Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. IV. Analysis of variant‐specific antigens by selection of antigen‐loss variants with cytolytic T cell clones

Mouse mastocytoma P815 tumor cell variants that express new individual antigens were subjected to immunoselection in vitro with specific cytolytic T lymphocyte (CTL) clones. From one variant (P35) a number of resistant clones were isolated after a single-step selection. These immunoselected clones (P35.iscA-) proved to have acquired a stable and complete resistance to lysis by the selecting CTL, but remained sensitive to lysis by a CTL clone that recognizes a common P815 antigen. However, when these P35.iscA- clones were used to stimulate in vitro spleen cells from mice immunized with variant P35, the cytolytic activity was significantly higher on P35 and P35.iscA- targets than on the original P815 clone (P1). This indicated that P35.iscA- cells, which had lost a P35-specific determinant (P35A), had nevertheless retained another P35-specific determinant (P35B). CTL clones directed against the residual P35B determinant were then isolated. When P35 cells were submitted to selection with anti-P35B CTL, resistant clones (P35.iscB-) were obtained that were still lysed by anti-P35A and anti-P815 CTL clones. Tum- variant P35 therefore carries at least two specific determinants that can be lost independently. These results show the possibility of using selection with CTL clones to separate different components of complex cell surface antigens. The correlation between antigen expression and tumorigenicity was also examined. Antigen-loss P35.iscA- clones were found to be more tumorigenic than variant P35 which has a greatly reduced tumorigenic capacity compared to the original P815 tumor. This correlation suggests that the P35 determinant that was no longer expressed by the P35.iscA- clones is recognized on the original P35 tum- variant as a tumor rejection antigen.