LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells.

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

Major transcriptomic, epigenetic and metabolic changes underlie the pluripotency continuum in rabbit preimplantation embryos.

Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit preimplantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis, and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic and metabolic map of the pluripotency continuum in rabbit preimplantation embryos, and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with those of humans and non-human primates.

Unexpected regulatory functions of cyprinid Viperin on inflammation and metabolism.

BACKGROUND: Viperin, also known as radical S-adenosyl-methionine domain containing protein 2 (RSAD2), is an interferon-inducible protein that is involved in the innate immune response against a wide array of viruses. In mammals, Viperin exerts its antiviral function through enzymatic conversion of cytidine triphosphate (CTP) into its antiviral analog ddhCTP as well as through interactions with host proteins involved in innate immune signaling and in metabolic pathways exploited by viruses during their life cycle. However, how Viperin modulates the antiviral response in fish remains largely unknown. RESULTS: For this purpose, we developed a fathead minnow (Pimephales promelas) clonal cell line in which the unique viperin gene has been knocked out by CRISPR/Cas9 genome-editing. In order to decipher the contribution of fish Viperin to the antiviral response and its regulatory role beyond the scope of the innate immune response, we performed a comparative RNA-seq analysis of viperin-/- and wildtype cell lines upon stimulation with recombinant fathead minnow type I interferon. CONCLUSIONS: Our results revealed that Viperin does not exert positive feedback on the canonical type I IFN but acts as a negative regulator of the inflammatory response by downregulating specific pro-inflammatory genes and upregulating repressors of the NF-κB pathway. It also appeared to play a role in regulating metabolic processes, including one carbon metabolism, bone formation, extracellular matrix organization and cell adhesion.

Toll-like receptors control the accumulation of neutrophils in lymph nodes that expand CD4+ T cells during experimental autoimmune encephalomyelitis.

Toll-like receptors (TLR) control the activation of dendritic cells that prime CD4+ T cells in draining lymph nodes, where these T cells then undergo massive clonal expansion. The mechanisms controlling this clonal T cell expansion are poorly defined. Using the CD4+ T cell-mediated disease experimental autoimmune encephalomyelitis (EAE), we show here that this process is markedly suppressed when TLR9 signaling is increased, without noticeably affecting the transcriptome of primed T cells, indicating a purely quantitative effect on CD4+ T cell expansion. Addressing the underpinning mechanisms revealed that CD4+ T cell expansion was preceded and depended on the accumulation of neutrophils in lymph nodes a few days after immunization. Underlying the importance of this immune regulation pathway, blocking neutrophil accumulation in lymph nodes by treating mice with a TLR9 agonist inhibited EAE progression in mice with defects in regulatory T cells or regulatory B cells, which otherwise developed a severe chronic disease. Collectively, this study demonstrates the key role of neutrophils in the quantitative regulation of antigen-specific CD4+ T cell expansion in lymph nodes, and the counter-regulatory role of TLR signaling in this process.

Long-term exposure from perinatal life to food-grade TiO2 alters intestinal homeostasis and predisposes to food allergy in young mice.

BACKGROUND: Food allergy (FA) is an inappropriate immunological response to food proteins resulting from an impaired induction of oral tolerance. Various early environmental factors can affect the establishment of intestinal homeostasis, predisposing to FA in early life. In this context, we aimed to assess the effect of chronic perinatal exposure to food-grade titanium dioxide (fg-TiO2 ), a common food additive. METHODS: Dams were fed a control versus fg-TiO2 -enriched diet from preconception to weaning, and their progeny received the same diet at weaning. A comprehensive analysis of baseline intestinal and systemic homeostasis was performed in offspring 1 week after weaning by assessing gut barrier maturation and microbiota composition, and local and systemic immune system and metabolome. The effect of fg-TiO2 on the susceptibility of progeny to develop oral tolerance versus FA to cow's milk proteins (CMP) was performed starting at the same baseline time-point, using established models. Sensitization to CMP was investigated by measuring ß-lactoglobulin and casein-specific IgG1 and IgE antibodies, and elicitation of the allergic reaction by measuring mouse mast cell protease (mMCP1) in plasma collected after an oral food challenge. RESULTS: Perinatal exposure to fg-TiO2 at realistic human doses led to an increased propensity to develop FA and an impaired induction of oral tolerance only in young males, which could be related to global baseline alterations in intestinal barrier, gut microbiota composition, local and systemic immunity, and metabolism. CONCLUSIONS: Long-term perinatal exposure to fg-TiO2 alters intestinal homeostasis establishment and predisposes to food allergy, with a clear gender effect.

Clonotypic IgH Response against Systemic Viral infection in Pronephros and Spleen of a Teleost Fish.

Upon infection, B lymphocytes develop clonal responses. In teleost fish, which lack lymph nodes, the kinetics and location of B cell responses remain poorly characterized. Fish pronephros is the site of B cell differentiation and the main niche for persistence of plasma cells. In this study, we undertook the analysis of the rainbow trout IgHµ repertoire in this critical tissue for humoral adaptive immunity after primary immunization and boost with a rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We used a barcoded 5' RACE-cDNA sequencing approach to characterize modifications of the IgHµ repertoire, including VH usage in expressed V(D)J rearrangements, clonal diversity, and clonotype sharing between individual fish and treatments. In the pronephros, our approach quantified the clonotype frequency across the whole IgH repertoire (i.e., with all VH), measuring the frequency of Ag-responding clonotypes. Viral infection led to extensive modifications of the pronephros B cell repertoire, implicating several VH subgroups after primary infection. In contrast, only modest changes in repertoire persisted 5 mo later, including VHSV-specific public expansions. The IgM public response implicating IgHV1-18 and JH5, previously described in spleen, was confirmed in pronephros in all infected fish, strongly correlated to the response. However, the distribution of top clonotypes showed that pronephros and spleen B cells constitute distinct compartments with different IgH repertoires. Unexpectedly, after boost, the frequency of anti-VHSV clonotypes decreased both in pronephros and spleen, raising questions about B cell circulation. A better monitoring of B cell response kinetics in lymphoid tissues will be an essential step to understand B memory and plasmocyte formation mechanisms in fish.

Antigen receptor-engineered Tregs inhibit CNS autoimmunity in cell therapy using nonredundant immune mechanisms in mice.

CD4+ FOXP3+ Tregs are currently explored to develop cell therapies against immune-mediated disorders, with an increasing focus on antigen receptor-engineered Tregs. Deciphering their mode of action is necessary to identify the strengths and limits of this approach. Here, we addressed this issue in an autoimmune disease of the CNS, EAE. Following disease induction, autoreactive Tregs upregulated LAG-3 and CTLA-4 in LNs, while IL-10 and amphiregulin (AREG) were increased in CNS Tregs. Using genetic approaches, we demonstrated that IL-10, CTLA-4, and LAG-3 were nonredundantly required for the protective function of antigen receptor-engineered Tregs against EAE in cell therapy whereas AREG was dispensable. Treg-derived IL-10 and CTLA-4 were both required to suppress acute autoreactive CD4+ T-cell activation, which correlated with disease control. These molecules also affected the accumulation in the recipients of engineered Tregs themselves, underlying complex roles for these molecules. Noteworthy, despite the persistence of the transferred Tregs and their protective effect, autoreactive T cells eventually accumulated in the spleen of treated mice. In conclusion, this study highlights the remarkable power of antigen receptor-engineered Tregs to appropriately provide multiple suppressive factors nonredundantly necessary to prevent autoimmune attacks.

Cutting Edge: Neutralizing Public Antibody Responses Are an Ancient Form of Defense Conserved in Fish and Mammals.

The repertoire of Abs is generated by genomic rearrangements during B cell differentiation. Although V(D)J rearrangements lead to repertoires mostly different between individuals, recent studies have shown that they contain a substantial fraction of overrepresented and shared "public" clones. We previously reported a strong public IgHµ clonotypic response against the rhabdovirus viral hemorrhagic septicemia virus in a teleost fish. In this study, we identified an IgL chain associated with this public response that allowed us to characterize its functionality. We show that this public Ab response has a potent neutralizing capacity that is typically associated with host protection during rhabdovirus infections. We also demonstrate that the public response is not restricted to a particular trout isogenic line but expressed in multiple genetic backgrounds and may be used as a marker of successful vaccination. Our work reveals that public B cell responses producing generic Abs constitute a mechanism of protection against infection conserved across vertebrates.

Maternal age affects equine day 8 embryo gene expression both in trophoblast and inner cell mass.

BACKGROUND: Breeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mare's age on blastocysts' gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bisection, were RNA sequenced. Deconvolution algorithm was used to discriminate gene expression in the ICM from that in the trophoblast. Differential expression was analyzed with embryo sex and diameter as cofactors. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed. RESULTS: Maternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity. In both compartments, the expression of genes involved in mitochondria and protein metabolism were disturbed by maternal age, although more genes were affected in the ICM. Mitosis, signaling and adhesion pathways and embryo development were decreased in the ICM of embryos from old mares. In trophoblast, ion movement pathways were affected. CONCLUSIONS: This is the first study showing that maternal age affects gene expression in the equine blastocyst, demonstrating significant effects as early as 10 years of age. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

Prepubertal nutritional modulation in the bull and its impact on sperm DNA methylation.

Enhanced pre-pubertal nutrition in Holstein bulls increased reproductive hormone production and sperm production potential with no negative effects on sperm quality. However, recent trends in human epigenetic research have identified pre-pubertal period to be critical for epigenetic reprogramming in males. Our objective was to evaluate the methylation changes in sperm of bulls exposed to different pre-pubertal diets. One-week-old Holstein bull calves (n = 9), randomly allocated to 3 groups, were fed either a high, medium or low diet (20%, 17% or 12.2% crude protein and 67.9%, 66% or 62.9% total digestible nutrients, respectively) from 2 to 32 weeks of age, followed by medium nutrition. Semen collected from bulls at two specific time points, i.e. 55-59 and 69-71 weeks, was diluted, cryopreserved and used for reduced representation bisulfite sequencing. Differential methylation was detected for dietary treatment, but minimal differences were detected with age. The gene ontology term, "regulation of Rho protein signal transduction", implicated in sperm motility and acrosome reaction, was enriched in both low-vs-high and low-vs-medium datasets. Furthermore, several genes implicated in early embryo and foetal development showed differential methylation for diet. Our results therefore suggest that sperm epigenome keeps the memory of diet during pre-pubertal period in genes important for spermatogenesis, sperm function and early embryo development.

Ovine fetal testis stage-specific sensitivity to environmental chemical mixtures.

Exposure of the fetal testis to numerous individual environmental chemicals (ECs) is frequently associated with dysregulated development, leading to impaired adult reproductive competence. However, 'real-life' exposure involves complex mixtures of ECs. Here we test the consequences, for the male fetus, of exposing pregnant ewes to EC mixtures derived from pastures treated with biosolids fertiliser (processed human sewage). Fetal testes from continuously exposed ewes were either unaffected at day 80 or exhibited a reduced area of testis immunostained for CYP17A1 protein at day 140. Fetal testes from day 140 pregnant ewes that were exposed transiently for 80-day periods during early (0-80 days), mid (30-110 days), or late (60-140 days) pregnancy had fewer Sertoli cells and reduced testicular area stained for CYP17A1. Male fetuses from ewes exposed during late pregnancy also exhibited reduced fetal body, adrenal and testis mass, anogenital distance, and lowered testosterone; collectively indicative of an anti-androgenic effect. Exposure limited to early gestation induced more testis transcriptome changes than observed for continuously exposed day 140 fetuses. These data suggest that a short period of EC exposure does not allow sufficient time for the testis to adapt. Consequently, testicular transcriptomic changes induced during the first 80 days of gestation may equate with phenotypic effects observed at day 140. In contrast, relatively fewer changes in the testis transcriptome in fetuses exposed continuously to ECs throughout gestation are associated with less severe consequences. Unless corrected by or during puberty, these differential effects would predictably have adverse outcomes for adult testicular function and fertility.

Dual role of DMXL2 in olfactory information transmission and the first wave of spermatogenesis.

Gonad differentiation is a crucial step conditioning the future fertility of individuals and most of the master genes involved in this process have been investigated in detail. However, transcriptomic analyses of developing gonads from different animal models have revealed that hundreds of genes present sexually dimorphic expression patterns. DMXL2 was one of these genes and its function in mammalian gonads was unknown. We therefore investigated the phenotypes of total and gonad-specific Dmxl2 knockout mouse lines. The total loss-of-function of Dmxl2 was lethal in neonates, with death occurring within 12 hours of birth. Dmxl2-knockout neonates were weak and did not feed. They also presented defects of olfactory information transmission and severe hypoglycemia, suggesting that their premature death might be due to global neuronal and/or metabolic deficiencies. Dmxl2 expression in the gonads increased after birth, during follicle formation in females and spermatogenesis in males. DMXL2 was detected in both the supporting and germinal cells of both sexes. As Dmxl2 loss-of-function was lethal, only limited investigations of the gonads of Dmxl2 KO pups were possible. They revealed no major defects at birth. The gonadal function of Dmxl2 was then assessed by conditional deletions of the gene in gonadal supporting cells, germinal cells, or both. Conditional Dmxl2 ablation in the gonads did not impair fertility in males or females. By contrast, male mice with Dmxl2 deletions, either throughout the testes or exclusively in germ cells, presented a subtle testicular phenotype during the first wave of spermatogenesis that was clearly detectable at puberty. Indeed, Dmxl2 loss-of-function throughout the testes or in germ cells only, led to sperm counts more than 60% lower than normal and defective seminiferous tubule architecture. Transcriptomic and immunohistochemichal analyses on these abnormal testes revealed a deregulation of Sertoli cell phagocytic activity related to germ cell apoptosis augmentation. In conclusion, we show that Dmxl2 exerts its principal function in the testes at the onset of puberty, although its absence does not compromise male fertility in mice.

Nasal Vaccination Drives Modifications of Nasal and Systemic Antibody Repertoires in Rainbow Trout.

Bony fish represent the most basal vertebrate branch with a dedicated mucosal immune system, which comprises immunologically heterogeneous microenvironments armed with innate and adaptive components. In rainbow trout (Oncorhynchus mykiss), a nasopharynx-associated lymphoid tissue (NALT) was recently described as a diffuse network of myeloid and lymphoid cells located in the olfactory organ of fish. Several studies have demonstrated high levels of protection conferred by nasal vaccines against viral and bacterial pathogens; however, the mechanisms underlying the observed protection are not well understood. We applied 5'RACE and a deep sequencing-based approach to investigate the clonal structure of the systemic and mucosal rainbow trout B cell repertoire. The analysis of Ig repertoire in control trout suggests different structures of IgM and IgT spleen and NALT repertoires, with restricted repertoire diversity in NALT. Nasal and injection vaccination with a bacterial vaccine revealed unique dynamics of IgM and IgT repertoires at systemic and mucosal sites and the remarkable ability of nasal vaccines to induce spleen Ig responses. Our findings provide an important immunological basis for the effectiveness of nasal vaccination in fish and other vertebrate animals and will help the design of future nasal vaccination strategies.

IFN-Stimulated Genes in Zebrafish and Humans Define an Ancient Arsenal of Antiviral Immunity.

The evolution of the IFN system, the major innate antiviral mechanism of vertebrates, remains poorly understood. According to the detection of type I IFN genes in cartilaginous fish genomes, the system appeared 500 My ago. However, the IFN system integrates many other components, most of which are encoded by IFN-stimulated genes (ISGs). To shed light on its evolution, we have used deep RNA sequencing to generate a comprehensive list of ISGs of zebrafish, taking advantage of the high-quality genome annotation in this species. We analyzed larvae after inoculation of recombinant zebrafish type I IFN, or infection with chikungunya virus, a potent IFN inducer. We identified more than 400 zebrafish ISGs, defined as being either directly induced by IFN or induced by the virus in an IFNR-dependent manner. Their human orthologs were highly enriched in ISGs, particularly for highly inducible genes. We identified 72 orthology groups containing ISGs in both zebrafish and humans, revealing a core ancestral ISG repertoire that includes most of the known signaling components of the IFN system. Many downstream effectors were also already present 450 My ago in the common ancestor of tetrapods and bony fish and diversified as multigene families independently in the two lineages. A large proportion of the ISG repertoire is lineage specific; around 40% of protein-coding zebrafish ISGs had no human ortholog. We identified 14 fish-specific gene families containing multiple ISGs, including finTRIMs. This work illuminates the evolution of the IFN system and provides a rich resource to explore new antiviral mechanisms.

Viral Resistance and IFN Signaling in STAT2 Knockout Fish Cells.

IFN belong to a group of cytokines specialized in the immunity to viruses. Upon viral infection, type I IFN is produced and alters the transcriptome of responding cells through induction of a set of IFN stimulated genes (ISGs) with regulatory or antiviral function, resulting in a cellular antiviral state. Fish genomes have both type I IFN and type II IFN (IFN-γ), but no type III (λ) IFN has been identified. Their receptors are not simple counterparts of the mammalian type I/II IFN receptors, because alternative chains are used in type I IFN receptors. The mechanisms of the downstream signaling remain partly undefined. In mammals, members of the signal transducer and activator of family of transcription factors are responsible for the transmission of the signal from cytokine receptors, and STAT2 is required for type I but not type II IFN signaling. In fish, its role in IFN signaling in fish remains unclear. We isolated a Chinook salmon (Oncorhynchus tshawytscha) cell line, GS2, with a stat2 gene knocked out by CRISPR/Cas9 genome editing. In this cell line, the induction of ISGs by stimulation with a recombinant type I IFN is completely obliterated as evidenced by comparative RNA-seq analysis of the transcriptome of GS2 and its parental counterpart, EC. Despite a complete absence of ISGs induction, the GS2 cell line has a remarkable ability to resist to viral infections. Therefore, other STAT2-independent pathways may be induced by the viral infection, illustrating the robustness and redundancy of the innate antiviral defenses in fish.

IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases.

B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM(+)CD138(hi)TACI(+)CXCR4(+)CD1d(int)Tim1(int) plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138(+) plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.

Spleen and head kidney differential gene expression patterns in trout infected with Lactococcus garvieae correlate with spleen granulomas.

Lactococcus garvieae is a significant pathogen in aquaculture with a potential zoonotic risk. To begin to characterize the late immune response of trout to lactococcosis, we selected infected individuals showing clinical signs of lactococcosis. At the time lactococcosis clinical signs appeared, infection by L. garvieae induced a robust inflammatory response in the spleen of rainbow trout, which correlated with abundant granulomatous lesions. The response in kidney goes in parallel with that of spleen, and most of the gene regulations are similar in both organs. A correlation existed between the early inflammatory granulomas in spleen (containing macrophages with internalized L. garvieae) and up-regulated gene sets, which defined the presence of macrophages and neutrophils. This is the first analysis of the immune transcriptome of rainbow trout following L. garvieae infection during the initiation of adaptive immune mechanisms and shows a transcriptome induction of antibody response by both IgM (+) and IgT (+) spleen B cells to respond to systemic infection. These results increase our understanding of lactococcosis and pave the way for future research to improve control measures of lactococcosis on fish farms.

B Cells Producing Type I IFN Modulate Macrophage Polarization in Tuberculosis.

RATIONALE: In addition to their well-known function as antibody-producing cells, B lymphocytes can markedly influence the course of infectious or noninfectious diseases via antibody-independent mechanisms. In tuberculosis (TB), B cells accumulate in lungs, yet their functional contribution to the host response remains poorly understood. OBJECTIVES: To document the role of B cells in TB in an unbiased manner. METHODS: We generated the transcriptome of B cells isolated from Mycobacterium tuberculosis (Mtb)-infected mice and validated the identified key pathways using in vitro and in vivo assays. The obtained data were substantiated using B cells from pleural effusion of patients with TB. MEASUREMENTS AND MAIN RESULTS: B cells isolated from Mtb-infected mice displayed a STAT1 (signal transducer and activator of transcription 1)-centered signature, suggesting a role for IFNs in B-cell response to infection. B cells stimulated in vitro with Mtb produced type I IFN, via a mechanism involving the innate sensor STING (stimulator of interferon genes), and antagonized by MyD88 (myeloid differentiation primary response 88) signaling. In vivo, B cells expressed type I IFN in the lungs of Mtb-infected mice and, of clinical relevance, in pleural fluid from patients with TB. Type I IFN expression by B cells induced an altered polarization of macrophages toward a regulatory/antiinflammatory profile in vitro. In vivo, increased provision of type I IFN by B cells in a murine model of B cell-restricted Myd88 deficiency correlated with an enhanced accumulation of regulatory/antiinflammatory macrophages in Mtb-infected lungs. CONCLUSIONS: Type I IFN produced by Mtb-stimulated B cells favors macrophage polarization toward a regulatory/antiinflammatory phenotype during Mtb infection.

Restricting nonclassical MHC genes coevolve with TRAV genes used by innate-like T cells in mammals.

Whereas major histocompatibility class-1 (MH1) proteins present peptides to T cells displaying a large T-cell receptor (TR) repertoire, MH1Like proteins, such as CD1D and MR1, present glycolipids and microbial riboflavin precursor derivatives, respectively, to T cells expressing invariant TR-α (iTRA) chains. The groove of such MH1Like, as well as iTRA chains used by mucosal-associated invariant T (MAIT) and natural killer T (NKT) cells, respectively, may result from a coevolution under particular selection pressures. Herein, we investigated the evolutionary patterns of the iTRA of MAIT and NKT cells and restricting MH1Like proteins: MR1 appeared 170 Mya and is highly conserved across mammals, evolving more slowly than other MH1Like. It has been pseudogenized or independently lost three times in carnivores, the armadillo, and lagomorphs. The corresponding TRAV1 gene also evolved slowly and harbors highly conserved complementarity determining regions 1 and 2. TRAV1 is absent exclusively from species in which MR1 is lacking, suggesting that its loss released the purifying selection on MR1. In the rabbit, which has very few NKT and no MAIT cells, a previously unrecognized iTRA was identified by sequencing leukocyte RNA. This iTRA uses TRAV41, which is highly conserved across several groups of mammals. A rabbit MH1Like gene was found that appeared with mammals and is highly conserved. It was independently lost in a few groups in which MR1 is present, like primates and Muridae, illustrating compensatory emergences of new MH1Like/Invariant T-cell combinations during evolution. Deciphering their role is warranted to search similar effector functions in humans.

Genetic and transcriptomic analyses provide new insights on the early antiviral response to VHSV in resistant and susceptible rainbow trout.

BACKGROUND: The viral hemorrhagic septicemia virus (VHSV) is a major threat for salmonid farming and for wild fish populations worldwide. Previous studies have highlighted the importance of innate factors regulated by a major quantitative trait locus (QTL) for the natural resistance to waterborne VHSV infection in rainbow trout. The aim of this study was to analyze the early transcriptomic response to VHSV inoculation in cell lines derived from previously described resistant and susceptible homozygous isogenic lines of rainbow trout to obtain insights into the molecular mechanisms responsible for the resistance to the viral infection. RESULTS: We first confirmed the presence of the major QTL in a backcross involving a highly resistant fish isogenic line (B57) and a highly susceptible one (A22), and were able to define the confidence interval of the QTL and to identify its precise position. We extended the definition of the QTL since it controls not only resistance to waterborne infection but also the kinetics of mortality after intra-peritoneal injection. Deep sequencing of the transcriptome of B57 and A22 derived cell lines exposed to inactivated VHSV showed a stronger response to virus inoculation in the resistant background. In line with our previous observations, an early and strong induction of interferon and interferon-stimulated genes was correlated with the resistance to VHSV, highlighting the major role of innate immune factors in natural trout resistance to the virus. Interestingly, major factors of the antiviral innate immunity were much more expressed in naive B57 cells compared to naive A22 cells, which likely contributes to the ability of B57 to mount a fast antiviral response after viral infection. These observations were further extended by the identification of several innate immune-related genes localized close to the QTL area on the rainbow trout genome. CONCLUSIONS: Taken together, our results improve our knowledge in virus-host interactions in vertebrates and provide novel insights in the molecular mechanisms explaining the resistance to VHSV in rainbow trout. Our data also provide a collection of potential markers for resistance and susceptibility of rainbow trout to VHSV infection.