During terminal differentiation of B lymphocytes, natural killer (NK) cells recognize target structure (NKTS) expressed by (or uncovered on) B cells prior to the plasma cell stage. The objectives of this study are to identify and characterize NKTS expressed on lymphocytic target cells and to determine how expression of NKTS is regulated. NK-resistant lymphoid cell lines will be selected following low-dose irradiation of NK-sensitive lines, multiple rounds of NK, and cloning the surviving cells. The NK- resistant lines will be compared to the parental, sensitive lines by analytical, 2-D-polyacrylamide gel electrophoresis to detect polypeptide differences, i.e., candidate NKTS. Monoclonal and polyclonal antibody reagents will be produced for detailed analyses of the structure and function of candidate NKTS. One such molecule, p115, has been identified on the surface of an NK-sensitive, T-cell line and is absent from the NK- resistant, variant line. Antibody reagents of p115 will be produced for studies to determine its cellular distribution and function in NK cell- target cell binding. The expression of NKTS is regulated, in part, by MHC class I molecules. B-cell lines will be transfected with the E3 gene of Adenovirus; E3 encodes (among other polypeptides) a p19 that complexes with class I molecules, preventing their cell-surface expression. Transfected B-cell lines should show an increase in NK sensitivity, if NKTS are constitutively produced, but masked, by class I molecules. Alternatively, they should show no increase in sensitivity, if NKTS are synthesized de novo during B-cell termial differentiation. NK-sensitive, class I- deficient, B-cell lines will be transfected with different class I gene constructs to map the domain(s) interacting with NKTS. Polypeptides coprecipitating with MHC class I molecules will be purified and tested as candidate NKTS; a polypeptide has been identified that coprecipitates with class I molecules from a NK-sensitive line but not from the NK-resistant line. These studies should clarify whether NK cells regulate terminal differentiation of B cells (and interact with late-stage B-cell malignancies) and should identify both the phenotype of the NK-sensitive B cells and NKTS.
Showing the most recent 10 out of 13 publications
