microbiome, including the associated profile of metabolites that modulate immune development and barrier function. Prebiotics are non-digestible food ingredients that beneficially influence the health of the host by 1) acting as a fermentable substrate for some specific commensal host bacteria leading to the release of short-chain fatty acids in the gut intestinal tract influencing many molecular and cellular processes; 2) acting directly on several compartments and specifically on different patterns of cells (epithelial and immune cells). Nutrients with prebiotic properties are therefore of central interest in allergy prevention for their potential to promote a more tolerogenic environment through these multiple pathways. Both observational studies and experimental models lend further credence to this hypothesis. In this review, we describe both the mechanisms and the therapeutic evidence from preclinical and clinical studies exploring the role of prebiotics in allergy prevention.

Regulation of the Immune Response by the Inflammatory Metabolic Microenvironment in the Context of Allotransplantation.

Antigen challenge induced by allotransplantation results in the activation of T and B cells, followed by their differentiation and proliferation to mount an effective immune response. Metabolic fitness has been shown to be crucial for supporting the major shift from quiescent to active immune cells and for tuning the immune response. Metabolic reprogramming includes regulation of the balance between glycolysis and mitochondrial respiration processes. Recent research has shed new light on the functions served by the end products of metabolism such as lactate, acetate, and ATP. At enhanced local concentrations, these metabolites have complex effects in which they not only induce T and B cell responses, cell mobility, and cytokine secretion but also favor the resolution of inflammation by promoting regulatory functions. Such mechanisms are instrumental in the context of the immune response in transplantation, not only to protect the graft and/or eliminate cells targeting it but also to maintain cell homeostasis per se. Metabolic adaptation thus plays an instrumental role on the outcome of the cellular and humoral responses. This, of course, raises the possibility of drugs that would interfere in these metabolic pathways to control the immune response but also highlights the risk that some drugs may perturb this metabolism and cell homeostasis and be deleterious for graft outcome. This review focuses on how metabolic alterations of the local immune microenvironment regulate the immune response and the impact of metabolic manipulation in allotransplantation.

CD9 Tetraspanin: A New Pathway for the Regulation of Inflammation?

CD9 belongs to the tetraspanin superfamily. Depending on the cell type and associated molecules, CD9 has a wide variety of biological activities such as cell adhesion, motility, metastasis, growth, signal transduction, differentiation, and sperm-egg fusion. This review focuses on CD9 expression by hematopoietic cells and its role in modulating cellular processes involved in the regulation of inflammation. CD9 is functionally very important in many diseases and is involved either in the regulation or in the mediation of the disease. The role of CD9 in various diseases, such as viral and bacterial infections, cancer and chronic lung allograft dysfunction, is discussed. This review focuses also on its interest as a biomarker in diseases. Indeed CD9 is primarily known as a specific exosome marker however, its expression is now recognized as an anti-inflammatory marker of monocytes and macrophages. It was also described as a marker of murine IL-10-competent Breg cells and IL-10-secreting CD9+ B cells were associated with better allograft outcome in lung transplant patients, and identified as a new predictive biomarker of long-term survival. In the field of cancer, CD9 was both identified as a favorable prognostic marker or as a predictor of metastatic potential depending on cancer types. Finally, this review discusses strategies to target CD9 as a therapeutic tool. Because CD9 can have opposite effects depending on the situation, the environment and the pathology, modulating CD9 expression or blocking its effects seem to be a new promising therapeutic strategy.

CD9+ Regulatory B Cells Induce T Cell Apoptosis via IL-10 and Are Reduced in Severe Asthmatic Patients.

CD9 was recently identified as a marker of murine IL-10-competent regulatory B cells. Functional impairments or defects in CD9+ IL-10-secreting regulatory B cells are associated with enhanced asthma-like inflammation and airway hyperresponsiveness. In mouse models, all asthma-related features can be abrogated by CD9+ B cell adoptive transfer. We aimed herein to decipher the profiles, features, and molecular mechanisms of the regulatory properties of CD9+ B cells in human and mouse. The profile of CD9+ B cells was analyzed using blood from severe asthmatic patients and normal and asthmatic mice by flow cytometry. The regulatory effects of mouse CD9+ B cells on effector T cell death, cell cycle arrest, apoptosis, and mitochondrial depolarization were determined using yellow dye, propidium iodide, Annexin V, and JC-1 staining. MAPK phosphorylation was analyzed by western blotting. Patients with severe asthma and asthmatic mice both harbored less CD19+CD9+ B cells, although these cells displayed no defect in their capacity to induce T cell apoptosis. Molecular mechanisms of regulation of CD9+ B cells characterized in mouse showed that they induced effector T cell cycle arrest in sub G0/G1, leading to apoptosis in an IL-10-dependent manner. This process occurred through MAPK phosphorylation and activation of both the intrinsic and extrinsic pathways. This study characterizes the molecular mechanisms underlying the regulation of CD9+ B cells to induce effector T cell apoptosis in mice and humans via IL-10 secretion. Defects in CD9+ B cells in blood from patients with severe asthma reveal new insights into the lack of regulation of inflammation in these patients.

High circulating CD4+CD25hiFOXP3+ T-cell sub-population early after lung transplantation is associated with development of bronchiolitis obliterans syndrome.

BACKGROUND: Chronic bronchiolitis obliterans syndrome (BOS) remains a major limitation for long-term survival after lung transplantation. The immune mechanisms involved and predictive biomarkers have yet to be identified. The purpose of this study was to determine whether peripheral blood T-lymphocyte profile could predict BOS in lung transplant recipients. METHODS: An in-depth profiling of CD4+ and CD8+ T cells was prospectively performed on blood cells from stable (STA) and BOS patients with a longitudinal follow-up. Samples were analyzed at 1 and 6 months after transplantation, at the time of BOS diagnosis, and at an intermediate time-point at 6 to 12 months before BOS diagnosis. RESULTS: Although no significant difference was found for T-cell compartments at BOS diagnosis or several months beforehand, we identified an increase in the CD4+CD25hiFoxP3+ T-cell sub-population in BOS patients at 1 and 6 months after transplantation (3.39 ± 0.40% vs 1.67 ± 0.22% in STA, p < 0.001). A CD4+CD25hiFoxP3+ T-cell threshold of 2.4% discriminated BOS and stable patients at 1 month post-transplantation. This was validated on a second set of patients at 6 months post-transplantation. Patients with a proportion of CD4+CD25hiFoxP3+ T cells up to 2.4% in the 6 months after transplantation had a 2-fold higher risk of developing BOS. CONCLUSIONS: This study is the first to report an increased proportion of circulating CD4+CD25hiFoxP3+ T cells early post-transplantation in lung recipients who proceed to develop BOS within 3 years, which supports its use as a BOS predictive biomarker.

Cell-surface C-type lectin-like receptor CLEC-1 dampens dendritic cell activation and downstream Th17 responses.

Dendritic cells (DCs) represent essential antigen-presenting cells that are critical for linking innate and adaptive immunity, and influencing T-cell responses. Among pattern recognition receptors, DCs express C-type lectin receptors triggered by both exogenous and endogenous ligands, therefore dictating pathogen response, and also shaping T-cell immunity. We previously described in rat, the expression of the orphan C-type lectin-like receptor-1 (CLEC-1) by DCs and demonstrated in vitro its inhibitory role in downstream T helper 17 (Th17) activation. In this study, we examined the expression and functionality of CLEC-1 in human DCs, and show a cell-surface expression on the CD16- subpopulation of blood DCs and on monocyte-derived DCs (moDCs). CLEC-1 expression on moDCs is downregulated by inflammatory stimuli and enhanced by transforming growth factor ß. Moreover, we demonstrate that CLEC-1 is a functional receptor on human moDCs and that although not modulating the spleen tyrosine kinase-dependent canonical nuclear factor-κB pathway, represses subsequent Th17 responses. Interestingly, a decreased expression of CLEC1A in human lung transplants is predictive of the development of chronic rejection and is associated with a higher level of interleukin 17A (IL17A). Importantly, using CLEC-1-deficient rats, we showed that disruption of CLEC-1 signaling led to an enhanced Il12p40 subunit expression in DCs, and to an exacerbation of downstream in vitro and in vivo CD4+ Th1 and Th17 responses. Collectively, our results establish a role for CLEC-1 as an inhibitory receptor in DCs able to dampen activation and downstream effector Th responses. As a cell-surface receptor, CLEC-1 may represent a useful therapeutic target for modulating T-cell immune responses in a clinical setting.