ABSTRACT
Food allergy affects an estimated 8% of children in the United States. Oral immunotherapy (OIT) is a recently approved treatment, with outcomes ranging from sustained tolerance to food allergens to no apparent benefit. The immunological underpinnings that influence clinical outcomes of OIT remain largely unresolved. Using single-cell RNA-Seq and paired T cell receptor α/ß (TCRα/ß) sequencing, we assessed the transcriptomes of CD154+ and CD137+ peanut-reactive T helper (Th) cells from 12 patients with peanut allergy longitudinally throughout OIT. We observed expanded populations of cells expressing Th1, Th2, and Th17 signatures that further separated into 6 clonally distinct subsets. Four of these subsets demonstrated a convergence of TCR sequences, suggesting antigen-driven T cell fates. Over the course of OIT, we observed suppression of Th2 and Th1 gene signatures in effector clonotypes but not T follicular helper-like (Tfh-like) clonotypes. Positive outcomes were associated with stronger suppression of Th2 signatures in Th2A-like cells, while treatment failure was associated with the expression of baseline inflammatory gene signatures that were present in Th1 and Th17 cell populations and unmodulated by OIT. These results demonstrate that differential clinical responses to OIT are associated with both preexisting characteristics of peanut-reactive CD4+ T cells and suppression of a subset of Th2 cells.
Subject(s)
Arachis , Desensitization, Immunologic , Peanut Hypersensitivity , RNA-Seq , Receptors, Antigen, T-Cell, alpha-beta , Single-Cell Analysis , T-Lymphocytes, Helper-Inducer/immunology , Child , Female , Humans , Male , Peanut Hypersensitivity/genetics , Peanut Hypersensitivity/immunology , Peanut Hypersensitivity/therapy , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunologyABSTRACT
Eosinophilic esophagitis (EoE) is an allergic disorder characterized by the recruitment of eosinophils to the esophagus, resulting in chronic inflammation. We sought to understand the cellular populations present in tissue biopsies of patients with EoE and to determine how these populations are altered between active disease and remission. To this end, we analyzed cells obtained from esophageal biopsies, duodenal biopsies, and peripheral blood of patients with EoE diagnosed with active disease or remission with single-cell RNA and T cell receptor (TCR) sequencing. Pathogenic effector TH2 (peTH2) cells present in the esophageal biopsies of patients with active disease expressed distinct gene signatures associated with the synthesis of eicosanoids. The esophageal tissue-resident peTH2 population also exhibited clonal expansion, suggesting antigen-specific activation. Peripheral CRTH2+CD161- and CRTH2+CD161+ memory CD4+ T cells were enriched for either a conventional TH2 phenotype or a peTH2 phenotype, respectively. These cells also exhibited substantial clonal expansion and convergence of TCR sequences, suggesting that they are expanded in response to a defined set of antigens. The esophagus-homing receptor GPR15 was up-regulated by peripheral peTH2 clonotypes that were also detected in the esophagus. Finally, GPR15+ peTH2 cells were enriched among milk-reactive CD4+ T cells in patients with milk-triggered disease, suggesting that these cells are an expanded, food antigen-specific population with enhanced esophagus homing potential.
Subject(s)
Eosinophilic Esophagitis/immunology , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/genetics , CD4-Positive T-Lymphocytes/immunology , Eosinophilic Esophagitis/pathology , Humans , Receptors, G-Protein-Coupled/immunology , Receptors, Peptide/immunology , Th2 Cells/immunologyABSTRACT
Recent advances in high-throughput T cell receptor (TCR) sequencing have allowed for new insights into the human TCR repertoire. However, methods for capturing antigen-specific repertoires remain an area of development. Here, we describe a potentially novel approach that utilizes both a biological and statistical enrichment to define putatively antigen-specific complementarity-determining region 3 (CDR3) repertoires in unselected individuals. The biological enrichment entailed FACS of in vitro antigen-activated memory CD4+ T cells, followed by TCRß sequencing. The resulting TCRß sequences were then filtered by selecting those that are statistically enriched when compared with their frequency in the autologous resting T cell compartment. Applying this method to define putatively peanut protein-specific repertoires in 27 peanut-allergic individuals resulted in a library of 7345 unique CDR3ß amino acid sequences that had similar characteristics to other validated antigen-specific repertoires in terms of homology and diversity. In-depth analysis of these CDR3ßs revealed 36 public sequences that demonstrated high levels of convergent recombination. In a network analysis, the public CDR3ßs were shown to be core sequences with more edges than their private counterparts. This method has the potential to be applied to a wide range of T cell-mediated disorders and to yield new biomarkers and biological insights.
Subject(s)
Amino Acid Sequence , Complementarity Determining Regions , Peanut Hypersensitivity/immunology , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , High-Throughput Nucleotide Sequencing/methods , Homologous Recombination , Humans , Immunologic Memory , Immunologic Tests/methods , Lymphokines , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunologyABSTRACT
BACKGROUND: Individuals with peanut allergy range in clinical sensitivity: some can consume grams of peanut before experiencing any symptoms, whereas others suffer systemic reactions to 10 mg or less. Current diagnostic testing only partially predicts this clinical heterogeneity. OBJECTIVE: We sought to identify characteristics of the peanut-specific CD4+ T-cell response in peanut-allergic patients that correlate with high clinical sensitivity. METHODS: We studied the T-cell receptor ß-chain (TCRß) usage and phenotypes of peanut-activated, CD154+ CD4+ memory T cells using fluorescence-activated cell sorting, TCRß sequencing, and RNA-Seq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. RESULTS: TCRß analysis of the CD154+ and CD154- fractions revealed more than 6000 complementarity determining region 3 sequences and motifs that were significantly enriched in the activated cells and 17% of the sequences were shared between peanut-allergic individuals, suggesting strong convergent selection of peanut-specific clones. These clones were more numerous among the reactive patients, and this expansion was identified within effector, but not regulatory T-cell populations. The transcriptional profile of CD154+ T cells in the reactive group skewed toward a polarized TH2 effector phenotype, and expression of TH2 cytokines strongly correlated with peanut-specific IgE levels. There were, however, also non-TH2-related differences in phenotype. Furthermore, the ratio of peanut-specific clones in the effector versus regulatory T-cell compartment, which distinguished the clinical groups, was independent of specific IgE concentration. CONCLUSIONS: Expansion of the peanut-specific effector T-cell repertoire is correlated with clinical sensitivity, and this observation may be useful to inform our assessment of disease phenotype and to monitor disease longitudinally.
Subject(s)
Cytokines/immunology , Immunologic Memory , Peanut Hypersensitivity/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Th2 Cells/immunology , Adult , Female , Humans , Male , Peanut Hypersensitivity/pathology , Th2 Cells/pathologyABSTRACT
High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Peanut Hypersensitivity/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Th2 Cells/immunology , 2S Albumins, Plant/immunology , Animals , Antigens, Plant/immunology , Cells, Cultured , Complementarity Determining Regions/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Immunization , Immunoglobulin E/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Papillomavirus E7 Proteins/immunology , Single-Cell Analysis , T-Cell Antigen Receptor Specificity/genetics , TranscriptomeABSTRACT
Staphylococcus aureus causes severe disease in humans for which no licensed vaccine exists. A novel S. aureus vaccine (SA4Ag) is in development, targeting the capsular polysaccharides (CPs) and two virulence-associated surface proteins. Vaccine-elicited antibody responses to CPs are efficacious against serious infection by other encapsulated bacteria. Studies of natural S. aureus infection have also shown a role for TH17 and/or TH1 responses in protection. Single-antigen vaccines, including CPs, have not been effective against S. aureus; a multiantigen vaccine approach is likely required. However, the impact of addition of protein antigens on the immune response to CPs has not been studied. Here, the immune response induced by a bivalent CP conjugate vaccine (to model the established mechanism of action of vaccine-induced protection against Gram-positive pathogens) was compared to the response induced by SA4Ag, which contains both CP conjugates and protein antigens, in cynomolgus macaques. Microengraving, flow cytometry, opsonophagocytic assays, and Luminex technology were used to analyze the B-cell, T-cell, functional antibody, and innate immune responses. Both the bivalent CP vaccine and SA4Ag induced cytokine production from naive cells and antigen-specific memory B-cell and functional antibody responses. Increases in levels of circulating, activated T cells were not apparent following vaccination, nor was a TH17 or TH1 response evident. However, our data are consistent with a vaccine-induced recruitment of T follicular helper (TFH) cells to lymph nodes. Collectively, these data suggest that the response to SA4Ag is primarily mediated by B cells and antibodies that abrogate important S. aureus virulence mechanisms.IMPORTANCEStaphylococcus aureus causes severe disease in humans for which no licensed vaccine exists. A novel vaccine is in development that targets multiple elements of the bacteria since single-component vaccines have not shown efficacy to date. How these multiple components alter the immune response raised by the vaccine is not well studied. We found that the addition of two protein components did not alter substantially the antibody responses raised with respect to function or mobilization of B cells. There was also not a substantial change in the activity of T cells, another part of the adaptive response. This study showed that protection by this vaccine may be mediated primarily by antibody protection.
Subject(s)
B-Lymphocytes/immunology , Cytokines/immunology , Immunity, Cellular , Staphylococcal Infections/immunology , Staphylococcal Infections/prevention & control , Staphylococcal Vaccines/immunology , Animals , Antibody Formation , Cells, Cultured , Humans , Staphylococcus aureus , T-Lymphocytes, Helper-Inducer/immunology , Vaccination , Vaccines, ConjugateABSTRACT
MOTIVATION: This work addresses two common issues in building classification models for biological or medical studies: learning a sparse model, where only a subset of a large number of possible predictors is used, and training in the presence of missing data. This work focuses on supervised generative binary classification models, specifically linear discriminant analysis (LDA). The parameters are determined using an expectation maximization algorithm to both address missing data and introduce priors to promote sparsity. The proposed algorithm, expectation-maximization sparse discriminant analysis (EM-SDA), produces a sparse LDA model for datasets with and without missing data. RESULTS: EM-SDA is tested via simulations and case studies. In the simulations, EM-SDA is compared with nearest shrunken centroids (NSCs) and sparse discriminant analysis (SDA) with k-nearest neighbors for imputation for varying mechanism and amount of missing data. In three case studies using published biomedical data, the results are compared with NSC and SDA models with four different types of imputation, all of which are common approaches in the field. EM-SDA is more accurate and sparse than competing methods both with and without missing data in most of the experiments. Furthermore, the EM-SDA results are mostly consistent between the missing and full cases. Biological relevance of the resulting models, as quantified via a literature search, is also presented. AVAILABILITY AND IMPLEMENTATION: A Matlab implementation published under GNU GPL v.3 license is available at http://web.mit.edu/braatzgroup/links.html . CONTACT: braatz@mit.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Computational Biology/methods , Models, Biological , Software , Bacterial Infections/classification , Bacterial Infections/genetics , Classification , Genes , Humans , Leukemia/classification , Leukemia/genetics , Machine LearningABSTRACT
Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.
Subject(s)
DNA Damage , DNA Topoisomerases, Type II/metabolism , Phosphoric Diester Hydrolases/chemistry , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/chemistry , Animals , Catalytic Domain , DNA/chemistry , DNA/metabolism , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA End-Joining Repair , DNA Topoisomerases, Type II/chemistry , DNA-Binding Proteins , Humans , Magnesium/chemistry , Mice , Mice, Knockout , Models, Molecular , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Phosphotyrosine/metabolism , Polymorphism, Single Nucleotide , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/genetics , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolismABSTRACT
We investigate the immobilization of a model system of functionalized yeast that surface-display enhanced green fluorescent protein (eGFP) within chemically crosslinked polyvinyl alcohol (PVA) nanofibers. Yeast is incorporated into water insoluble nanofibrous materials by direct electrospinning with PVA followed by vapor phase chemical crosslinking of the polymer. Incorporation of yeast into the fibers is confirmed by elemental analysis and the viability is indicated by live/dead staining. Following electrospinning and crosslinking, we confirm that the yeast maintains its viability as well as the ability to express eGFP in the correct conformation. This method of processing functionalized yeast may thus be a powerful tool in the direct immobilization of properly folded, active enzymes within electrospun nanofibers with potential applications in biocatalysis.