Traditional vaccine efficacy evaluation predominantly relies on antibody levels, while the assessment of T-cell responses remains underexplored. In this study, we employed multi-parameter flow cytometry to comprehensively analyze T-cell responses in cows vaccinated against foot-and-mouth disease (FMD). We categorized the cows into high and low vaccine potency groups based on antibody levels and investigated differences in T-cell responses between these groups. Phenotypic analysis revealed a significant reduction in CD4, CD8, and γδ T cells in peripheral blood following FMD vaccine inoculation, concomitant with altered CD44 expression. Intriguingly, FMD vaccination induced a marked increase in the percentage of CD4
+
CD8
+
double-positive (DP) T cells in cow peripheral blood. Notably, cows with high vaccine potency exhibited a significantly higher proportion of CD4
+
CD8
+
DP T cells compared to those with low potency post-vaccination, suggesting their potential involvement in FMD vaccine-induced immune protection, possibly through the regulation of B-cell antibody secretion. Additionally, FMD vaccination led to the generation of central memory CD4 and CD8 T cells but not γδ T cells. Our groundbreaking findings shed light on the latent function of CD4
+
CD8
+
DP T cells in FMD vaccine-induced immune protection and established a novel method for evaluating FMD vaccine efficacy based on T-cell responses.
IMPORTANCE
Vaccination plays a crucial role in the prevention and control of FMD; however, outbreaks persist occurring worldwide. Assessing the immune response to FMD vaccines is essential for effective prevention of FMD. In this study, a seven-color flow cytometry protocol was developed to systematically evaluate the T-cell response of Chinese Holstein cows vaccinated with FMD bivalent inactivated vaccine. Our findings showed that while most T-cell subsets (%) decreased post-vaccination, a significant increase was observed in CD4
+
CD8
+
DP T cells, which was consistent with the levels of specific foot-and-mouth disease virus (FMDV) antibodies. These findings suggested that CD4
+
CD8
+
DP T cells could serve as a potential biomarker for the evaluation of cellular and humoral responses to FMDV vaccination. Additionally, we should be aware of the potential decline in cellular immunity among cattle during FMD vaccination, as this may increase the risk of other pathogen-related issues.