Generation of long-term T-lymphoid cell lines with specific cytotoxic reactivity for a syngeneic murine lymphoma

The long-term in vitro growth in T-cell growth factor (TCGF) of murine cytotoxic T-lymphoid cells directed against syngeneic tumor antigens was investigated. C57BL/6 mice were immunized to syngeneic FBL-3 lymphoma by two ip injections of irradiated FBL-3 lymphoma cells. Splenocytes from these animals were injected into mice with disseminated lethal FBL-3 tumor. The injection of cyclophosphamide (Cy) plus immunized lymphocytes significantly improved survival with cure of 53% of 38 animals. In comparison, treatment with Cy alone resulted in 0 of 31 cured and treatment with Cy plus unimmunized cells resulted in 0 of 40 cured (P < 0.000). These in vivo immunized lymphocytes were reexposed to FBL-3 tumor in vitro for 5 days in lectin-free TCGF (LF-TCGF). Although in vivo and in vitro sensitized lymphocytes exhibited no cytotoxicity toward fresh FBL-3 tumor cells in an 18-hour ⁵¹Cr release assay, expansion of appropriately sensitized cells in LF-TCGF resulted in significant lysis of fresh FBL-3 tumor cells. This significant lysis was specific and lysed syngeneic FBL-3 but not syngeneic MCA-103 fresh tumor targets. This maximal specific cytotoxicity was maintained for 2.5 months. A screening assay was developed that permitted rapid identification and isolation of low-frequency cytotoxic clones with reactivity specific for FBL-3 tumor. Several of these cloned cells were grown for almost 3 months with maintenance of high degrees of specific lysis (as much as 4,500 lytic U/10⁶ cells). These cytotoxic lines and clones will be of value for the study of tumor-host immunologic interactions and perhaps for use in adoptive immunotherapy.