Preparing and cloning a natural killer cell hybridoma /

Abstract

NK cells, a component of our innate immune system, are responsible for the elimination of virus-infected and tumor cells which bypass detection by T cells. In these studies, we attempted to create a NK cell hybridoma clone, which would provide a testable pool of NK cells. Mouse splenocytes were fused with P3X mouse non-secreting myeloma cells in the presence of polyethylene glycol (PEG). Following HAT selection, the cells were tested for the presence of the mouse NK cell surface antigen DX5. After labeling with biotinylated anti-mouse DX5 and Strepavidin-FITC in the presence of rat anti-mouse CD16/CD32 (Fe Block), the mixed lymphocyte cell pool which yielded the highest amount of mean fluorescence (257.17), was cell pool C6. C6 was then subcloned, and the clones were retested for the presence ofDX5 and absence ofCD3. The data analysis, however, was hindered by the presence of autofluorescence in the hybridoma cell line. In an attempt to correct the autofluorescence of the hybridomas, two permeabilzing detergents, n-Octyl-B-D-Glucopyr anoside (OG), and Cytoperm/Ctyofix™ were used with various concentrations (.01 f.!g/ml to 10f.!g/ml) of the quenching dye trypan blue. Because of the low binding affinity of the anti-DX5 antibody and the inability to compensate for cellular autofluorescence a pure NK cell hybridoma clone could not be identified.