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⁺surfaceCD3^-CD7⁺CD56^- aberrant cell population. However, RCDII pathogenesis is ill-defined. Here, we aimed at single-cell characterization 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 intratumoral cell heterogeneity within the duodenal and peripheral aberrant cell population present in RCDII. Phenotypic discrepancy 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 aberrant cells with CD163⁺ antigen-presenting cells (APCs) in situ. In addition, we observed phenotypic discrepancy between peripheral and duodenal aberrant cells. Conclusions: Novel high-dimensional single-cell technologies show substantial intertumoral and intratumoral heterogeneity in the aberrant cell population in RCDII. This may underlie variability in refractory disease status between patients and responsiveness to therapy, pointing to the need for personalized therapy in RCDII based on patient-specific immune profiles.

Chronic intestinal inflammation underlies inflammatory bowel disease (IBD). Previous studies indicated alterations in the cellular immune system; however, it has been challenging to interrogate the role of all immune cell subsets simultaneously. Therefore, we aimed to identify immune cell types associated with inflammation in IBD using high-dimensional mass cytometry. We analyzed 188 intestinal biopsies and paired blood samples of newly-diagnosed, treatment-naive patients (n=42) and controls (n=26) in two independent cohorts. We applied mass cytometry (36-antibody panel) to resolve single cells and analyzed the data with unbiased Hierarchical-SNE. In addition, imaging-mass cytometry (IMC) was performed to reveal the spatial distribution of the immune subsets in the tissue. We identified 44 distinct immune subsets. Correlation network analysis identified a network of inflammation-associated subsets, including HLA-DR⁺CD38⁺ EM CD4⁺ T cells, T regulatory-like cells, PD1⁺ EM CD8⁺ T cells, neutrophils, CD27⁺ TCRγδ cells and NK cells. All disease-associated subsets were validated in a second cohort. This network was abundant in a subset of patients, independent of IBD subtype, severity or intestinal location. Putative disease-associated CD4⁺ T cells were detectable in blood. Finally, imaging-mass cytometry revealed the spatial colocalization of neutrophils, memory CD4⁺ T cells and myeloid cells in the inflamed intestine. Our study indicates that a cellular network of both innate and adaptive immune cells colocalizes in inflamed biopsies from a subset of patients. These results contribute to dissecting disease heterogeneity and may guide the development of targeted therapeutics in IBD.