Initial characterization of ruminal T lymphocytes in lactating dairy cows

γδ T cells may regulate immune responses through interleukin-10 secretion and reduce cytotoxic and helper T cell proliferation, in vitro. γδ T cells are prominent in ruminant peripheral blood, but their presence in a recently defined ruminal immune cell population remains unclear. Our study aimed to determine the relative proportions of T cell types within rumen tissue (heterogeneous tissue consisting of lamina propria, stratified epithelium, and vasculature) of healthy dairy cows through spectral flow cytometry, and we hypothesized that the largest proportion of ruminal T cells would be γδ T cells. We used spectral flow cytometry to assess ruminal and peripheral blood T cell proportions. The panel included CD45 (leukocytes), CD3 (T lymphocytes), CD4 (helper T cells), and TCRN-24 (γδ T cells). Results were analyzed with a mixed linear model that included the effect of blood versus rumen tissue and the random effect of cow. Cows were clinically healthy throughout the experiment, as indicated by their DMI (26.0 ± 3.44 kg/d), milk yield (50.6 ± 12.99 kg/d; mean ± SD), and ruminal pH (6.06 ± 0.55). CD3+ cells constituted 83.9% ± 2.85% (mean ± SEM) of ruminal CD45+ cells compared with 3.3% ± 2.85% of CD45+ cells in blood. On a CD3+ basis, TCRN24+ were the most prominent subset of T lymphocytes in rumen tissue samples and were enriched compared with blood (78.7% ± 6.92% of CD3+ vs. 38.1 ± 6.92% of CD3+). The CD4+ were reduced as a proportion of T cells in the rumen compared with blood (1.4% ± 1.95% of CD3+ vs. 21.4 ± 1.95% of CD3+). We observed a TCRN24+CD4+ population isolated from the rumen which was lesser in blood (9.0% ± 2.72% of CD3+ vs. 1.5 ± 2.72% of CD3+). We confirmed our hypothesis that γδ T cells are the most prominent proportion of T cells within the rumen tissue in healthy lactating dairy cows. Furthermore, we observed a unique population that was both TCRN24+ and CD4+; investigations of this potential T cell subtype are needed to confirm their presence and determine their function. Understanding the role of T lymphocytes in rumen immunity may guide strategies that strengthen rumen health and digestion while reducing gut-derived inflammation.