Single-Cell Analysis of Refractory Celiac Disease Demonstrates Inter- and Intra-Patient Aberrant Cell Heterogeneity

BACKGROUND & AIMS: Refractory celiac disease type II (RCDII) is a rare indolent lymphoma in the small intestine characterized by a clonally expanded intraepithelial intracellular CD3 thorn surfa- ceCD3-CD7 thorn CD56- aberrantcell population. However, RCDII pathogenesis is ill-defined. Here, we aimed at single-cell charac-terization of the innate and adaptive immune system in RCDII. METHODS: Paired small intestinal and blood samples from 12 RCDII patients and 6 healthy controls were assessed by single-cell mass cytometry with a 39-cell surface marker antibody panel, designed to capture heterogeneity of the innate and adaptive immune system. A second single-cell mass cytometry panel that included transcription factors and immune checkpoints was used for analysis of paired samples from 5 RCDII patients. Single-cell RNA sequencing analysis was performed on duodenal samples from 2 RCDII patients. Finally, we developed a 40-marker imaging mass cytometry antibody panel to evaluate cell-cell interactions in duodenal biopsy specimens of RCDII patients. RESULTS: We provide evidence for intertumoral and intra-tumoral cell heterogeneity within the duodenal and peripheral aberrant cell population present in RCDII. Phenotypic discrep-ancy was observed between peripheral and duodenal aberrant cells. In addition, we observed that part of the aberrant cell population proliferated and observed co-localization of aber-rant cells with CD163 thorn antigen-presenting cells (APCs) in situ. In addition, we observed phenotypic discrepancy between pe-ripheral and duodenal aberrant cells. CONCLUSIONS: Novel high-dimensional single-cell technolo-gies show substantial intertumoral and intratumoral hetero-geneity in the aberrant cell population in RCDII. This may underlie variability in refractory disease status between pa-tients and responsiveness to therapy, pointing to the need for personalized therapy in RCDII based on patient-specific im-mune profiles.Molecular Technology and Informatics for Personalised Medicine and Healt