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Asthma is a chronic disease most commonly associated with allergy and type 2 inflammation. However, the mechanisms that link airway inflammation to the structural changes that define asthma are incompletely understood. Using a human model of allergen-induced asthma exacerbation, we compared the lower airway mucosa in allergic asthmatics and allergic non-asthmatic controls using single-cell RNA sequencing. In response to allergen, the asthmatic airway epithelium was highly dynamic and up-regulated genes involved in matrix degradation, mucus metaplasia, and glycolysis while failing to induce injury-repair and antioxidant pathways observed in controls. IL9 -expressing pathogenic T H 2 cells were specific to asthmatic airways and were only observed after allergen challenge. Additionally, conventional type 2 dendritic cells (DC2 that express CD1C ) and CCR2 -expressing monocyte-derived cells (MCs) were uniquely enriched in asthmatics after allergen, with up-regulation of genes that sustain type 2 inflammation and promote pathologic airway remodeling. In contrast, allergic controls were enriched for macrophage-like MCs that up-regulated tissue repair programs after allergen challenge, suggesting that these populations may protect against asthmatic airway remodeling. Cellular interaction analyses revealed a T H 2–mononuclear phagocyte–basal cell interactome unique to asthmatics. These pathogenic cellular circuits were characterized by type 2 programming of immune and structural cells and additional pathways that may sustain and amplify type 2 signals, including TNF family signaling, altered cellular metabolism, failure to engage antioxidant responses, and loss of growth factor signaling. Our findings therefore suggest that pathogenic effector circuits and the absence of proresolution programs drive structural airway disease in response to type 2 inflammation.

Therapeutic blockade of co-inhibitory immune receptors PD-1 and CTLA-4 has revolutionized oncology, but treatments are limited by immune-related adverse events (IRAEs). IRAE Colitis (irColitis) is the most common, severe IRAE affecting up to 25% of patients on dual PD-1 and CTLA-4 inhibition. Here, we present a systems biology approach to define the cell populations and transcriptional programs driving irColitis. We collected paired colon mucosal biopsy and blood specimens from 13 patients with irColitis, 8 healthy individuals, and 8 controls on immune checkpoint inhibitors (ICIs), and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis (Luminex). We profiled 299,407 cells from tissue and blood and identified 105 cell subsets that revealed significant tissue remodeling in active disease. Colon mucosal immune populations were dominated by tissue-resident memory (TRM) ITGAE-expressing CD8 T cells representing a phenotypic spectrum defined by gene programs associated with T cell activation, cytotoxicity, cycling, and exhaustion. CD8 TRM and effector CD4 T cells upregulated type 17 immune programs (IL17A, IL26) and Tfh-like programs (CXCL13, PDCD1). We also identified for the first time an increased abundance of two KLRG1 and ITGB2-expressing CD8 T cell populations with circulatory cell markers, including a GZMK TRM-like population and a CX3CR1 population that is predicted to be intravascular. These two populations were more abundant in irColitis patients treated with dual PD-1/CTLA-4 inhibition than those receiving anti-PD-1 monotherapy. They also had significant TCR sharing with PBMCs, suggesting a circulatory origin. In irColitis we observed significant epithelial turnover marked by fewer LGR5-expressing stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs such as the cGAS-STING pathway. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Mesenchymal remodeling was defined by increased endothelial cells, both in irColitis patients and specifically in patients on dual PD-1/CTLA-4 blockade. Cell-cell communication analysis identified putative receptor-ligand pairs that recruit CD8 T cells from blood to inflamed endothelium and positive feedback loops such as the CXCR3 chemokine system that retain cells in tissue. This study highlights the cellular and molecular drivers underlying irColitis and provides new insights into the role of CTLA-4 and PD-1 signaling in maintaining CD8 TRM homeostasis, regulating CD8 T recruitment from blood, and promoting epithelial-immune crosstalk critical to gastrointestinal immune tolerance and intestinal barrier function.

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.

Food allergy affects an estimated 8% of children in the US, with increasing severity and global prevalence1. Oral immunotherapy (OIT) is a recently approved treatment with outcomes ranging from sustained tolerance to food allergen to no apparent benefit2,3. The immunological underpinnings that influence clinical outcomes of OIT still remain largely unresolved. Using single-cell RNA sequencing and paired TCRα/β sequencing, we assessed the transcriptomes of CD154+ and CD137+ peanut-reactive T helper cells from 12 peanut-allergic patients longitudinally throughout OIT. We observed expanded populations of cells expressing Th1, Th2, and Th17 signatures that further separated into six clonally distinct subsets, including a Tfh1-like, a Tfh2-like, a Th2A-like, and a Th2reg-like subset. Four of these subsets demonstrated convergence of TCR sequences, suggesting antigen-driven T cell fate. Although we observed suppression during OIT of Th2 and Th1 gene signatures within effector clonotypes, Tfh clonotypes were unaffected. We also did not observe significant clonal deletion or induction among the antigen-reactive T cells characterized. Positive outcomes were associated with larger decrease of Th2 signatures in Th2A-like cells, while treatment failure was associated with high baseline inflammatory gene signatures that were unmodulated by OIT. These signatures, including expression of OX40, OX40L, STAT1, and GPR15, were most clearly present in Th1 and Th17 clonotypes, but were also more broadly detected across the CD154+ CD4 population. These results demonstrate that differential clinical response is associated both with pre-existing trait characteristics of the CD4 immune compartment and with susceptibility to modulation by OIT.Conflict of Interest StatementA.A.T., T.M.G., J.C.L., and the Massachusetts Institute of Technology have filed patents related to the single-cell sequencing methods used in this work. J.C.L. has interests in Sunflower Therapeutics PBC, Pfizer, Honeycomb Biotechnologies, OneCyte Biotechnologies, SQZ Biotechnologies, Alloy Therapeutics, QuantumCyte, Amgen, and Repligen. J.C.L.’s interests are reviewed and managed under Massachusetts Institute of Technology’s policies for potential conflicts of interest. J.C.L. receives sponsored research support at MIT from Amgen, the Bill & Melinda Gates Foundation, Biogen, Pfizer, Roche, Takeda, and Sanofi. The spouse of J.C.L. is an employee of Sunflower Therapeutics PBC. T.M.G. is currently an employee of Honeycomb Biotechnologies, Inc. A.A.T. is currently an employee of Immunitas Therapeutics, Inc. W.G.S. is a consultant of Aimmune Therapeutics.

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/β (TCRα/β) 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.

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 T

Recent advances in high-throughput T cell receptor (TCR) sequencing have allowed for new insights into the TCR repertoire. However, methods for capturing antigen-specific repertoires remain an area of development. Here, we describe a novel approach that utilizes both a biological and statistical enrichment to define putatively antigen-specific complementarity-determining region 3 (CDR3) repertoires in unrelated individuals. The biological enrichment entails fluorescence-activated cell sorting ofin vitroantigen-activated memory CD4+T cells followed by TCRβ sequencing. The resulting TCRβ sequences are then filtered by selecting those that are statistically enriched when compared to 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 CDR3 amino acid sequences that had similar characteristics to validated antigen-specific repertoires in terms of homology and diversity. In-depth analysis of these CDR3s revealed 36 public sequences that demonstrated high levels of convergent recombination. In a network analysis, the public CDR3s unveiled themselves as 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.

© 2019 American Academy of Allergy, Asthma & Immunology 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 β-chain (TCRβ) usage and phenotypes of peanut-activated, CD154+ CD4+ memory T cells using fluorescence-activated cell sorting, TCRβ sequencing, and RNA-Seq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. Results: TCRβ 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.

BACKGROUND: Individuals with peanut allergy range in clinical sensitivity: some can consume grams of peanut before experiencing any symptoms, while 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 β-chain (TCRβ) usage and phenotypes of peanut-activated, CD154(+) CD4(+) memory T cells using fluorescence-activated cell sorting, TCRβ sequencing, and RNASeq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. RESULTS: TCRβ analysis of the CD154(+) and CD154(−) fractions revealed >6,000 complementarity determining region 3 (CDR3) sequences and CDR3 motifs that were significantly enriched in the activated cells and 17% 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 towards 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. CONCLUSION: 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.

Background Peanut is a potent inducer of proallergenic TH2 responses in susceptible individuals. Antigen-presenting cells (APCs) including dendritic cells and monocytes instruct naive T cells to differentiate into various effector cells, determining immune responses such as allergy and tolerance. Objective We sought to detect peanut protein (PN)-induced changes in gene expression in human myeloid dendritic cells (mDCs) and monocytes, identify signaling receptors that mediate these changes, and assess how PN-induced genes in mDCs impact their ability to promote T-cell differentiation. Methods mDCs, monocytes, and naive CD4+ T cells were isolated from blood bank donors and peanut-allergic patients. APCs were incubated with PN and other stimulants, and gene expression was measured using microarray and RT quantitative PCR. To assess T-cell differentiation, mDCs were cocultured with naive TH cells. Results PN induced a unique gene expression profile in mDCs, including the gene that encodes retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in the retinoic acid (RA)-producing pathway. Stimulation of mDCs with PN also induced a 7-fold increase in the enzymatic activity of RALDH2. Blocking antibodies against Toll-like receptor (TLR)1/TLR2, as well as small interfering RNA targeting TLR1/TLR2, reduced the expression of RALDH2 in PN-stimulated APCs by 70%. Naive TH cells cocultured with PN-stimulated mDCs showed an RA-dependent 4-fold increase in production of IL-5 and expression of integrin α4β7. Conclusions PN induces RALDH2 in human APCs by signaling through the TLR1/TLR2 heterodimer. This leads to production of RA, which acts on TH cells to induce IL-5 and gut-homing integrin. RALDH2 induction by PN in APCs and RA-promoted TH2 differentiation could be an important factor determining allergic responses to peanut.