Abstract
In coeliac disease (CeD), immune‐mediated small intestinal damage is precipitated by gluten, leading to variable symptoms and complications, occasionally including aggressive T‐cell lymphoma. Diagnosis, based primarily on histopathological examination of duodenal biopsies, is confounded by poor concordance between pathologists and minimal histological abnormality if insufficient gluten is consumed. CeD pathogenesis involves both CD4+ T‐cell‐mediated gluten recognition and CD8+ and γδ T‐cell‐mediated inflammation, with a previous study demonstrating a permanent change in γδ T‐cell populations in CeD. We leveraged this understanding and explored the diagnostic utility of bulk T‐cell receptor (TCR) sequencing in assessing duodenal biopsies in CeD. Genomic DNA extracted from duodenal biopsies underwent sequencing for TCR‐δ (TRD) (CeD, n = 11; non‐CeD, n = 11) and TCR‐γ (TRG) (CeD, n = 33; non‐CeD, n = 21). We developed a novel machine learning‐based analysis of the TCR repertoire, clustering samples by diagnosis. Leave‐one‐out cross‐validation (LOOCV) was performed to validate the classification algorithm. Using TRD repertoire, 100% (22/22) of duodenal biopsies were correctly classified, with a LOOCV accuracy of 91%. Using TCR‐γ (TRG) repertoire, 94.4% (51/54) of duodenal biopsies were correctly classified, with LOOCV of 87%. Duodenal biopsy TRG repertoire analysis permitted accurate classification of biopsies from patients with CeD following a strict gluten‐free diet for at least 6 months, who would be misclassified by current tests. This result reflects permanent changes to the duodenal γδ TCR repertoire in CeD, even in the absence of gluten consumption. Our method could complement or replace histopathological diagnosis in CeD and might have particular clinical utility in the diagnostic testing of patients unable to tolerate dietary gluten, and for assessing duodenal biopsies with equivocal features. This approach is generalisable to any TCR/BCR locus and any sequencing platform, with potential to predict diagnosis or prognosis in conditions mediated or modulated by the adaptive immune response. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Highlights
Coeliac disease (CeD) is a gluten-sensitive enteropathy that develops in genetically susceptible individuals who are exposed to cereal gluten proteins, found in in wheat, rye, and barley
Proteins encoded by these genes bind gluten peptides, those peptides post-translationally modified by tissue transglutaminase
We obtained duodenal biopsies from CeD patients, with a previous biopsy showing at least Marsh 3b features, who had been on a strict gluten-free diet (GFD) for at least 6 months with normal duodenal biopsy histopathology (n=4)
Summary
Coeliac disease (CeD) is a gluten-sensitive enteropathy that develops in genetically susceptible individuals who are exposed to cereal gluten proteins, found in in wheat, rye, and barley. Much of the genetic susceptibility is contributed by possession of the MHC class II molecules HLADQ2 and HLA-DQ8 Proteins encoded by these genes bind gluten peptides, those peptides post-translationally modified by tissue transglutaminase (tTG). A recent study elegantly demonstrated that do the γδ T-cell numbers increase permanently, once tolerance to gluten is lost, but that there is an irreversible alteration of the functional subtypes of γδ T-cells present in the duodenum. They demonstrated depletion of naturally occurring, innate-like Vγ4+/Vδ1+ intraepithelial lymphocytes (IELs) with specificity for the butyrophilin-like (BTNL) molecules BTNL3/BTNL8, expressed in duodenum. They observed expansion of gluten-sensitive, interferon-γ-producing Vδ1+ IELs bearing T-cell receptors (TCR) with a shared non-germline-encoded motif that failed to recognize BTNL3/BTNL8 and were phenotypically more akin to adaptive T-cells [4]
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