Abstract
The intestinal epithelium is continually exposed to food-derived antigens and microbiota. This continual exposure requires a delicate immune homeostasis for food tolerance and protection against infection. Unconventional T lymphocytes, including γδT cells, Natural killer T (NKT) cells, Mucosal-Associated Invariant T (MAIT) cells, and Double-Negative T (DNT) cells, typically reside in the mucosal tissue, such as the colon. These cells play crucial roles in maintaining the integrity of the mucosal barrier and immune homeostasis through cytokine secretion and direct cell-mediated effects. Understanding the proportions and functional status of unconventional T lymphocytes in the colon is crucial for elucidating disease mechanisms. In this study, we developed a 22-color flow cytometry panel for comprehensive immunophenotyping of unconventional T lymphocytes in the murine colon. Our optimized protocol included antibody titration and customized gating strategies. We identified distinct populations of unconventional T lymphocytes, including γδT cells, NKT cells and DNT cells, and compared them with conventional T lymphocyte subsets (CD4(+) T, CD8αα(+) T, and CD8αβ(+) T). We assessed their proliferation, cytotoxicity, cytokine production, and immune checkpoint molecule expression. Inhibitory receptor levels on intraepithelial and lamina propria unconventional T lymphocytes differed, suggesting distinct local environments and regulatory mechanisms. Our findings elucidate the status and function characteristics of unconventional T cells in colonic tissues, providing insights for mechanistic studies and the development of therapies for gastrointestinal diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43657-025-00237-6.