Natural killer (Nk) cells can not only mediate non-specific lysis of tumor cells but also specific lysis of allogeneic non-transformed cells. This is noted both in the NK-mediated rejection of allogeneic bone marrow cells in vivo and lysis of T cells blasts in vitro. The basis of allospecific recognition by Nk cells is not clear. Recent evidence indicates that class I MHC receptors belonging to the Ly49 family of genes expressed on subsets of adult splenic murine NK cells are likely to be involved. The expression of Ly49 molecules seems to be regulated by the MHC class l genotype of the host. In particular Ly49A, known to bind to H-2(d) and transduce a negative signal to NK cells, is down regulated in mice. NK cells in such mice express low levels of Ly49. The hypothesis to be tested in this project is that during development of NK cells there is an ordered and sequential expression of cell surface receptors. Positive signalling molecules, whose ligands are not MHC molecules appear first. The subsequent expression of Ly49 molecules is regulated by interaction with host MHC class molecules expressed on some component of the bone marrow. This hypothesis is supported by the preliminary observation that 14 day fetal liver and fetal thymus derived NK cells lack Ly49 molecules, and by the finding that Ly49 molecules first appear on bone marrow NK-1.1 + cells after 7th post natal day. We propose to test the influence of MHC class I molecules in the modulation of NK cell Ly49 receptor repertoire. For these studies, availability of well characterized clonal populations of murine NK cells are critical. Hence attempts will be made to clone and characterize immature NK1.1+, Ly49-cells from fetal thymus or fetal liver at different days of gestation. Cloning will also be attempted from adult splenic and marrow NK-1.1 + cells that express Ly49 molecules. Several strategies including cytokine mixtures, feeder layers, fusion with tumor cells, transfection with retroviral constructs will be utilized. To investigate the regulation of NK cell repertoire, Ly49A will be chosen as the model receptor. Attempts will be made to modulate the intensity of Ly49A expression on H-2(b) derived NK-1.1+, Ly49- fetal liver cells by exposure to H-2(b) or H-2(d) class l molecules expressed on bone marrow cells in vivo and in vitro. The in vivo studies will involve transfer of fetal liver derived Ly49- cells from H-2(b) donors into H-2(d) hosts. In vitro studies will involve co-culture of Ly49- cells with irradiated bone marrow cells or marrow derived stromal cells. These experiments will provide an insight into the regulation of class I MHC receptor development on NK cells.
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