We have recently demonstrated that human peripheral blood lymphocytes (PBL) contain a population of large granular lymphocytes which preferentially lyse HeLa cells infected with Shigella flexneri. In this proposal we will investigate the effector cells involved in this preferential killing of bacteria- infected cells, and the mechanism by which Shigella pretreatment leads to increased susceptibility to cytotoxic effector cell activity. The surface phenotype of cytotoxic effector cells present in human PBL, and in mouse spleen and gut associated lymphoid tissues (GALT) will be determined using different well characterized monoclonal antisera. The specificity of these cytotoxic effector cells will be addressed by using cold target inhibition and monolayer absorption types of experiments using different tumor cells and virus-infected cells. The susceptibility of bacteria-infected cells to soluble factors produced by natural killer (NK) cells will be investigated and compared to other tumor cells and virus-infected cells. To investigate the mechanism(s) by which Shigella renders a cell susceptible to natural cytotoxic effector cell activity, we will investigate the following: 1) requirement for bacteria invasion, 2) requirement for cell membrane alterations and/or presence of bacteria antigens, and 3) require- ment for cell versus bacteria metabolism. Using recombinant DNA methodology, we will isolate the bacterial gene(s) and define the gene product(s) important for demonstrating natural cytotoxic activity. Finally, we will investigate whether natural cytotoxic activity can be demonstrated against cells infected with other facultative intracellular bacteria. Results from these studies will not only contribute significantly to our understanding of immunity to facultative intracellular bacteria but will also provide the information necessary for establishing in vitro models for immune recognition of bacteria-infected cells.
