Biology subjects

Shreffler, W. G.

Publications and source records attributed to Shreffler, W. G..

6 recordsLinked to original sources

bioRxiv2025

GPR15 and CD38 define a subset of peripheral blood pathogenic effector Th2 cells associated with active eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a chronic allergic disease driven by exposure to culprit antigens. Due to the local nature of the inflammation, diagnosis and assessment are limited to invasive procedures. Based on prior single-cell RNA sequencing (scRNA-seq) data linking peripheral GPR15+ pathogenic effector Th2 (peTh2) cells to esophageal tissue peTh2s, we hypothesized the direct involvement of GPR15+ peTh2 cells in EoE pathogenesis and aimed to further evaluate their association with EoE disease status. We subjected samples from subjects with or without EoE to flow cytometry (n = 74 peripheral blood, 17 biopsy) and scRNA-seq (n = 27 peripheral blood, 10 biopsy). Expression of GPR15 by peripheral peTh2 cells was increased in EoE, and these cells expressed increased CD38 in active EoE--findings recapitulated in esophageal biopsies. We also identified a peTh2-associated, CD38-containing gene expression program that peripheral GPR15+ peTh2 cells upregulated in active EoE. The level of upregulation was distinct from other circulating peTh2 cells and was more similar to that seen in esophageal peTh2 cells. An association between expression of GPR15 by peripheral peTh2 cells, the aryl hydrocarbon receptor was strongest in subjects with EoE, suggesting an environmental exposure or susceptibility. The magnitude of GPR15 expression by peripheral peTh2 cells could effectively in discriminate active EoE from no EoE in our study population (AUC 0.93). Our data suggest that EoE-related peTh2 cells are identifiable and accessible in the peripheral blood, and could be exploited in both clinical practice as a non-invasive biomarker and continued investigation into mechanisms driving EoE. One sentence summaryGPR15 marks a subset of peripheral blood pathogenic effector Th2 cells associated with eosinophilic esophagitis (EoE) that upregulate CD38 during active disease - an observation that has potential to be used for non-invasive diagnosis and monitoring of EoE and that has suggests new mechanisms driving this increasingly prevalent allergic disease.

immunology↗

bioRxiv2025

Microbial metabolism of food allergens determines the severity of IgE-mediated anaphylaxis

Anaphylaxis is an acute, potentially life-threatening reaction, often triggered by foods and largely mediated by IgE. Critically important to anaphylaxis are the factors that modulate its severity. The human microbiota is known to influence oral tolerance, but the microbial mechanisms directly involved in IgE-mediated anaphylaxis remain unknown. Here, we demonstrate that human saliva and jejunum harbor peanut-degrading bacteria that metabolize immunodominant allergens (Ara h 1 and 2) and alter IgE-binding. Additionally, we provide in vivo evidence that oral bacteria metabolize peanut allergens, influencing systemic allergen exposure and anaphylaxis severity. Finally, in clinical studies, we observe that common peanut-degrading bacteria, such as Rothia, from the oral cavity, are more abundant in peanut-allergic patients who exhibit better tolerance to allergen exposure. Altogether, these results demonstrate that human microbiota modulates IgE-mediated reactions through allergen metabolism. We reveal a novel microbial mechanism with potential to prevent, or reduce, the severity of IgE-mediated anaphylaxis.

immunology↗

bioRxiv2024

Companion to: A molecular basis for milk allergen immune recognition in eosinophilic esophagitis

BackgroundEosinophilic esophagitis (EoE) is a chronic, type 2 inflammatory disease that is increasing in incidence and has substantial morbidity. Despite being clinically defined as a food allergy, the molecular details of food antigen presentation and recognition by the immune system are unknown. ObjectiveThe objective of this study was to identify and characterize the molecular basis of milk antigen presentation and T cell recognition in a patient with EoE milk allergy. MethodsMilk-expanded TCR clonotypes were identified using ex vivo stimulation followed by single cell RNA with linked, paired TRA and TRB sequencing. HLA restriction and antigen specificity of TCRs were identified using a combination of lentiviral expression, HLA sequencing, antibody blockade, and a peptide library screen. ResultsWe isolated a {beta}-casein specific TCR clonotype (eoeTCR-4) and determined its HLA restriction (HLA-DRB1*07:01) and cognate antigen ({beta}-casein AA 59-78). EoeTCR-4 was not detected among a larger group of subjects and is likely private to EoE Subject 1. ConclusionIn conjunction with the parent manuscript, this companion article provides the first molecular identity of food antigen presentation and immune recognition in EoE. SUMMARYThis manuscript is a companion to the parent manuscript "A molecular basis for milk allergen immune recognition in eosinophilic esophagitis". It provides additional data and detailed methods not provided in the parent manuscript. It is designed to supplement and support the findings and conclusions of the parent manuscript.

immunology↗

bioRxiv2024

Interferon-γ signaling in eosinophilic esophagitis has implications for epithelial barrier function and programmed cell death.

ObjectiveEosinophilic esophagitis (EoE) is a chronic esophageal inflammatory disorder characterized by eosinophil-rich mucosal inflammation and tissue remodeling. Transcriptional profiling of esophageal biopsies has previously revealed upregulation of type I and II interferon (IFN) response genes. We aim to unravel interactions between immune and epithelial cells and examine functional significance in esophageal epithelial cells. Design We investigated epithelial gene expression from EoE patients using single-cell RNA sequencing and a confirmatory bulk RNA-sequencing experiment of isolated epithelial cells. The functional impact of interferon signaling on epithelial cells was investigated using in vitro organoid models. ResultsWe observe upregulation of interferon response signature genes (ISGs) in the esophageal epithelium during active EoE compared to other cell types, single-cell data, and pathway analyses, identified upregulation in ISGs in epithelial cells isolated from EoE patients. Using an esophageal organoid and air-liquid interface models, we demonstrate that IFN-{gamma} stimulation triggered disruption of esophageal epithelial differentiation, barrier integrity, and induced apoptosis via caspase upregulation. We show that an increase in cleaved caspase-3 is seen in EoE tissue and identify interferon gamma (IFNG) expression predominantly in a cluster of majority-CD8+ T cells with high expression of CD69 and FOS. ConclusionThese findings offer insight into the interplay between immune and epithelial cells in EoE. Our data illustrate the relevance of several IFN-{gamma}-mediated mechanisms on epithelial function in the esophagus, which have the potential to impact epithelial function during inflammatory conditions. Key MessagesWhat is already known about this topic: O_LIThe transcriptome of esophageal biopsy tissue reproducibly distinguishes eosinophilic esophagitis from histologically normal tissue, with evidence of mixed inflammatory signals. C_LIO_LIInterferon response signature genes are elevated in EoE biopsy tissue compared to controls, suggesting T1 in addition to T2 cytokine signaling within EoE mucosa. C_LI What this study adds: O_LIWe observe reproducible, robust upregulation of interferon signature genes in esophageal epithelium, and we confirm that esophageal epithelium expresses functional IFN-a and IFN-{gamma} receptors. C_LIO_LIIFN-{gamma} treatment of epithelial organoids has several detrimental effects, including decreased proliferation, organoid formation, and increased caspase activation. C_LIO_LIAnalysis of single-cell RNA-sequencing data from of EoE biopsy tissue during active disease and remission identified that a CD8+ population expressing high levels of FOS, ITGAE, and ITGA1 expresses high levels of IFNG C_LI How this study might affect research, practice, or policy: O_LIWe identify esophageal epithelium as the cellular source for interferon response gene signature in EoE and a CD8+ tissue-resident memory T-cell population was the main source of IFNG. C_LIO_LIFurther mechanistic studies are required to identify how non-T2 signaling mechanisms like IFN-{gamma} signaling contribute to EoE pathogenesis, and if this pathway can be targeted as an adjunctive therapy for EoE. C_LI

immunology↗

bioRxiv2020

Identification of antigen-specific TCR sequences using a strategy based on biological and statistical enrichment in unselected subjects

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 of in vitro antigen-activated memory CD4+ T cells followed by TCR{beta} sequencing. The resulting TCR{beta} 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.

immunology↗

bioRxiv2019

BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy: an ephemeral existence?

Immunoglobulin (Ig) E is the critical effector molecule in allergic reactions. Consequently, research efforts to understand the biology of IgE-expressing cells is of paramount importance. In particular, the role of IgE+ memory B cells (MBCs) in the perpetuation of allergic reactivity has been the subject of intense research. Studies in mice have convincingly established that IgE+ B cells are rare and transient and, therefore, an unlikely candidate to maintain allergic disease. In contrast, IgE+ MBCs have been detected by flow cytometry in the sputum and peripheral blood of humans and have been proposed as a clinical marker of allergic disease. We established a method to genetically validate, at the single-cell level, the putative IgE+ MBCs identified by flow cytometry from humans. We, then used this information to develop an enhanced flow cytometry protocol that more accurately identifies bona fide IgE+ MBCs. We found that IgE+ MBCs were detected in some patients with atopic dermatitis, but at a frequency that was ~100 times lower than previously reported. We also found that IgE+ MBCs were undetectable in PBMCs from peanut allergic patients. These findings provide tools to identify bona fide IgE+ MBCs, demonstrate their extreme rarity in circulation and are consistent with the lack of a central role for IgE+ MBCs in the maintenance of allergic sensitivity.\n\nOne Sentence SummaryThe frequency of IgE+ MBCs in the peripheral circulation of humans is orders of magnitude lower than previously reported and comparable between allergic and healthy donors, which cautions about the clinical utility of their assessment.

immunology↗