# Initial characterization of ruminal T lymphocytes in lactating dairy cows

## TL;DR

This study identifies γδ T cells as the most common T cell type in the rumen of healthy dairy cows, with a unique CD4+TCRN24+ population observed.

## Contribution

The study is the first to characterize T lymphocyte subsets in the rumen of lactating dairy cows using spectral flow cytometry.

## Key findings

- γδ T cells constitute the majority of T lymphocytes in the rumen tissue.
- A novel TCRN24+CD4+ T cell population was identified in the rumen.
- CD4+ T cells are significantly reduced in the rumen compared to peripheral blood.

## Abstract

Summary: Our study aimed to determine the relative proportions of T cell types in the rumen tissue of healthy dairy cows through spectral flow cytometry, hypothesizing that the largest proportion would be γδ T cells. We used spectral flow cytometry to assess ruminal T cell proportions. The panel included CD45+ (leukocytes), CD3+ (T lymphocytes), CD4+ (helper T cells), and TCRN-24+ (γδ T cells). CD3+ cells constituted 83.9% ± 2.85% (mean ± standard error of the mean) of ruminal CD45+ cells. On a CD3+ basis, TCRN24+ cells were the most prominent subset of T lymphocytes in rumen tissue (78.7% ± 6.92% of CD3+). CD4+ were reduced as a proportion of T cells in the rumen (1.4% ± 1.95% of CD3+). We observed a TCRN24+CD4+ population in the rumen (9.0% ± 2.72% of CD3+). Understanding the role of T lymphocytes in rumen immunity may guide strategies that strengthen rumen health and digestion while reducing gut-derived inflammation. Created in BioRender. Krogstad, K. (2025) https://BioRender.com/amyibh6.

Summary: Our study aimed to determine the relative proportions of T cell types in the rumen tissue of healthy dairy cows through spectral flow cytometry, hypothesizing that the largest proportion would be γδ T cells. We used spectral flow cytometry to assess ruminal T cell proportions. The panel included CD45+ (leukocytes), CD3+ (T lymphocytes), CD4+ (helper T cells), and TCRN-24+ (γδ T cells). CD3+ cells constituted 83.9% ± 2.85% (mean ± standard error of the mean) of ruminal CD45+ cells. On a CD3+ basis, TCRN24+ cells were the most prominent subset of T lymphocytes in rumen tissue (78.7% ± 6.92% of CD3+). CD4+ were reduced as a proportion of T cells in the rumen (1.4% ± 1.95% of CD3+). We observed a TCRN24+CD4+ population in the rumen (9.0% ± 2.72% of CD3+). Understanding the role of T lymphocytes in rumen immunity may guide strategies that strengthen rumen health and digestion while reducing gut-derived inflammation. Created in BioRender. Krogstad, K. (2025) https://BioRender.com/amyibh6.

•T lymphocytes are the most prominent leukocytes in the rumen tissue.•γδ T cells are the most prominent T-cell subset in the rumen tissue.•A novel cell type (CD4+TCRN24+) may exist within the rumen.

T lymphocytes are the most prominent leukocytes in the rumen tissue.

γδ T cells are the most prominent T-cell subset in the rumen tissue.

A novel cell type (CD4+TCRN24+) may exist within the rumen.

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.