Single Cell RNA Sequencing Unveils Distinct Cellular Dynamics in Celiac Disease Duodenal Biopsies

Celiac disease (CeD) is the most common autoimmune disorder in the U.S., affecting at least 1% of the population. The ingestion of gluten-containing proteins triggers an adaptive immune response, causing intestinal damage and leading to both gastrointestinal and systemic symptoms. Despite a strong genetic predisposition, the mechanistic understanding of CeD remains limited. The lack of approved therapy, other than dietary avoidance of gluten which is difficult and often unsuccessful, underscores the need to identify new mechanisms that can provide insights for developing therapeutics. To gain an unbiased view of the transcriptional landscape of the duodenum, we compared duodenal biopsies showing active CeD to normal controls using single-cell RNA sequencing. We analyzed fresh research biopsies from 4 patients with active CeD, confirmed by diagnostic biopsies from the same endoscopy, as well as 2 healthy control biopsies without specific pathological changes. Our analysis captured every major intestinal mucosal cell type including all subsets of immune cells, epithelial cells and stromal cells. Among these, enterocytes and activated T cells exhibited the most significant differences between CeD biopsies and controls. In CeD biopsies, CD8+TCR aß and TCR ?d T cells were the primary producers of IFNG, a feature that was largely absent in the control biopsies. Moreover, enterocytes that express villous defining marker APOA4+ showed elevated levels of IFNGR1, suggesting an increased responsiveness to IFN? produced by CeD T cells. We assessed effects of IFN? on potential target molecules expressed on enterocytes that could activate and trigger killing by CTL. Bioinformatic ligand receptor analyses showed frequent interactions between intraepithelial CTLs and enterocyte HLA-E or HLA-B, suggesting that IFN? production by the CTLs likely promotes further CTL recruitment and antigen-dependent killing of the villous epithelium. Overall design: Duodenal mucosal biopsies for research were obtained with informed consent from 4 patients with CeD with mild villous blunting (Marsh grade 3A) or severe villous blunting (Marsh grade 3C) and 2 patients with normal mucosa undergoing diagnostic endoscopy. All six samples were deidentified prior to transfer to the laboratory under a protocol approved by the Yale Institutional Review Board. Research biopsies were processed for droplet-based single-cell RNA-seq (scRNA-seq) using 10x Chromium GEM technology.