Intestinal natural killer (NK) cells contribute to maintaining gut homeostasis but may also play a role in driving local inflammation, leading to inflammatory bowel disease (IBD). However, the role of intestinal NK cells in healthy and inflamed gut tissues remains poorly understood. Employing high-parameter flow cytometry and a publicly available RNA-seq dataset, we performed an in-depth characterization of NK cells in ileum, caecum, and colon derived from organ donors as well as individuals with and without IBD, with a particular focus on effector molecule expression. Comparison with matched organs such as liver, spleen, lung, and lymph nodes further highlighted the unique features of intestinal NK cells.

Effector molecule expression in CD16- and CD16+ NK cells varied across tissues. Notably, CD16- ileal NK cells expressed strikingly high levels of granzyme A (GzmA), with levels exceeding those observed in CD8+ T cells. These GzmA^hi ileal NK cells predominantly comprised CD49a+ tissue-resident NK cells that strongly co-expressed CD39. Despite lacking Eomes expression, RNA-seq data validated their identity as NK cells rather than innate lymphoid cells. In mild IBD, ileal CD16- NK cells displayed a trend towards reduced GzmA and elevated perforin expression compared to non-IBD controls. Functional assays demonstrated reduced TNF production specifically in GzmA^hi NK cells, suggesting a distinct functional capacity for this subset.

Together, these findings underscore a previously unrecognized heterogeneity in tissue-resident NK cells across organs and between inflamed and non-inflamed conditions in the human gut. They suggest a potential role for GzmA^hi tissue-resident NK cells in ileal homeostasis and inflammation. Gaining further insights into site-specific features of NK cells will enhance our current understanding of their roles in IBD and other diseases such as gastrointestinal cancer.