Characterization of Bovine Intraepithelial T Lymphocytes in the Gut

Intraepithelial T lymphocytes (T-IELs), which constitute over 50% of the total T lymphocytes in the animal, patrol the mucosal epithelial lining to defend against pathogen invasion while maintaining gut homeostasis. In addition to expressing T cell markers such as CD4 and CD8, T-IELs display T cell receptors (TCR), including either TCRαβ or TCRγδ. Both humans and mice share similar T-IEL subsets: TCRγδ⁺, TCRαβ⁺CD8αα⁺, TCRαβ⁺CD4⁺, and TCRαβ⁺CD8αβ⁺. Among these subsets, human T-IELs are predominantly TCRαβ⁺ (over 80%), whereas those in mice are mostly TCRγδ⁺ (~60%). Of note, the majority of the TCRγδ⁺ subset expresses CD8αα in both species. Although T-IELs have been extensively studied in humans and mice, their profiles in cattle have not been well examined. Our study is the first to characterize bovine T-IELs using flow cytometry, where we identified several distinct features. The percentage of TCRγδ⁺ was comparable to that of TCRαβ⁺ T-IELs (both ~50% of CD3⁺), and the majority of bovine TCRγδ⁺ T-IELs did not express CD8 (CD8^-) (above 60%). Furthermore, about 20% of TCRαβ⁺ T-IELs were CD4⁺CD8αβ⁺, and the remaining TCRαβ⁺ T-IELs were evenly distributed between CD4⁺ and CD8αβ⁺ (~40% of TCRαβ⁺ T-IELs each) with no TCRαβ⁺CD8αα⁺ identified. Despite these unique properties, bovine T-IELs, similar to those in humans and mice, expressed a high level of CD69, an activation and tissue-retention marker, and a low level of CD62L, a lymphoid adhesion marker. Moreover, bovine T-IELs produced low levels of inflammatory cytokines such as IFNγ and IL17A, and secreted small amounts of the immune regulatory cytokine TGFβ1. Hence, bovine T-IELs' composition largely differs from that of human and mouse, with the dominance of the CD8^- population among TCRγδ⁺ T-IELs, the substantial presence of TCRαβ⁺CD4⁺CD8αβ⁺ cells, and the absence of TCRαβ⁺CD8αα⁺ T-IELs. These results provide the groundwork for conducting future studies to examine how bovine T-IELs respond to intestinal pathogens and maintain the integrity of the gut epithelial barrier in animals.