Metabolic and functional changes in T helper cells during the periparturient period of dairy cows

The periparturient period in dairy cows is marked by major metabolic and physiological changes that increase disease susceptibility and challenge immune function. However, the function of T helper (CD4⁺) cells across the periparturient period remains poorly understood. This study evaluated the in vitro metabolic function, proliferation capacity and phenotype of T helper cells across the periparturient period. Multiparous Holstein cows (n = 22) having mean ± SD lactation of 4.16 ± 1.34 were sampled at -28, +3, and +28 d relative to calving. Peripheral blood mononuclear cells were isolated and T helper cells were obtained using magnetic sorting. A Seahorse XF HS Mini analyzer was used to assess the metabolic function through a T cell metabolic persistence assay, measuring glycolysis and oxidative phosphorylation via extracellular acidification and oxygen consumption rates respectively. Cells were treated either with assay medium (nonactivated) or phorbol 12-myristate 13-acetate and ionomycin (activated) for metabolic measurements, as this combination induces rapid, receptor-independent activation. For proliferation capacity, isolated T helper cells were incubated with concanavalin A, as it provides sustained stimulation to assess clonal expansion, for 72h and tracked using cell trace in a flow cytometer. Cytokine production was measured in the cell culture supernatant using a MILLIPLEX Bovine Cytokine/Chemokine kit. RNA was extracted from isolated cells, and gene expression of immune-related and metabolic markers were evaluated by quantitative PCR. Data were analyzed using a linear mixed effect model in R 4.4.3 using day relative to calving, activation status and block as fixed effect and cow as a random effect. When properly, time of the assay and its interaction with day relative to calving was included as a fixed effect. Activated T helper cells showed greater glycolytic and oxidative metabolism at +3 and +28 d compared with -28. Expansion index was highest at +3 (2.65), followed by +28 (2.38) and -28 (1.91). Proinflammatory cytokine production, like IL-1 α, IL-1 β and IL-6 also increased during +3 and +28 compared with -28. However, no significant differences in gene expression were detected across time points. These findings suggest that T helper cell metabolism and proliferation capacity are upregulated after calving and that these cells express a more proinflammatory phenotype, indicating that after parturition, immune function on this subset of cells does not appear to be suppressed.