Lactation stage-dependent profiles of T lymphocyte subpopulations and immunoregulatory cytokines in mammary secretions

Immunofluorescent techniques and flow cytometry analysis were used to phenotype milk mononuclear cells. At any stage of the lactation cycle (i.e., involution or dry stage, colostral stage of parturition, and the normal stage of milk production), the majority ($\sim$90%) of milk lymphocytes were CD2$\sp+$ T cells. In the dry period, 50-60% of T lymphocytes were CD4$\sp+$ and less than 30-40% were CD8$\sp+$. In normal mature milk, however, the proportion of CD4$\sp+$ T cells decreased to less than 20%, whereas CD8$\sp+$ T cells were the predominant T cell subpopulation constituting 30-40% of T lymphocytes. Whereas all T lymphocytes in the dry period were CD5$\sp+$ T cells, only 50-60% were CD5$\sp+$ in normal mature milk, suggesting that normal mature milk contained 30-40% CD2$\sp+$/CD5$\sp-$ immature T cells. Although in mastitic milk CD2$\sp+$ T cells constituted the majority (60-70%) of lymphocytes, the proportion of CD4$\sp+$ T cells increased to $\sim$30%, the proportion of CD8$\sp+$ T cells decreased to $\sim$25%, and the proportion of CD5$\sp+$ T cells increased to $\sim$60%.^ Analysis of milk showed that transforming growth factor-beta (TGF-$\beta$), which is a potent immunoregulatory cytokine, was present in high concentrations in its active (1.88-99.0 ng/ml) and latent (923-10,737 ng/ml) forms in dry milk secretions. Mastitic milk contained significantly higher levels of TGF-$\beta$ (1,015 $\pm$ 366 ng/ml) as compared to normal milk (105 $\pm$ 22 ng/ml). Milk did not contain detectable levels of interleukin-2 (IL-2), and caused $\sim$50-80% inhibition on the growth of IL-2 dependent CD4$\sp+$ bovine lymphoblastoid T cell line (BLTC). Furthermore, TGF-$\beta$ inhibited the lectin-induced blastogenesis and IL-2 production by milk mononuclear cells. These results suggest that TGF-$\beta$ is a potent immunosuppressive cytokine for T cells in milk. Further in vitro analysis showed that TGF-$\beta$ inhibited stationary BLTC cells through decreasing their IL-2 receptor expression, arresting these cells in the $\rm G\sb0/G\sb1$ phase of the cell cycle, and inducing these cells to undergo apoptosis. These inhibitory events, however, were reversed by the addition of exogenous IL-2. In fact, when present together, IL-2 and TGF-$\beta$ synergized to promote the growth of BLTC. In conclusion, this study showed that T cells selectively migrate to the mammary gland under various physiological and pathological conditions, and that TGF-$\beta$ is a potent immunoregulatory cytokine in the mammary gland.