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Background Mucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and therefore virus shedding. Parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior mucosal infection compared with injections of a parenteral vaccine in a mucosally naive subject. We investigated whether this was also the case for the BNT162b2 coronavirus disease 2019 (COVID-19) messenger RNA vaccine. Methods Twenty recovered COVID-19 subjects (RCSs) and 23 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–naive subjects were vaccinated with, respectively, 1 and 2 doses of the BNT162b2 COVID-19 vaccine. Nasal epithelial lining fluid (NELF) and plasma were collected before and after vaccination and assessed for immunoglobulin G (IgG) and IgA antibody levels to Spike and for their ability to neutralize binding of Spike to angiotensin-converting enzyme-2 receptor. Blood was analyzed 1 week after vaccination for the number of Spike-specific antibody-secreting cells (ASCs) with a mucosal tropism. Results All RCSs had both nasal and blood SARS-CoV-2–specific antibodies at least 90 days after initial diagnosis. In RCSs, a single dose of vaccine amplified preexisting Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including Delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2–naive subjects after 2 vaccine doses compared with RCSs after 1 dose. Conclusions Mucosal immune response to the SARS-CoV-2 Spike protein is higher in RCSs after a single vaccine dose compared with SARS-CoV-2–naive subjects after 2 doses.

BackgroundMucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and hence virus shedding. Preclinical and clinical studies have shown that a parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior infection compared to two injections of a parenteral vaccine. We investigated whether this was also the case for a COVID-19 mRNA vaccine.MethodsTwenty-three COVID-19 convalescent patients and 20 SARS-CoV-2-naive subjects were vaccinated with respectively one and two doses of the Pfizer-BioNTech COVID-19 RNA vaccine. Nasal Epithelial Lining Fluid (NELF) and plasma were collected before and after vaccination and assessed for Immunoglobulin (Ig)G and IgA to Spike and for their ability to inhibit the binding of Spike to its ACE-2 receptor. Blood was analyzed one week after vaccination for the number of Spike-specific Antibody Secreting Cells (ASCs) with a mucosal tropism.ResultsIn COVID-19 convalescent patients, a single dose of vaccine amplified pre-existing Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2-naive subjects after two vaccine dosesConclusionThis study showed that a parenteral booster injection of a COVID-19 RNA vaccine promoted stronger mucosal immune responses in COVID-19 convalescent patients compared to SARS-CoV-2 naive subjects who had received a first vaccine dose.

International audience; Developing protocols aimed at inhibiting effector T cells would be key for the treatment of T cell-dependent autoimmune diseases including type 1 autoimmune diabetes (T1D) and multiple sclerosis (MS). While heme oxygenase-1 (HO-1) inducers are clinically approved drugs for non-immune-related diseases, they do have immunosuppressive properties when administered systemically in rodents. Here we show that HO-1 inducers inhibit antigen-specific effector T cells when injected intradermally together with the T cell cognate antigens in mice. This phenomenon was observed in both a CD8 þ T cell-mediated model of T1D and in a CD4 þ T cell-dependent MS model. Intradermal injection of HO-1 in-ducers induced the recruitment of HO-1 þ monocyte-derived dendritic cell (MoDCs) exclusively to the lymph nodes (LN) draining the site of intradermal injection. After encountering HO-1 þ MoDCs, effector T-cells exhibited a lower velocity and a reduced ability to migrate towards chemokine gradients resulting in impaired accumulation to the inflamed organ. Intradermal co-injection of a clinically approved HO-1 inducer and a specific antigen to non-human primates also induced HO-1 þ MoDCs to accumulate in dermal draining LN and to suppress delayed-type hypersensitivity. Therefore, in both mice and non-human primates, HO-1 inducers delivered locally inhibited effector T-cells in an antigen-specific manner, paving the way for repositioning these drugs for the treatment of immune-mediated diseases.

Background:Vagus nerve (VN) stimulation has shown the potential to improve the disease development in animal models of arthritis and in patients with RA. However, the VN can affect respiratory, cardiovascular, endocrine and gastro-intestinal physiology. The splenic nerve (SpN) has been confirmed to be the principal effector nerve for the VN-mediated immune control. Previous studies have shown that stimulating the splenic nerve resulted in an increase of norephiniphrine in the spleen, as well as a significant reduction in LPS-induced TNF (1).Objectives:To test the therapeutic efficiency of splenic nerve stimulation (SNS) in collagen-induced arthritis (CIA) in mice alone or in combination with anti-TNF treatment.Methods:CIA was induced in DBA1/J mice by immunization with bovine type II collagen at days 0 and 21. At day 11, mice were implanted with micro-cuff electrode (CorTec) onto the SpN or VN. From day 16 to day 45, SNS were applied as rectangular charged-balanced biphasic pulses with 650 μA pulse amplitude, 200 µs pulse width at 10 Hz frequency for 2 min 1 or 6 times a day using a Plexon stimulator. In order to investigatedthe mechanism of action in more detail, propranolol, a beta-adrenergic receptor (β-AR) antagonist, was added to the drinking water of mice receiving SNS. In addition, a control group was treated with anti-TNF (etanercept, 3 times/week; 10mg/kg i.p.). In curative settings, SNS and/or anti-TNF treatment was applied starting when mice scored positive for 3 consecutive days. Clinical arthritis was determined by visual examination of swelling and redness of the paws and measurement of paw thickness. Sham mice were undergoing the same procedure but did not receive stimulation.Results:In CIA in mice all sham animals developed arthritis, compared to only 14% following six times per day SNS (p Conclusion:These studies demonstrate that SNS suppresses pro-inflammatory cytokine production, and reduces clinical symptoms in mice with CIA which is at least partially mediated by the β-AR. The additive effect of anti-TNF in reducing the clinical scores demonstrates that that mechanism of action of SNS is not primarilys mediated by reducing TNF levels. Moreover, anti-TNF potentiating the inhibitory effect of SNS is supporting a combined use of these treatments, or even a combination of SNS with other biologicals, to treat RA, potentially getting more patients closer to remission. In conclusion, the data is providing compelling scientific rationale and pre-clinical evidence that splenic neuromodulation might be a new treatment modality for RA.References:[1] Guyot M et al, Brain Behav Immun. 2019;80:238.Disclosure of Interests:Thomas Simon Grant/research support from: research grant from Galvani Bioelectronics, Clara Panzolini Grant/research support from: Working on research grant Galvani bioelectronics, Julien Lavergne Grant/research support from: working on research grant Galvani Bioelectrocnics, Nicolas Hypolite Grant/research support from: Working on research grant Galvani Bioelectronics, Nicolas Glaichenhaus: None declared, Margriet Vervoordeldonk Employee of: I am an employee of Galvani Bioelectronics, Philippe Blancou Grant/research support from: Received research grant from Galvani Bioelectronics

Background Both the parasympathetic and sympathetic nervous system exert control over innate immune responses. In inflammatory bowel disease, sympathetic innervation in intestinal mucosa is reduced. Our aim was to investigate the role of sympathetic innervation to the intestine on regulation of the innate immune responses. Methods In lipopolysaccharide (LPS)-stimulated macrophages, we evaluated the effect of adrenergic receptor activation on cytokine production and metabolic profile. In vivo, the effect of sympathetic denervation on mucosal innate immune responses using 6-hydroxydopamine (6-OHDA), or using surgical transection of the superior mesenteric nerve (sympathectomy) was tested in Rag1−/− mice that lack T- and B-lymphocytes. Results In murine macrophages, adrenergic β2 receptor activation elicited a dose-dependent reduction of LPS-induced cytokines, reduced LPS-induced glycolysis and increased maximum respiration. Sympathectomy led to a significantly decreased norepinephrine concentration in intestinal tissue. Within 14 days after sympathectomy, mice developed clinical signs of colitis, colon oedema and excess colonic cytokine production. Both 6-OHDA and sympathectomy led to prominent goblet cell depletion and histological damage of colonic mucosa. Conclusions We conclude that the sympathetic nervous system plays a regulatory role in constraining innate immune cell reactivity towards microbial challenges, likely via the adrenergic β2 receptor. Electronic supplementary material The online version of this article (10.1186/s10020-018-0068-8) contains supplementary material, which is available to authorized users.

International audience; Antigen-presenting cells (APCs) including dendritic cells (DCs) play a critical role in the development of autoimmune diseases by presenting self-antigen to T-cells. Different signals modulate the ability of APCs to activate or tolerize autoreactive T-cells. Since the expression of heme oxygenase-1 (HO-1) by APCs has been associated with the tolerization of autoreactive T-cells, we hypothesized that HO-1 expression might be altered in APCs from autoimmune-prone non-obese diabetic (NOD) mice. We found that, compared to control mice, NOD mice exhibited a lower percentage of HO-1-expressing cells among the splenic DCs, suggesting an impairment of their tolerogenic functions. To investigate whether restored expression of HO-1 in APCs could alter the development of diabetes in NOD mice, we generated a transgenic mouse strain in which HO-1 expression can be specifically induced in DCs using a tetracycline-controlled transcriptional activation system. Mice in which HO-1 expression was induced in DCs exhibited a lower Type 1 Diabetes (T1D) incidence and a reduced insulitis compared to non-induced mice. Upregulation of HO-1 in DCs also prevented further increase of glycemia in recently diabetic NOD mice. Altogether, our data demonstrated the potential of induction of HO-1 expression in DCs as a preventative treatment, and potential as a curative approach for T1D.

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)-treated DCs loaded with pancreatic β-cell peptides protect mice from disease. This protection is peptide-restricted, independent of IL-10 secretion by DCs and of CD4+ T cells. Although no differences were observed in autoreactive CD8+ T-cell function from gCO-treated versus untreated DC-immunized groups, gCO-treated DCs strongly inhibited accumulation of autoreactive CD8+ T cells in the pancreas. Interestingly, induction of β1-integrin was curtailed when CD8+ T cells were primed with gCO-treated DCs, and the capacity of these CD8+ T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO-treated DCs appears to be an original strategy to control autoimmune disease.

Heme oxygenase-1 (HO-1) exerts its functions via the catabolism of heme into carbon monoxide (CO), Fe2+, and biliverdin, as well as by depletion of free heme. We have recently described that overexpression of HO-1 is associated with the tolerogenic capacity to dendritic cells (DCs) stimulated by LPS. In this study, we demonstrate that treatment of human monocyte-derived DCs with CO blocks TLR3 and 4-induced phenotypic maturation, secretion of proinflammatory cytokines, and alloreactive T cell proliferation, while preserving IL-10 production. Treatment of DCs with biliverdin, bilirubin, and deferoxamine or replenishing intracellular heme stores had no effect on DC maturation. HO-1 and CO inhibited LPS-induced activation of the IFN regulatory factor 3 pathway and their effects were independent of p38, ERK, and JNK MAPK. HO-1 and CO treatment also inhibited mouse DC maturation in vitro and mouse DC immunogenic properties in vivo, as shown by adoptive cell transfer in a transgenic model of induced diabetes. Thus, for the first time, our data show that CO treatment inhibits DC immunogenicity induced by TLR ligands and that blockade of IFN regulatory factor 3 is associated with this effect.

The results of yeast and mammalian two-hybrid assays previously indicated complex formation between prenylated Rab acceptor 1 (PRA1) and the cytoplasmic domain of gp41 (gp41CD) for both the human and simian immunodeficiency viruses [Evans, D. T., Tilman, K. C. & Desrosiers, R. C. (2002). J Virol 76, 327-337]. The assembly and release of infectious virus particles was studied under conditions of PRA1 overexpression in a transient transfection assay or suppression by RNA interference. Although a clear pattern of co-localization of PRA1 and gp41 was observed, no changes in virion release, infectivity or envelope content were observed as a result of either PRA1 suppression or overexpression. These data show that PRA1 co-localizes with gp41 inside cells and they are consistent with a direct or indirect interaction between these proteins. However, variation in the levels of PRA1 expression did not influence virion production, infectivity or envelope incorporation under the conditions of these assays.

Despite vigorous cell-mediated immune responses to human and simian immunodeficiency viruses (HIV/SIV) the immune system is unable to clear latently infected resting T cells. These infected cells are reactivated by antigenic stimulation, leading to viral replication. By using the SIV/macaque model of HIV pathogenesis, the dynamics of T cell infiltration into delayed type hypersensitivity sites specific for the purified protein derivative of bacillus Calmette–Guérin have been studied. Early viral mRNA synthesis coincided with the infiltration of antigen-specific T cells. When the infiltration of anti-SIV-specific T cells was rapid compared with the kinetics of viral assembly, the sites were sterilized before the transition to late viral mRNA synthesis occurred. When their infiltration was slow, ephemeral foci of replication were identified. These findings were paralleled by plasma viremia; low viremia coincided with rapid sterilization of the delayed type hypersensitivity sites, whereas high load was found in association with local replication and delayed sterilization. These data suggest that although effective local control of SIV is possible once antiviral T lymphocytes have arrived on site, the slower deployment of these T cells may allow the virus to escape and thus to reseed the pool of memory T cells.

Accumulating evidence indicates that heme oxygenase-1 expression in dendritic cells could be the key step for providing immune suppression through Tregs in many diseases or disorders.

A key obstacle limiting development of an effective AIDS vaccine is the inability to deliver antigen for a sufficient period of time resulting in weak and transient protection. HIV transmission occurs predominantly across mucosal surfaces; therefore, an ideal vaccine strategy would be to target HIV at mucosal entry sites to prevent infection. Such a novel strategy relies on the activation of mucosal immune response via presentation of viral antigens by the mucosal epithelial cells. The use of a terminally differentiated epithelial cell promoter to drive expression of antigens leading to viral protein production in the upper layers of the epithelium is central to the success of this approach. Our results show that when administered intradermally to mice, a GFP-reporter gene under the transcriptional control of the involucrin promoter is expressed in the upper layers of the epidermis and, although transduced cells were very low in number, high and sustained anti-GFP antibody production is observed in vivo. A subsequent experiment investigates the effectiveness of GFP-tagged replication-competent SIVdeltaNef and GFP-tagged replication-deficient SIVdeltaVifdeltaNef constructs under the transcriptional control of the involucrin promoter. Optimal conditions for production of pseudotyped VSV-G viral particles destined to transduce basal epithelial stem cells at the mucosal sites of entry of SIV in our animal model were determined. Altogether, the data demonstrate the feasibility of an epithelium-based vaccine containing involucrin-driven viral antigen encoding sequences that integrate into epithelial stem cells and show long-term expression in the upper layer of the epithelium even after multiple cycle of epithelia renewal. Such epithelium-based vaccine should elicit a long-term immunity against HIV/SIV infection at the site of entry of the virus.

International audience; Heme oxygenase-1 (HO-1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide (CO), Fe 2+ , and biliverdin. We have previously shown that either induction of HO-1 or treatment with exogenous CO inhibits LPS-induced maturation of dendritic cells (DCs) and protects in vivo and in vitro antigen-specific inflammation. Here, we evaluated the capacity of HO-1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS-treated DCs. We observed that HO-1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA-derived peptides, bead-bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO-1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO-1 and CO can inhibit the ability of LPS-treated DCs to present exogenous soluble antigens to naïve T cells by blocking antigen trafficking at the level of late endosome-lysosome fusion.

Despite widespread usage of β-adrenergic receptor (AR) agonists and antagonists in current clinical practice, our understanding of their interactions with the immune system is surprisingly sparse. Among the AR expressed by dendritic cells (DC), β2-AR can modify in vitro cytokine release upon stimulation. Because DC play a pivotal role in CD8+ T cell immune responses, we examined the effects of β2-AR stimulation on MHC class I exogenous peptide presentation and cross-presentation capacities. We demonstrate that β2-AR agonist-exposed mature DC display a reduced ability to cross-present protein Ags while retaining their exogenous peptide presentation capability. This effect is mediated through the nonclassical inhibitory G (Gαi/0) protein. Moreover, inhibition of cross-presentation is neither due to reduced costimulatory molecule expression nor Ag uptake, but rather to impaired phagosomal Ag degradation. We observed a crosstalk between the TLR4 and β2-AR transduction pathways at the NF-κB level. In vivo, β2-AR agonist treatment of mice inhibits Ag protein cross-presentation to CD8+ T cells but preserves their exogenous MHC class I peptide presentation capability. These findings may explain some side effects on the immune system associated with stress or β-agonist treatment and pave the way for the development of new immunomodulatory strategies.

International audience; Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to b-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 mg/ml60.1), CD80 (LOGEC50 = 4.88 mg/ml60.15) and CD86 (LOGEC50 = 5.36 mg/ml60.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.765.1% cells versus 1262.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed that PBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.

International audience; Heme oxygenases (HOs) are the rate-limiting intracellular enzymes that degrade heme into carbon monoxide (CO), biliverdin and free divalent iron. Among HOs, HO-1 is the only isoform that is highly inducible in response to numerous stress factors and proinflammatory cytokines. This enzyme has shown cytoprotective, antioxidant and anti-inflammatory properties. Moreover, HO-1 and, in particular, CO also have tolerogenic actions in adaptive immune responses. HO-1 can provide immunosuppression through its expression by regulatory T cells or antigen-presenting cells. The physiological importance of HO-1 has been demonstrated in both mice and humans, and modulation of HO-1 expression has therapeutic effects in a variety of disorders involving inflammation and immune responses, including organ transplantation and cancer. Consistently, upregulation of the HO-1 pathway has a significant protective effect against spontaneous or induced autoimmune diseases, allergy and can be beneficial to graft survival. However, HO-1 may also play a role in tumorigenesis by lowering antitumor innate immune responses that control tumor growth or reduce tumor expansion. Thus, controlling HO-1 expression may be of great interest in immune intervention protocols where tolerance is desirable, such as in transplantation, or where enhanced immunogenicity is needed in the case of cancer.

Heme oxygenase-1 (HO-1) is one of the three isoforms of the heme oxygenase enzyme that catabolyzes the degradation of heme into biliverdin with the production of free iron and CO. HO-1 is induced by its substrate and by other stimuli, including agents involved in oxidative stress and proinflammatory cytokines as well as several anti-inflammatory stimuli. A growing body of evidence points toward the capacity of this molecule to inhibit immune reactions and the pivotal role of HO-1 in inflammatory diseases. We will first review the physiological role of HO-1 as determined by the analysis of HO-1-deficient individuals. This will be followed by an examination of the effect of HO-1 within immunopathological contexts such as immune disorders (autoimmunity and allergy) or infections. A section will be devoted to the use of an HO-1 inducer as an immunosuppressive molecule in transplantation. Finally, we will review the molecular basis of HO-1 actions on different immune cells.

Heme oxygenase-1 (HO-1) is one of the three isoforms of the heme oxygenase enzyme that catabolyzes the degradation of heme into biliverdin with the production of free iron and CO. HO-1 is induced by its substrate and by other stimuli, including agents involved in oxidative stress and proinflammatory cytokines as well as several anti-inflammatory stimuli. A growing body of evidence points toward the capacity of this molecule to inhibit immune reactions and the pivotal role of HO-1 in inflammatory diseases. We will first review the physiological role of HO-1 as determined by the analysis of HO-1-deficient individuals. This will be followed by an examination of the effect of HO-1 within immunopathological contexts such as immune disorders (autoimmunity and allergy) or infections. A section will be devoted to the use of an HO-1 inducer as an immunosuppressive molecule in transplantation. Finally, we will review the molecular basis of HO-1 actions on different immune cells.

CD8+ T cells play an important role in the initiation of insulitis and in the destructive stage leading to insulin-dependent diabetes mellitus. A string of recent studies has led to the identification of numerous HLA-A2-restricted epitopes derived from pancreatic β cell Ags. It is hoped that assays detecting responses of patient PBMC to such epitopes might be instrumental for early diagnosis of β cell-directed autoimmunity and for monitoring trials of immunointervention. However, it remains unclear whether the results of assays studying PBMC reflect responses of islet-infiltrating lymphocytes, and to what extent they correlate with disease risk and/or activity. We have used female and male humanized NOD mice expressing HLA-A2 in addition to murine MHC class I molecules to study spontaneous responses of islet-infiltrating blood, spleen, and lymph node lymphocytes of various age groups to a panel of 16 epitopes. Twelve of these are restricted by HLA-A2, have previously been shown to be recognized by patient CTL, and have identical sequences in human and murine autoantigens. Using an IFN-γ ELISPOT assay, we find highly similar hierarchies of epitope immunodominance in the different T cell compartments, including peripheral blood and pancreatic islets. Moreover, we demonstrate that most of the epitopes eliciting dominant responses in humans display similar status in the mouse model. These results emphasize the potential of humanized mice as tools for studying spontaneous autoimmune CTL responses, and they provide a strong rationale for the development and use of assays monitoring responses of CD8+ PBMC in human type 1 diabetes.

OBJECTIVE—Islet-reactive CD8+ T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown.RESEARCH DESIGN AND METHODS—We took advantage of a recently validated islet-specific CD8+ T-cell γ-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2+ adult type 1 diabetic patients close to diagnosis and at a second time point 7–16 months later.RESULTS—CD8+ T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60–67 to 20% (P < 0.02). The previously subdominant IA-2206–214 and IGRP265–273 peptides were newly targeted, thus becoming the immunodominant epitopes.CONCLUSIONS—Shifts both in frequency and in immunodominance of CD8+ T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements.

Despite the understanding that type 1 diabetes pathogenesis is mediated by T-cells, detection of these rare lymphocytes remains largely elusive. Suitable T-cell assays are highly needed, since they could offer preclinical diagnoses and immune surrogate end points for clinical trials. Although CD4+ T-cell assays have met with limited success, CD8+ T-cells are increasingly recognized as key actors in the diabetes of the NOD mouse. CD8+ T-cells are likely to play a role also in humans and may provide new markers of β-cell autoimmunity. Taking advantage of a panel of HLA-A2–restricted β-cell epitopes derived from preproinsulin, GAD, and islet glucose-6-phosphatase catalytic subunit-related protein (IGRP), we have implemented an islet-specific CD8+ T-cell interferon-γ enzyme-linked immunospot (ISL8Spot) assay. The ISL8Spot assay is capable of detecting and quantifying β-cell–reactive CD8+ T-cells directly ex vivo, without any preliminary expansion, using either fresh or frozen samples. Positive ISL8Spot responses separate new-onset diabetic and healthy samples with high accuracy (86% sensitivity, 91% specificity), using as few as five immunodominant epitopes. Moreover, sensitivity reaches 100% when the ISL8Spot assay is complemented by antibody determinations. Combination of CD8+ T-cell measurements with immune intervention strategies may open new avenues toward type 1 diabetes prediction and prevention.

Immunology of Diabetes IV: Progress in Our Understanding; International audience; Type 1 diabetes mellitus (T1DM) results from the destruction of β cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention.

Simian immunodeficiency virus (SIV) infection of rhesus macaques is widely used to explore the immunopathology of AIDS and to evaluate potential candidate vaccines in the face of challenge virus, whether they be SIVs or simian/human immunodeficiency virus (SHIV) chimeras (17). The model is essential when initially evaluating immunogens presented by way of viral vectors (29, 31, 36, 38, 39, 42). To date subunit vaccines, DNA vaccination, or various combinations have not proved too effective at controlling challenge virus (2, 4, 10, 14, 22). In contrast, the greatest degree of protection has been achieved with live attenuated strains of the macaque virus (SIVmac) harboring gene deletions. They can elicit strong protection even against challenge with an uncloned pathogenic isolate (12, 18, 46). By associating deletions in the vpx, vpr, and nef genes the recombinant SIVs became more and more attenuated. However, this resulted in an inverse correlation between the level of attenuation and the degree of protection against homologous challenge (18). Safety is the overriding problem for a live attenuated vaccine of any sort and, in the case of SIVmac, all attempts have failed to demonstrate a safety level commensurate with use in humans. The SIVmacΔnef strain which confers strongest protection can both induce AIDS in neonates (3, 37) and, over time, revert to a pathogenic phenotype (1, 8, 37, 45). In contrast to the attenuation resulting from the introduction of deletions into SIV genes, deletions engineered into the SIV promoter, or redesigning the long terminal repeat (LTR), attenuated little SIV in vivo (16, 30). An alternative to modifying the SIV promoter is to exchange it for that of a DNA virus. Given that the promoter would have been optimized in a totally different context, exchanging promoters may alter far more profoundly the replication of the resulting SIV chimera. The human cytomegalovirus major immediate-early (CMV-IE) promoter is widely used in molecular biology for driving high gene expression in transfection assays. However, in a more biological context, that of a recombinant adenovirus vector with a reporter gene under the control of the CMV-IE promoter, expression is far more restricted than anticipated from transfection assays (41). Given such restriction of the CMV-IE promoter in vivo, it was reasoned that exchanging the core enhancer/promoter sequences of SIV by those of CMV-IE might be strong enough to drive the expression of viral mRNA but might at the same time attenuate the resulting chimera. Not only was replication of the chimera, called SIVmegalo, in rhesus macaques highly attenuated but the infection also conferred strong protection to challenge virus. This finding shows that promoter exchange may indeed constitute a novel way of attenuating SIV.

We are interested in elucidating how the brain controls the immune system. To this aim, we have established and validated an environmental enriched (EE) paradigm in which C57BL/6 mice experience higher levels of sensory, motor, social, and cognitive stimuli, compared to animals housed in a standard environment (SE). In agreement with previous studies, we found that EE increased hippocampal neurogenesis and synaptogenesis, decreased anxiety and increases exploratory activity. EE housing did not result in gross alterations in the frequency of neutrophils, dendritic cells (DC), macrophages, T cells and B lymphocytes in immunologically naive animals. In contrast, upon immunization with ovalbumin (OVA), the number of OVA-specific CD8+ T cells in spleen was higher in EE mice compared to SE mice. Because the activation of CD8+ T cells is known to be dependent on antigen cross-presentation by lymphoid CD8+ DCs, we compared the phenotype of these DCs in SE and EE mice and found that lymphoid CD8+ DCs from EE mice expressed reduced levels of PD-L1, a molecule that regulate the activation of CD8+ T cells. PDL-1 down-regulation in EE mice was associated with altered serum levels of corticosterone, therefore providing a possible link between the environment, the Hypothalamo–Pituitary–Adrenal (HPA) axis and DC function. Altogether, our results suggest that environment conditions could impact adaptive immune responses and therefore the ability of the host to deal with pathogens.

Introduction Bien que les agonistes et antagonistes des recepteurs adrenergiques soient largement utilises en pratique, peu de donnees sont disponibles concernant les consequences potentielles de leur utilisation sur le systeme immunitaire en general et les maladies auto-immunes en particulier. Pourtant, les cellules immunitaires, dont les cellules dendritiques (DC), expriment les adrenorecepteurs. Dans ce contexte, nous avons etudie les effets de la stimulation β2-adrenergique des DC sur leur dialogue avec les lymphocytes T CD8+. Materiels et methodes En utilisant des approches pharmacologiques et genetiques, nous avons analyse les effets de la stimulation β2-adrenergique des DC sur leur phenotype, leur capacite a prendre en charge et a processer des antigenes particulaires et solubles afin de les presenter sur les molecules de CMH-I et sur leur efficacite a induire une reponse T cytotoxique in vitro et in vivo Resultats Nous avons demontre que la stimulation β2-adrenergique des DC leur confere un potentiel tolerogene caracterise par une inhibition de leur capacite a cross-presenter des antigenes aux lymphocytes T CD8+. Cet effet majeur est medie par l’activation d’une voie de signalisation dependante de la proteine Gαi/0 aboutissant a l’inhibition de la translocation nucleaire du complexe NFkB. De plus, cette inhibition de cross-presentation n’est due ni a une diminution de l’expression des molecules de co-stimulation ni a un defaut de captation des antigenes, mais a une diminution de la degradation des antigenes et de leur chargement sur les molecules de CMH-I. In vivo, l’administration de β2-agonistes a des souris inhibe la cross-presentation des antigenes et l’activation des lymphocytes T CD8+. Conclusion Ainsi, la stimulation β2-adrenergique des DC inhibe leur capacite a induire des reponses immunitaires specifiques d’antigene en interferant avec la voie de la cross-presentation. Ces resultats pourraient permettre d’identifier de nouvelles cibles therapeutiques du diabete auto-immun.

A search through the archives clears early vaccines of starting the AIDS pandemic. It has been suggested that chimpanzee kidney cultures may have been used in the preparation of oral polio vaccine stocks used in Africa during the late 1950s, and so could have introduced the primate precursor of the immunodeficiency virus HIV-1 into humans1,2. Here we analyse frozen samples of the suspect vaccine by using the polymerase chain reaction (PCR) to amplify any HIV-1-related nucleic acids or chimpanzee mitochondrial DNA that might be present, but we have failed to detect either. Our findings do not support the hypothesis that HIV-1 was introduced by oral vaccination against poliovirus.

The carotid bodies (CB) are located bilaterally at the carotid artery bifurcation. They are polymodal sensors capable of detecting various physiological stimuli – blood gas concentration and blood pressure. The carotid body, via its innervating nerve – the carotid sinus nerve (CSN), signals to the brain to modulate these physiological stimuli through efferent activity. Recent evidence has suggested that there is a relationship between the immune system and the carotid body. It has been shown that the carotid body detects inflammation and functionally responds. Additionally, there is in vivo data, demonstrating that bilateral removal of the CSN decreases survival to endotoxemic shock. We hypothesized that activation of the carotid body would attenuate inflammation. To carry out this study, we electrostimulated the CSN instead of using a non-specific pharmacological approach. A surgical method was developed to isolate the CSN, implant electrodes and then stimulate the nerve. We found that electrical stimulation of CSN attenuated the expression of proinflammatory cytokines induced by lipopolysaccharide (LPS) injection, including Tumor Necrosis Factor (TNF). The mechanism by which electrical activation of the CSN attenuated inflammation was unknown. It was investigated if the effect of CSN stimulation was mediated via efferent parasympathetic or sympathetic nerves - it was found that it was not mediated via these nerves. In contrast, it was discovered, that CSN stimulation increased corticosterone – an anti-inflammatory hormone, which activates the glucocorticoid receptor. An antagonist for the glucocorticoid receptor abolished the attenuation of pro-inflammatory cytokines by CSN electrostimulation. This indicated that corticosterone is the mediator of the effect of CSN stimulation. In addition, we found that the stimulation of the CSN led to the increase of the spontaneous discharge activity of the hypothalamic paraventricular nucleus likening the CSN activity to the hypothalamic-pituitary adrenal axis. Using transgenic mice with no glucocorticoid receptor on their myeloid immune cell, it was found that effect of CSN stimulation was additionally prevented. Finally, we were able to show that the stimulation of the CSN led to the increase of the spontaneous discharge activity of the hypothalamic neurons suggesting a link between the CSN and the hypothalamus-pituitary axis. Overall, these results indicate that electrostimulation of CSN attenuates inflammation by acting on the hypothalamus which, by increasing the secretion of glucocorticoids, leads to an inhibition of the secretion of inflammatory cytokines by myeloid cells. From a clinical perspective, using a high dose of lipopolysaccharide, it was found that mice who had received electrostimulation of the CSN were more likely to survive than control mice. This result is particularly interesting as it demonstrates that electrostimulation of the CSN may be a potential therapeutic in bioelectronic medicine for immune-mediated inflammatory diseases.; Les corps carotidiens sont des structures bilatérales situées au niveau de la bifurcation de l'artère carotide. Ils sont capables de détecter divers stimuli physiologiques tels que la concentration de gaz dans le sang et la pression sanguine. Suite à la variation de ces paramètres au-delà d’une valeur consigne, le corps carotidien, via un nerf afférent appelé nerf sinuso-carotidien (NSC), déclenche une réponse riposte par des voies nerveuses ou hormonales efférentes. De récentes études ont suggéré l’existence d’une relation entre le système immunitaire et le corps carotidien. Il a en particulier été démontré que le corps carotidien est capable de détecter des effecteurs de l’inflammation tels que les cytokines. En outre, des données in vivo démontrent que la résection bilatérale du NSC diminue la survie suite à un choc endotoxémique. Notre étude a porté sur les capacités immunomodulatrices du corps carotidien. Pour mener à bien cette étude, nous avons utilisé une approche d’électrostimulation du NSC plutôt qu’une une approche pharmacologique moins spécifique. Nous avons en particulier développé une méthode chirurgicale pour isoler le NSC, implanter des électrodes puis stimuler le nerf. Nous avons pu constater que la stimulation électrique du NSC atténuait l'expression des cytokines pro-inflammatoires induites par injection de lipopolysaccharide (LPS), en particuliers le Tumor Necrosis Factor (TNF). Par la suite nous avons pu montrer que l'effet de la stimulation du nerf sinusal de la carotide n’était médié ni par des nerfs sympathiques, ni par des nerfs parasympathiques efférents. En revanche, nous avons découvert que la stimulation du NSC augmentait le taux de corticostérone, une hormone anti-inflammatoire qui active le récepteur des glucocorticoïdes. L’antagoniste du récepteur des glucocorticoïdes, abolit les effets de l’atténuation des cytokines proinflammatoires par l’électrostimulation du NSC. De plus, l’utilisation de souris transgéniques ayant des cellules immunitaires myéloïdes dépourvues de récepteurs aux glucocorticoïdes, a montré que l’effet de la stimulation nerveuse du NSC était également perdu indiquant le rôle crucial joué par ces cellules et ce récepteur. Enfin, nous avons pu montrer que la stimulation du NSC conduisait à l’augmentation de l’activité de décharge spontanée des neurones hypothalamiques évoquant un lien entre le NSC et l’axe hypotalamo-hypophysaire. Au final, ces résultats indiquent que l’électrostimulation du NSC atténue l’inflammation en agissant sur l’hypothalamus qui, en augmentant la sécrétion de glucocorticoïdes, conduit à une inhibition de la sécrétion de cytokines inflammatoires par les cellules myéloïdes. D’un point de vu thérapeutique, nous avons pu mettre en évidence que les souris qui avaient reçu une électrostimulation du NSC avaient un taux de survive plus important après injection d’une dose élevée de LPS. Ces résultats suggèrent que l’électrostimulation du NSC pourrait être un traitement potentiel considéré par la médecine bioélectronique pour les maladies inflammatoires immunomédiées en agissant sur l’axe hypotalamo-hypophyso-surrénalien.

The carotid bodies (CB) are located bilaterally at the carotid artery bifurcation. They are polymodal sensors capable of detecting various physiological stimuli – blood gas concentration and blood pressure. The carotid body, via its innervating nerve – the carotid sinus nerve (CSN), signals to the brain to modulate these physiological stimuli through efferent activity. Recent evidence has suggested that there is a relationship between the immune system and the carotid body. It has been shown that the carotid body detects inflammation and functionally responds. Additionally, there is in vivo data, demonstrating that bilateral removal of the CSN decreases survival to endotoxemic shock. We hypothesized that activation of the carotid body would attenuate inflammation. To carry out this study, we electrostimulated the CSN instead of using a non-specific pharmacological approach. A surgical method was developed to isolate the CSN, implant electrodes and then stimulate the nerve. We found that electrical stimulation of CSN attenuated the expression of proinflammatory cytokines induced by lipopolysaccharide (LPS) injection, including Tumor Necrosis Factor (TNF). The mechanism by which electrical activation of the CSN attenuated inflammation was unknown. It was investigated if the effect of CSN stimulation was mediated via efferent parasympathetic or sympathetic nerves - it was found that it was not mediated via these nerves. In contrast, it was discovered, that CSN stimulation increased corticosterone – an anti-inflammatory hormone, which activates the glucocorticoid receptor. An antagonist for the glucocorticoid receptor abolished the attenuation of pro-inflammatory cytokines by CSN electrostimulation. This indicated that corticosterone is the mediator of the effect of CSN stimulation. In addition, we found that the stimulation of the CSN led to the increase of the spontaneous discharge activity of the hypothalamic paraventricular nucleus likening the CSN activity to the hypothalamic-pituitary adrenal axis. Using transgenic mice with no glucocorticoid receptor on their myeloid immune cell, it was found that effect of CSN stimulation was additionally prevented. Finally, we were able to show that the stimulation of the CSN led to the increase of the spontaneous discharge activity of the hypothalamic neurons suggesting a link between the CSN and the hypothalamus-pituitary axis. Overall, these results indicate that electrostimulation of CSN attenuates inflammation by acting on the hypothalamus which, by increasing the secretion of glucocorticoids, leads to an inhibition of the secretion of inflammatory cytokines by myeloid cells. From a clinical perspective, using a high dose of lipopolysaccharide, it was found that mice who had received electrostimulation of the CSN were more likely to survive than control mice. This result is particularly interesting as it demonstrates that electrostimulation of the CSN may be a potential therapeutic in bioelectronic medicine for immune-mediated inflammatory diseases.; Les corps carotidiens sont des structures bilatérales situées au niveau de la bifurcation de l'artère carotide. Ils sont capables de détecter divers stimuli physiologiques tels que la concentration de gaz dans le sang et la pression sanguine. Suite à la variation de ces paramètres au-delà d’une valeur consigne, le corps carotidien, via un nerf afférent appelé nerf sinuso-carotidien (NSC), déclenche une réponse riposte par des voies nerveuses ou hormonales efférentes. De récentes études ont suggéré l’existence d’une relation entre le système immunitaire et le corps carotidien. Il a en particulier été démontré que le corps carotidien est capable de détecter des effecteurs de l’inflammation tels que les cytokines. En outre, des données in vivo démontrent que la résection bilatérale du NSC diminue la survie suite à un choc endotoxémique. Notre étude a porté sur les capacités immunomodulatrices du corps carotidien. Pour mener à bien cette étude, nous avons utilisé une approche d’électrostimulation du NSC plutôt qu’une une approche pharmacologique moins spécifique. Nous avons en particulier développé une méthode chirurgicale pour isoler le NSC, implanter des électrodes puis stimuler le nerf. Nous avons pu constater que la stimulation électrique du NSC atténuait l'expression des cytokines pro-inflammatoires induites par injection de lipopolysaccharide (LPS), en particuliers le Tumor Necrosis Factor (TNF). Par la suite nous avons pu montrer que l'effet de la stimulation du nerf sinusal de la carotide n’était médié ni par des nerfs sympathiques, ni par des nerfs parasympathiques efférents. En revanche, nous avons découvert que la stimulation du NSC augmentait le taux de corticostérone, une hormone anti-inflammatoire qui active le récepteur des glucocorticoïdes. L’antagoniste du récepteur des glucocorticoïdes, abolit les effets de l’atténuation des cytokines proinflammatoires par l’électrostimulation du NSC. De plus, l’utilisation de souris transgéniques ayant des cellules immunitaires myéloïdes dépourvues de récepteurs aux glucocorticoïdes, a montré que l’effet de la stimulation nerveuse du NSC était également perdu indiquant le rôle crucial joué par ces cellules et ce récepteur. Enfin, nous avons pu montrer que la stimulation du NSC conduisait à l’augmentation de l’activité de décharge spontanée des neurones hypothalamiques évoquant un lien entre le NSC et l’axe hypotalamo-hypophysaire. Au final, ces résultats indiquent que l’électrostimulation du NSC atténue l’inflammation en agissant sur l’hypothalamus qui, en augmentant la sécrétion de glucocorticoïdes, conduit à une inhibition de la sécrétion de cytokines inflammatoires par les cellules myéloïdes. D’un point de vu thérapeutique, nous avons pu mettre en évidence que les souris qui avaient reçu une électrostimulation du NSC avaient un taux de survive plus important après injection d’une dose élevée de LPS. Ces résultats suggèrent que l’électrostimulation du NSC pourrait être un traitement potentiel considéré par la médecine bioélectronique pour les maladies inflammatoires immunomédiées en agissant sur l’axe hypotalamo-hypophyso-surrénalien.