IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease.

Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.

Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host.

Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies1-7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2-/- mice8,9 and humans with TET2 mutations1,3,5-7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2-/- mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2-/- mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2-/- mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.

Sensitive and Quantitative Detection of MHC-I Displayed Neoepitopes Using a Semiautomated Workflow and TOMAHAQ Mass Spectrometry.

Advances in several key technologies, including MHC peptidomics, have helped fuel our understanding of basic immune regulatory mechanisms and the identification of T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. However, the current multistep analytical process is challenging, and improvements in throughput and reproducibility would enable rapid characterization of multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines such as MC38, can be enriched in a semiautomated fashion on reusable, dry-storage, customized antibody cartridges. Using this method, a researcher, with very little hands-on time and in a single day, can perform up to 96 simultaneous enrichments at a similar level of quality as a manual workflow. TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification), a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation. This unique combination of robust semiautomated MHC-I peptide isolation and high-throughput multiplexed targeted quantitation allows for both the routine analysis of >4000 unique MHC-I peptides from 250 million cells using nontargeted methods, as well as quantitative sensitivity down to the low amol/µl level using TOMAHAQ targeted MS.

HLA-A*01:03, HLA-A*24:02, HLA-B*08:01, HLA-B*27:05, HLA-B*35:01, HLA-B*44:02, and HLA-C*07:01 monochain transgenic/H-2 class I null mice: novel versatile preclinical models of human T cell responses.

We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α1α2 H chain domains fused with a mouse α3 domain and covalently linked to human ß2-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8(+) T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ-producing CD8(+) T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8(+) T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell-based vaccines.

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity.

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression.

Dietary antioxidant micronutrients alter mucosal inflammatory risk in a murine model of genetic and microbial susceptibility.

Inflammatory bowel diseases (IBD) are caused by the convergence of microbial, environmental, and genetic factors. Diet significantly alters these interactions by affecting both the host and microbiome. Using a mucosal inflammatory model that resembles the human condition of ileal pouchitis, we investigated the effects of Control (CONT) or Antioxidant (AOX) diet, containing pharmacologically relevant levels of 4 micronutrients, on disease risk in wild-type and IL-10-/- animals following surgical self-filling (SF) ileal blind loop placement. Although no differences were found in body weight change or survival, IL-10-/- CONT animals had significantly larger lymphoid organs compared with IL-10-/- AOX or with WT. SF loops from IL-10-/- CONT loop mucosa demonstrated histological inflammation, characterized by goblet cell depletion, increased mucosal myeloperoxidase (MPO), and elevated IFNγ, TNFα, and IL-17α gene expression, which AOX attenuated. AOX elevated luminal IgA in IL-10-/- animals, but not significantly in WT. In IL-10-/- animals, AOX significantly decreased the percentage of CD4 + T-bet and CD4 + RORγ T-cells compared with CONT, with no changes in CD4 + Foxp3+ Treg cells. 16S rRNA gene sequencing demonstrated AOX increased microbial alpha diversity compared with CONT in both genotypes. Notably, colonizing germ-free IL-10-/- hosts with CONT bacterial communities, but not AOX, recapitulated the inflammatory phenotype. Collectively, these findings highlight that common dietary antioxidant micronutrients reshape the gut microbial community to mitigate intestinal inflammatory profiles in genetically susceptible hosts. Insights into the dietary-immune-microbial nexus may improve understanding for recurrent inflammatory episodes in susceptible patient populations and opportunities for practical therapeutics to restore immune and microbial homeostasis.

Murine Norovirus Infection Induces TH1 Inflammatory Responses to Dietary Antigens.

Intestinal reovirus infection can trigger T helper 1 (TH1) immunity to dietary antigen, raising the question of whether other viruses can have a similar impact. Here we show that the acute CW3 strain of murine norovirus, but not the persistent CR6 strain, induces TH1 immunity to dietary antigen. This property of CW3 is dependent on its major capsid protein, a virulence determinant. Transcriptional profiling of mesenteric lymph nodes following infection reveals an immunopathological signature that does not segregate with protective immunity but with loss of oral tolerance, in which interferon regulatory factor 1 is critical. These data show that viral capacity to trigger specific inflammatory pathways at sites where T cell responses to dietary antigens take place interferes with the development of tolerance to an oral antigen. Collectively, these data provide a foundation for the development of therapeutic strategies to prevent TH1-mediated complex immune disorders triggered by viral infections.

Interleukin-15 promotes intestinal dysbiosis with butyrate deficiency associated with increased susceptibility to colitis.

Dysbiosis resulting in gut-microbiome alterations with reduced butyrate production are thought to disrupt intestinal immune homeostasis and promote complex immune disorders. However, whether and how dysbiosis develops before the onset of overt pathology remains poorly defined. Interleukin-15 (IL-15) is upregulated in distressed tissue and its overexpression is thought to predispose susceptible individuals to and have a role in the pathogenesis of celiac disease and inflammatory bowel disease (IBD). Although the immunological roles of IL-15 have been largely studied, its potential impact on the microbiota remains unexplored. Analysis of 16S ribosomal RNA-based inventories of bacterial communities in mice overexpressing IL-15 in the intestinal epithelium (villin-IL-15 transgenic (v-IL-15tg) mice) shows distinct changes in the composition of the intestinal bacteria. Although some alterations are specific to individual intestinal compartments, others are found across the ileum, cecum and feces. In particular, IL-15 overexpression restructures the composition of the microbiota with a decrease in butyrate-producing bacteria that is associated with a reduction in luminal butyrate levels across all intestinal compartments. Fecal microbiota transplant experiments of wild-type and v-IL-15tg microbiota into germ-free mice further indicate that diminishing butyrate concentration observed in the intestinal lumen of v-IL-15tg mice is the result of intrinsic alterations in the microbiota induced by IL-15. This reconfiguration of the microbiota is associated with increased susceptibility to dextran sodium sulfate-induced colitis. Altogether, this study reveals that IL-15 impacts butyrate-producing bacteria and lowers butyrate levels in the absence of overt pathology, which represent events that precede and promote intestinal inflammatory diseases.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease.

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells.

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti-human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1-specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

Design of a HIV-1-derived HLA-B07.02-restricted polyepitope construct.

OBJECTIVE: To design a vaccine construct containing various but conserved HIV-1-derived epitopes and generating broad CD8 T cell responses. METHODS: HLA-B7 transgenic H-2KD KO transgenic mice were used to identify potential new HLA-B07.02-restricted HIV-1-derived epitopes. Immunological recognition of these epitopes was confirmed by IFN-gamma ELISpot assays with PBMCs from HLA-B*0702 HIV-1-infected individuals. For these peptides as well as others previously identified, the capacity to induce cross-reactive responses against their frequent allelic variants was evaluated in the mouse model. A set of epitopes inducing strong T cell responses against various and conserved regions of HIV-1 was selected. A DNA vaccine was designed to express them as a unique antigen with or without a three amino acid ARY extension flanking each epitope. The spectrum of CD8 T responses generated by polyepitope constructs was tested in HLA-B7 transgenic mice. RESULTS: Five new epitopes were identified in accessory and regulatory HIV-1 proteins. Twelve HLA-B07.02-restricted epitopes were selected on the basis of their structural conservation and cross-reactive immunogenicity. The ARY N-terminal extension flanking each epitope markedly increases their affinity for TAP and the use of this flanking extension in polyepitope vaccine has a sizable advantage to induce CD8 T cell cytotoxic responses in mice following DNA immunization. CONCLUSION: The HLA-B7 mouse model allows to rapidly identify various HIV-1-derived epitopes of vaccine interest. Grouped in a polyepitope construct designed to increase their processing, this vaccine may be suitable for inducing multiple and relevant HIV-1-specific CTL responses in humans.