Decidual NK cells (dNK), the largest population of maternal immune cells at the maternal-fetal interface in the first trimester of pregnancy, directly contact fetal extravillous trophoblasts (EVT), which invade the decidua to remodel the vasculature to establish the blood supply to the placenta. The direct contact between dNK and EVT challenges the maternal immune system, which must tolerate fetal cells, but still protect against infection. How dNK protect the placenta and fetus from infection is not well understood. Most clinically significant infections of the placenta and fetus are caused by intracellular pathogens (bacteria, parasites and viruses), for which killer lymphocytes (NK and cytotoxic T lymphocytes) are key to systemic protective immunity. In the first trimester, when infection has the most serious fetal consequences, there are few T cells in the decidua. Although dNK have cytotoxic granules, express all the cytotoxic molecules, and kill conventional NK cell targets, their cytolytic activity is reduced compared to peripheral blood NK cells. Moreover, although dNK form contacts with EVT, they do not degranulate or kill human cytomegalovirus-infected EVT. These findings emphasize the difficulties of maternal immune cells to clear placental infections and prevent transmission of pathogens to the unborn child. This proposal investigates a novel and exciting mechanism we recently discovered by which dNK kill L. monocytogenes (Lm) inside trophoblasts, without killing the host cell. dNK express large amounts of granulysin (GNLY), an antimicrobial peptide found both in cytotoxic granules and the cytosol that preferentially disrupts microbial, relative to mammalian, membranes. Our preliminary data suggest that dNK establish nanotube cytoplasmic connections to EVT. Without forming a conventional immune synapse or degranulating, dNK transfer GNLY via nanotubes to EVT, but not other cytotoxic molecules (perforin, granzymes), which would kill the host cell. This mechanism provides an elegant solution to the immune dilemma of pregnancy – defense against infection while maintaining tolerance of the fetus and placenta. As far as we are aware, this is the first evidence for an immune function of nanotubes. Nanotube transfer of GNLY and potentially other bioactive molecules from dNK to EVT helps control intracellular infection and could regulate trophoblast functions. Our goals are to confirm our preliminary data showing that intracellular microbes, but not fetal cells, are killed by dNK transfer of GNLY, independently of perforin and granzymes; identify which infected maternal and fetal cells in the placenta dNK protect and by what mechanism; explore the mechanism responsible for nanotube formation, which molecules are transferred and which pathogens important in pregnancy are susceptible. The protective role of GNLY and dNK will also be evaluated in human placental tissue explants and in mice by comparing pregnancy outcomes following infection of G...