TNFSF13 insuffiency causes epithelial and immune disruption in the human colon I

Objective: Cytokines produced by epithelial and immune cells can act upon one another to promote mucosal healing- a process that goes awry with chronic inflammation as in inflammatory bowel disease. TNFSF13 is a cytokine important for B cell function, but its role in epithelial cell behavior and contribution to inflammatory bowel disease is poorly understood. Design: We performed histological and functional assays, single cell transcriptomics, and imaging mass cytometry on tissue-derived colonoids or biopsies from a patient with very early onset inflammatory bowel disease (VEO-IBD) harboring a novel TNFSF13 variant. To confirm causal, variant-driven phenotypes, parallel experiments were performed in induced pluripotent stem cell (iPSC)- derived colon organoids engineered with the same TNFSF13 variant. Results: TNFSF13 variant colonoids exhibited reduced TNFSF13 expression associated with increased proliferation and reduced apoptosis, which was confirmed in iPSC-derived colon organoids. Single cell RNA-sequencing and flow cytometry defined FAS as the predominant colonic epithelial receptor for TNFSF13. TNFSF13 variant colon biopsies demonstrated a shift in tissue B cell populations consistent with altered differentiation of memory B cells. Finally, imaging mass cytometry revealed an increase in epithelial-associated B cells in TNFSF13 variant colon tissue sections compared to healthy and non-monogenic VEO-IBD controls. Conclusions: The current study defines a previously unknown role for the cytokine TNFSF13 as a regulator of colonic epithelial proliferation and apoptosis. Furthermore, this study suggests that variant TNFSF13 can contribute to aberrant epithelial- B cell crosstalk in the human colon. Overall design: scRNA-seq analysis of colonoids from 2 controls, 2 patients with VEO-IBD and 1 patient with VEO-IBD and variant in TNFSF13 (APRIL). Samples included untreated colonoids as well as colonoisd treated with TNFa, IFNg or TRAIL. Cell hashing was used to allow for multiplexing of untreated and treated colonoids from the same sample ID. HTO barcode sequence hastagged sample TotalSeq-B0251 anti-human Hashtag 1 GTCAACTCTTTAGCG Untreated TotalSeq-B0252 anti-human Hashtag 2 TGATGGCCTATTGGG TNFa-treated TotalSeq-B0253 anti-human Hashtag 3 TTCCGCCTCTCTTTG IFNg-treated TotalSeq-B0254 anti-human Hashtag 4 AGTAAGTTCAGCGTA TRAIL-treated