A simple method for the quantitation of specific cytotoxic precursors to synrgeneic tumors

Abstract

Cytotoxic T lymphocytes (CTLs) to EL4, a syngeneic lymphocytic leukemia of C57BL/6 (B6) (H-2 b) mice, were obtained by culturing normal B6 spleen cells with irradiated EL4 tumor cells for 4 days in conical bottom mitrotiter trays. A much lower, though significant, amount of cytotoxicity towards EL4 was observed in B6 spleen cell cultures not stimulated with EL4. The cytotoxic effector cells were shown to be Thy-1 + and their cytolytic activity to EL4 tumor cells was inhibited by an anti-H-2 b serum. Cold target competition studies suggest that the B6 anti-EL4 CTLs could discriminate between EL4 tumor cells and a second H-2 b lymphoma, C1498. The converse experiment yielded similar results. Culture conditions limiting for CTL precursors (CLPs) to EL4 were attained by including interleukin 2 (IL2), a lymphokine obtained by stimulating murine or rat spleen cells with concanavalin A, in the culture medium. In the presence of IL2, the CLP frequencies in B6 spleen cells to EL4 and C1498 in antigen-stimulated cultures were 113 and 231 per 10 6 responder cells, respectively. In non-antigen stimulated B6 spleen cell cultures, the apparent CLP frequencies to EL4 and C1498 were 6 and 33 per 10 6 responder cells, respectively. In agreement with the cold target competition studies using CTLs generated in the absence of IL2, we found that the CTL clones produced in cultures stimulated with EL4 and C1498 in the presence of IL2 were specific for the stimulating antigen. The specificity of the CTL clones obtained from cultures stimulated with IL2 in the absence of antigen appears to be different from those derived from antigen-stimulated cultures. The potential applications and limitations of this culture system are discussed.