Factors responsible for resistance of cells to lytic attack by murine cytotoxic lymphocytes
Description
To isolate genes responsible for resistance of cytotoxic T lymphocytes (CTLs) to their own toxic proteins, a cDNA library from the murine CTL 2C was prepared in the expression vector pRC/CMV. A-20 lymphoma cells were transfected with the library and selected by culture in G418. Transfectants were further selected for resistance to lysis by perforin containing extracts of CTLs. Successive rounds of lysis with extract were followed by expansion of survivors in culture. The extract-selected line was twice as resistant to extract as the unselected control as measured by chromium release. This partially resistant line differs morphologically from transfected, but unselected, controls and also from non-transfected A-20. The resistant line consists predominantly of grape-like clusters, while the control contains mainly spreading adherent cells. Clones were isolated from this line by limiting dilution. Clustering clones were more resistant to lysis than spreading clones However, the growth rate of survivors from clustering clones and the selected line was initially reduced after extract treatment in comparison to survivors of spreading clones or controls which were not inhibited. Normal growth rates returned after three days in culture. Inhibition was not due to latent cytotoxic effect as the cloning efficiency of survivors from clustering clones was equal to that of survivors from spreading clones or controls. When proliferation of cells was arrested at the G1/S border with 2 mM Thymidine, cells became markedly resistant to perforin mediated lysis. Efforts to isolate the gene(s) responsible for the clustering phenotype and resistance are underway. These results also suggest that growth inhibition is associated with a defensive response to CTL-mediated injury