In these experiments, we have introduced a novel nonhuman primate model for studying breast milk immunity during HIV/SIV infection. The advantages of pharmacologic-induction of lactation in rhesus monkeys include independence from both primate breeding cycles and care of infant monkeys, and ease of sample collection. Lymphocyte subsets were similar in HIL and NL breast milk, indicating that antigen-specific cellular immune response in each type of breast milk should be comparable. Not surprisingly, the HIL breast milk displayed characteristics similar to colostrum or early milk with higher immunoglobulin (37
), macrophage/monocyte, and neutrophil content (32
) than mature milk, as the artificial lactation protocol likely induces breast milk that is not fully mature. However, human colostrum has a macrophage/monocyte predominance that was not observed in the HIL breast milk, suggesting that the HIL breast milk may be on the continuum between early milk and mature milk. Since breast milk viral load (41
) and rates of HIV transmission are higher in early lactation (42
), the apparent immaturity of the HIL breast milk is advantageous for modeling the virologic and immunologic factors contributing to this period of high risk for the breastfeeding infant.
The lactation model is a valuable tool to investigate antigen-specific mucosal immunity, as lymphocytes and antibody can be collected in the fluid phase at multiple time points and immune responses can be evaluated simultaneously in the mucosal compartment and the blood. In the lactation-induction SIV/rhesus monkey model, we were able to quantitate epitope-specific T lymphocytes, as well as characterize functional, ex vivo-stimulated cellular responses without the high background activation that often limits the ability to monitor cellular immune responses. While blood contamination of manually-collected breast milk is a concern, our data are consistent with the detection of breast milk-specific humoral and cellular immune responses, as the defined phenotype of the lymphocytes and the antibody content of the rhesus monkey breast milk were distinct from the blood and consistent with previous descriptions of human breast milk immune responses (26
While HIV-specific CD8+
T lymphocytes have previously been demonstrated in breast milk of HIV-infected women (44
), the kinetics and function of this population of cells has not been evaluated. This study has documented a robust SIV-specific cellular immunity in breast milk that parallels the kinetics of the CD8+
T lymphocyte response in the blood, but is two to three times higher at the time of both peak and set point of viremia. The robust SIV-specific cellular response in the breast milk was not related to high antigen burden, as the viral load in the breast milk remained at least one log lower than that in the blood throughout acute infection.
The high frequency of SIV-specific T lymphocytes in breast milk measured during acute and chronic SIV infection was not predicted. The SIV-specific CD8+
T lymphocyte response reported in the gastrointestinal and genital tracts during acute infection is less than or equal to the CD8+ T lymphocyte response in the blood (18
), despite intense viral replication in those mucosal compartments. The robust mucosal CD8+
T lymphocyte response in breast milk provides further evidence that the mucosal compartments are immunologically distinct (16
). Moreover, the high levels of cellular immunity in breast milk during acute infection may contribute to prevention of viral transmission from mothers to infants during breastfeeding.
It is well-established that the SIV-specific CD8+
T lymphocyte response is essential for the control of viral replication in the peripheral blood (47
). While control of viral replication in mucosal compartments are likely affected by both anatomic and immunologic factors, the breast milk SIV-specific CD8+
T lymphocytes likely play a key role in the containment of breast milk viral load because of several supporting observations. First, the breast milk viral load remained appreciably less than the viral load in plasma throughout acute and chronic SIV infection in association with a particularly high SIV-specific CD8+
T lymphocyte response in the breast milk. Second, the initial containment of breast milk viral load occurred near the time of the emergence of the Tat TL8-specific CD8+
T lymphocyte response in the breast milk, although this association may be explained by the concurrent reduction in plasma viral load. Finally, maintenance of low breast milk viral load after viral set point was coincident with a sustained high level Gag p11C-specific CD8+
T lymphocyte response in breast milk.
The potency of the breast milk SIV-specific cellular immune response during acute SIV infection may contribute to containing mother to child transmission of the virus. As high breast milk HIV RNA viral load is associated with infant breast milk transmission (9
), cellular immune containment of viral shedding in breast milk could contribute to a reduction in risk of transmission via breastfeeding. HIV-specific cytotoxic CD8+
T lymphocytes in breast milk are expected to reduce breast milk viral load by eliminating local cellular reservoirs of virus, including macrophage/dendritic cells and activated CD4+
T lymphocytes. These cell types are in high concentration in early milk (34
) and likely play an important role in the transmission of HIV via breastfeeding. Additionally, the infant is exposed to a high frequency of HIV-specific maternal CD8+
T lymphocytes that may be absorbed by the infant gastrointestinal tract, as animal studies have demonstrated the absorption of maternal lymphocytes into the gastrointestinal mucosa (50
), as well as the bloodstream (52
), of suckling neonates. Furthermore, maternal lymphocytes transmitted in breast milk may play an active role in the developing neonatal immune system, as evidence of acquisition of maternal mitogen and antigen-specific cellular responses via breastfeeding exists in both experimental animal (53
) and human (34
) studies. Further investigation of the function of the high frequency viral-specific CD8+ T lymphocytes in breast milk is certainly warranted. Through these studies, maternal intervention to enhance this breast milk immune response may be a viable strategy for prevention of mother to child transmission via breastfeeding.