The goals of our research are to determine if mononuclear cells mediating NK-like killing are effector cells within the human delayed type hypersensitivity (DTH) response, to characterize those cells and to investigate the cells and factors involved in their stimulation. Since the cells responsible for the tissue damage in the DTH response are likely to be effector cells in other cell mediated immune phenomena including transplant and tumor rejection, graft versus host disease, destruction of infected cells, and auto-immune disease, it is important to determine the nature of these cells and how they function. Skin test antigens (STA's) stimulate NK-like cytotoxicity in human peripheral blood mononuclear cells in vitro and there is a strong correlation between the amount of NK-like killing stimulated by a STA in vitro and the magnitude of the DTH response to that same antigen in vivo. When artificial skin blisters were used to obtain cells from positive and negative DTH reactions to STA stimulation in vivo, only those blisters associated with positive DTH responses contained mononuclear cells capable of NK-like killing. These finding suggest that cells mediating NK-like killing may be effector cells in the human DTH response and that STA stimulation of natural effector cell (NEC) activity in vitro may reflect events occurring within that response. We therefore plan to characterize and compare the DTH associated NEC to the NEC generated by STA stimulation in vitro. The morphology of these NEC's will be examined by performing conjugate analyses and single cell cytotoxicity assays on cells obtained from Percoll gradient fractions enriched in NK-like killing. They will be phenotyped using mouse monoclonal antibodies directed against human cell markers in conjunction with a variety of cell separation techniques including complement mediated cytolysis, fluorescense activated cell sorting and indirect panning. Experiments will be performed to determine if T-helper cells, interferon (IFN) and interleukin-2 (IL-2) are involved in STA stimulation of human NEC's in vitro. The levels of IFN and IL-2 within the DTH response will be investigated by measuring these factors in fluids obtained from DTH-associated artificial skin blisters. In order to assess the importance of NEC's to the DTH response we plan to investigate the effects on the DTH response of depleting and repleting NEC activity in mice. By knowing the nature of the DTH-effector and having a good in vitro model to for its activation we can determine the safest most specific methods for increasing or decreasing DTH reactivity in patients without having to expose patients to significant risk.
