Structure and Mechanisms of Assembly-Line Polyketide Synthases.

Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS. Looking forward, one anticipates major advances in our understanding of the mechanisms by which the free energy of a repetitive Claisen-like reaction is harnessed to guide the growing polyketide chain along the assembly line in a manner that is kinetically robust yet evolutionarily adaptable.

Past, Present, and Future of Noninvasive Tests to Assess Gluten Exposure, Celiac Disease Activity, and End-Organ Damage.

Although many biomarkers have been proposed, and several are in widespread clinical use, there is no single readout or combination of readouts that correlates tightly with gluten exposure, disease activity, or end-organ damage in treated patients with celiac disease. Challenges to developing and evaluating better biomarkers include significant interindividual variability-related to immune amplification of gluten exposure and how effects of immune activation are manifest. Furthermore, the current "gold standard" for assessment of end-organ damage, small intestinal biopsy, is itself highly imperfect, such that a marker that is a better reflection of the "ground truth" may indeed appear to perform poorly. The goal of this review was to analyze past and present efforts to establish robust noninvasive tools for monitoring treated patients with celiac disease and to highlight emerging tools that may prove to be useful in clinical practice.

Discovery and Characterization of the Fully Decorated Nocardiosis-Associated Polyketide Natural Product.

The genomes of 40 strains of Nocardia, most of which were associated with life-threatening human infections, encode a highly conserved assembly line polyketide synthase designated as the NOCAP (NOCardiosis-Associated Polyketide) synthase, whose product structure has been previously described. Here we report the structure and inferred biosynthetic pathway of the fully decorated glycolipid natural product. Its structure reveals a fully substituted benzaldehyde headgroup harboring an unusual polyfunctional tail and an O-linked disaccharide comprising a 3-α-epimycarose and 2-O-methyl-α-rhamnose whose installation requires flavin monooxygenase-dependent hydroxylation of the polyketide product. Production of the fully decorated glycolipid was verified in cultures of two patient-derived Nocardia species. In both E. coli and Nocardia spp., the glycolipid was only detected in culture supernatants, consistent with data from genetic knockout experiments implicating roles for two dedicated proteins in installing the second sugar substituent only after the monoglycosyl intermediate is exported across the bacterial cell membrane. With the NOCAP product in hand, the stage is set for investigating the evolutionary benefit of this polyketide biosynthetic pathway for Nocardia strains capable of infecting human hosts.

Celiac disease: mechanisms and emerging therapeutics.

Celiac disease (CeD) is a widespread, gluten-induced, autoimmune disorder that lacks any medicinal therapy. Towards the goal of developing non-dietary treatments for CeD, research has focused on elucidating its molecular and cellular etiology. A model of pathogenesis has emerged centered on interactions between three molecular families: specific class II MHC proteins on antigen-presenting cells (APCs), deamidated gluten-derived peptides, and T cell receptors (TCRs) on inflammatory CD4+ T cells. Growing evidence suggests that this pathogenic axis can be pharmacologically targeted to protect patients from some of the adverse effects of dietary gluten. Further studies have revealed the existence of additional host and environmental contributors to disease initiation and tissue damage. This review summarizes our current understanding of CeD pathogenesis and how it is being harnessed for therapeutic design and development.

Targeted Lysosomal Degradation of Secreted and Cell Surface Proteins through the LRP-1 Pathway.

Protein dysregulation has been characterized as the cause of pathogenesis in many different diseases. For proteins lacking easily druggable pockets or catalytically active sites, targeted protein degradation is an attractive therapeutic approach. While several methods for targeted protein degradation have been developed, there remains a demand for lower molecular weight molecules that promote efficient degradation of their targets. In this work, we describe the synthesis and validation of a series of heterobifunctional molecules that bind a protein of interest through a small molecule ligand while targeting them to the lysosome using a short gluten peptide that leverages the TG2/LRP-1 pathway. We demonstrate that this approach can be used to effectively endocytose and degrade representative secreted, cell surface, and transmembrane proteins, notably streptavidin, the vitamin B12 receptor, cubilin, and integrin αvß5. Optimization of these prototypical molecules could generate pharmacologically relevant LYTAC agents.

Genomic mining and diversity of assembly line polyketide synthases.

Assembly line polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for synthesizing many medicinally relevant natural products with remarkable structural variety and biological activity. The decrease in cost of genomic sequencing paired with development of computational tools like antiSMASH presents an opportunity to survey the vast diversity of assembly line PKS. Mining the genomic data in the National Center for Biotechnology Information database, our updated catalogue (https://orphanpkscatalog2022.stanford.edu/catalog) presented in this article revealed 8799 non-redundant assembly line polyketide synthase clusters across 4083 species, representing a threefold increase over the past 4 years. Additionally, 95% of the clusters are 'orphan clusters' for which natural products are neither chemically nor biologically characterized. Our analysis indicates that the diversity of assembly line PKSs remains vastly under-explored and also highlights the promise of a genomics-driven approach to natural product discovery.

Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase.

Fragment antigen-binding domains of antibodies (Fabs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of Fabs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two Fabs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediated reactions catalyzed by this module, albeit by distinct mechanisms. AC2 primarily affects the rate (kcat), whereas BB1 increases the KM of an ACP-mediated reaction. A third Fab, AA5, binds to the KS-AT fragment of DEBS Module 2 without altering either parameter; it is phenotypically reminiscent of a previously characterized Fab, 1B2, shown to principally recognize the N-terminal helical docking domain of DEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-AT fragment of Module 2 were solved to 2.70 and 2.65 Å resolution, respectively, and revealed entirely distinct recognition features of the two antibodies. The new tools and insights reported here pave the way toward advancing our understanding of the structure-function relationships of DEBS Module 2, arguably the most well-studied module of an assembly line PKS.

Evaluation of Acebilustat, a Selective Inhibitor of Leukotriene B4 Biosynthesis, for Treatment of Outpatients With Mild-Moderate Coronavirus Disease 2019: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

BACKGROUND: The vast majority of coronavirus disease 2019 (COVID-19) disease occurs in outpatients where treatment is limited to antivirals for high-risk subgroups. Acebilustat, a leukotriene B4 inhibitor, has potential to reduce inflammation and symptom duration. METHODS: In a single-center trial spanning Delta and Omicron variants, outpatients were randomized to 100 mg/d of oral acebilustat or placebo for 28 days. Patients reported daily symptoms via electronic query through day 28 with phone follow-up on day 120 and collected nasal swab samples on days 1-10. The primary outcome was sustained symptom resolution to day 28. Secondary 28-day outcomes included time to first symptom resolution, area under the curve (AUC) for longitudinal daily symptom scores, duration of viral shedding through day 10, and symptoms on day 120. RESULTS: Sixty participants were randomized to each study arm. At enrollment, the median duration was 4 days (interquartile range, 3-5 days), and the median number of symptoms was 9 (7-11). Most patients (90%) were vaccinated, with 73% having neutralizing antibodies. A minority of participants (44%; 35% in the acebilustat arm and 53% in placebo) had sustained symptom resolution at day 28 (hazard ratio, 0.6 [95% confidence interval, .34-1.04]; P = .07 favoring placebo). There was no difference in the mean AUC for symptom scores over 28 days (difference in mean AUC, 9.4 [95% confidence interval, -42.1 to 60.9]; P = .72). Acebilustat did not affect viral shedding or symptoms at day 120. CONCLUSIONS: Sustained symptoms through day 28 were common in this low-risk population. Despite this, leukotriene B4 antagonism with acebilustat did not shorten symptom duration in outpatients with COVID-19. Clinical Trials Registration. NCT04662060.

Challenges in Harnessing Shared Within-Host Severe Acute Respiratory Syndrome Coronavirus 2 Variation for Transmission Inference.

Background: The limited variation observed among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference. Methods: We performed tiled amplicon sequencing on 307 SARS-CoV-2 samples, including 130 samples from 32 individuals in 14 households and 47 longitudinally sampled individuals, from 4 prospective studies with household membership data, a proxy for transmission linkage. Results: Consensus sequences from households had limited diversity (mean pairwise distance, 3.06 single-nucleotide polymorphisms [SNPs]; range, 0-40). Most (83.1%, 255 of 307) samples harbored at least 1 intrahost single-nucleotide variant ([iSNV] median, 117; interquartile range [IQR], 17-208), above a minor allele frequency threshold of 0.2%. Pairs in the same household shared significantly more iSNVs (mean, 1.20 iSNVs; 95% confidence interval [CI], 1.02-1.39) than did pairs in different households infected with the same viral clade (mean, 0.31 iSNVs; 95% CI, .28-.34), a signal that decreases with increasingly stringent minor allele frequency thresholds. The number of shared iSNVs was significantly associated with an increased odds of household membership (adjusted odds ratio, 1.35; 95% CI, 1.23-1.49). However, the poor concordance of iSNVs detected across sequencing replicates (24.8% and 35.0% above a 0.2% and 1% threshold) confirms technical concerns that current sequencing and bioinformatic workflows do not consistently recover low-frequency within-host variants. Conclusions: Shared within-host variation may augment the information in consensus sequences for predicting transmission linkages. Improving sensitivity and specificity of within-host variant identification will improve the informativeness of within-host variation.

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for ß-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did ß-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

LRP-1 links post-translational modifications to efficient presentation of celiac disease-specific T cell antigens.

Celiac disease (CeD) is an autoimmune disorder in which gluten-derived antigens trigger inflammation. Antigenic peptides must undergo site-specific deamidation to be presentable to CD4+ T cells in an HLA-DQ2 or -DQ8 restricted manner. While the biochemical basis for this post-translational modification is understood, its localization in the patient's intestine remains unknown. Here, we describe a mechanism by which gluten peptides undergo deamidation and concentration in the lysosomes of antigen-presenting cells, explaining how the concentration of gluten peptides necessary to elicit an inflammatory response in CeD patients is achieved. A ternary complex forms between a gluten peptide, transglutaminase-2 (TG2), and ubiquitous plasma protein α2-macroglobulin, and is endocytosed by LRP-1. The covalent TG2-peptide adduct undergoes endolysosomal decoupling, yielding the expected deamidated epitope. Our findings invoke a pathogenic role for dendritic cells and/or macrophages in CeD and implicate TG2 in the lysosomal clearance of unwanted self and foreign extracellular proteins.

Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2).

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.

Early immune markers of clinical, virological, and immunological outcomes in patients with COVID-19: a multi-omics study.

Background: The great majority of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunological outcomes in SARS-CoV-2-infected patients. Methods: Leveraging longitudinal samples and data from a clinical trial (N=108) in SARS-CoV-2-infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients. We characterized the association between early immune markers and subsequent disease progression, control of viral shedding, and SARS-CoV-2-specific T cell and antibody responses measured up to 7 months after enrollment. We further compared associations between early immune markers and subsequent T cell and antibody responses following natural infection with those following mRNA vaccination. We developed machine-learning models to predict patient outcomes and validated the predictive model using data from 54 individuals enrolled in an independent clinical trial. Results: We identify early immune signatures, including plasma RIG-I levels, early IFN signaling, and related cytokines (CXCL10, MCP1, MCP-2, and MCP-3) associated with subsequent disease progression, control of viral shedding, and the SARS-CoV-2-specific T cell and antibody response measured up to 7 months after enrollment. We found that several biomarkers for immunological outcomes are shared between individuals receiving BNT162b2 (Pfizer-BioNTech) vaccine and COVID-19 patients. Finally, we demonstrate that machine-learning models using 2-7 plasma protein markers measured early within the course of infection are able to accurately predict disease progression, T cell memory, and the antibody response post-infection in a second, independent dataset. Conclusions: Early immune signatures following infection can accurately predict clinical and immunological outcomes in outpatients with COVID-19 using validated machine-learning models. Funding: Support for the study was provided from National Institute of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) (U01 AI150741-01S1 and T32-AI052073), the Stanford's Innovative Medicines Accelerator, National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA) DP1DA046089, and anonymous donors to Stanford University. Peginterferon lambda provided by Eiger BioPharmaceuticals.

In vivo visualization and molecular targeting of the cardiac conduction system.

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo, providing a proof of principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging, and arrhythmia management.

A Mouse Model of Celiac Disease.

The design and use of mouse models that reproduce key features of human diseases are critical to advance our understanding of the pathogenesis of autoimmune diseases and to test new therapeutic strategies. Celiac disease is a unique organ-specific autoimmune-like disorder occurring in genetically susceptible individuals carrying HLA-DQ2 or HLA-DQ8 molecules who consume gluten. The key histological characteristic of the disease in humans is the destruction of the lining of the small intestine, a feature that has been difficult to reproduce in immunocompetent animal models. This unit describes the DQ8-Dd -villin-IL-15 transgenic mouse model of CeD, which was engineered based on the knowledge acquired from studying CeD patients' intestinal samples, and which represents the first animal model that develops villous atrophy in an HLA- and gluten-dependent manner without administration of any adjuvant. We provide detailed protocols for inducing and monitoring intestinal tissue damage, evaluating the cytotoxic properties of intraepithelial lymphocytes that mediate enterocyte lysis, and assessing the activation of the enzyme transglutaminase 2, which contributes to the generation of highly immunogenic gluten peptides. Detailed protocols to prepare pepsin-trypsin digested gliadin (PT-gliadin) or chymotrypsin-digested gliadin (CT-gliadin), which allow antibody detection against native or deamidated gluten peptides, are also provided in this unit. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of celiac-like disease in DQ8-Dd -villin-IL-15tg mice Basic Protocol 2: Histological assessment of villous atrophy Support Protocol 1: Morphometric assessment of villous/crypt ratio Support Protocol 2: Evaluation of epithelial cells renewal Support Protocol 3: Evaluation of the density of intraepithelial lymphocytes Basic Protocol 3: Analysis of cytotoxic intraepithelial lymphocytes Basic Protocol 4: Transglutaminase 2 activation and measurement of antibodies against native and deamidated gluten peptides Support Protocol 4: Preparation of CT-gliadin Support Protocol 5: Preparation of PT-gliadin.

Latiglutenase Protects the Mucosa and Attenuates Symptom Severity in Patients With Celiac Disease Exposed to a Gluten Challenge.

BACKGROUND & AIMS: Gluten ingestion in patients with celiac disease can lead to gastrointestinal symptoms and small intestinal mucosal injury. METHODS: This gluten challenge phase 2 trial was double blind and placebo controlled, and it assessed the efficacy and safety of a 1200-mg dose of IMGX003 in patients with celiac disease exposed to 2 g of gluten per day for 6 weeks. The change in the ratio of villus height to crypt depth was the primary endpoint. Secondary endpoints included density of intraepithelial lymphocytes and symptom severity. These endpoints were evaluated by analysis of covariance. Additional endpoints included serology and gluten-immunogenic peptides in urine. RESULTS: Fifty patients were randomized, and 43 patients completed the study (IMGX003, n = 21; placebo, n = 22). The mean change in the ratio of villus height to crypt depth (primary endpoint) for IMGX003 vs placebo was -0.04 vs -0.35 (P = .057). The mean change in the density of intraepithelial lymphocytes (secondary endpoint) for IMGX003 vs placebo was 9.8 vs 24.8 cells/mm epithelium (P = .018). The mean change (worsening) in symptom severity in relative units (secondary endpoint) for IMGX003 vs placebo was 0.22 vs 1.63 (abdominal pain, P = .231), 0.96 vs 3.29 (bloating, P = .204), and 0.02 vs 3.20 (tiredness, P = .113). The 3 × 2-week trend line significance values for these symptoms, respectively, were P = .014, .030, and .002. CONCLUSIONS: IMGX003 reduced gluten-induced intestinal mucosal damage and symptom severity. (ClinicalTrials.gov, Number: NCT03585478).

Engineering site-selective incorporation of fluorine into polyketides.

Although natural products and synthetic small molecules both serve important medicinal functions, their structures and chemical properties are relatively distinct. To expand the molecular diversity available for drug discovery, one strategy is to blend the effective attributes of synthetic and natural molecules. A key feature found in synthetic compounds that is rare in nature is the use of fluorine to tune drug behavior. We now report a method to site-selectively incorporate fluorine into complex structures to produce regioselectively fluorinated full-length polyketides. We engineered a fluorine-selective trans-acyltransferase to produce site-selectively fluorinated erythromycin precursors in vitro. We further demonstrated that these analogs could be produced in vivo in Escherichia coli on engineering of the fluorinated extender unit pool. By using engineered microbes, elaborate fluorinated compounds can be produced by fermentation, offering the potential for expanding the identification and development of bioactive fluorinated small molecules.

Favipiravir for Treatment of Outpatients With Asymptomatic or Uncomplicated Coronavirus Disease 2019: A Double-Blind, Randomized, Placebo-Controlled, Phase 2 Trial.

BACKGROUND: Favipiravir, an oral, RNA-dependent RNA polymerase inhibitor, has in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite limited data, favipiravir is administered to patients with coronavirus disease 2019 (COVID-19) in several countries. METHODS: We conducted a phase 2, double-blind, randomized controlled outpatient trial of favipiravir in asymptomatic or mildly symptomatic adults with a positive SARS-CoV-2 reverse-transcription polymerase chain reaction assay (RT-PCR) within 72 hours of enrollment. Participants were randomized to receive placebo or favipiravir (1800 mg twice daily [BID] day 1, 800 mg BID days 2-10). The primary outcome was SARS-CoV-2 shedding cessation in a modified intention-to-treat (mITT) cohort of participants with positive enrollment RT-PCRs. Using SARS-CoV-2 amplicon-based sequencing, we assessed favipiravir's impact on mutagenesis. RESULTS: We randomized 149 participants with 116 included in the mITT cohort. The participants' mean age was 43 years (standard deviation, 12.5 years) and 57 (49%) were women. We found no difference in time to shedding cessation overall (hazard ratio [HR], 0.76 favoring placebo [95% confidence interval {CI}, .48-1.20]) or in subgroups (age, sex, high-risk comorbidities, seropositivity, or symptom duration at enrollment). We detected no difference in time to symptom resolution (initial: HR, 0.84 [95% CI, .54-1.29]; sustained: HR, 0.87 [95% CI, .52-1.45]) and no difference in transition mutation accumulation in the viral genome during treatment. CONCLUSIONS: Our data do not support favipiravir at commonly used doses in outpatients with uncomplicated COVID-19. Further research is needed to ascertain if higher favipiravir doses are effective and safe for patients with COVID-19. CLINICAL TRIALS REGISTRATION: NCT04346628.

KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19.

In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease.

Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we develop a liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detect over 600 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes.