In vitro induction of cell-mediated immunity to murine leukemia cells. VII. Methods for augmenting the induction and expression of cytotoxic response in vitro to syngeneic tumors

Transplantable murine leukemias induced by chemical carcinogens or oncogenic viruses often show a limited capacity to elicit in vitro cytotoxic effector T lymphocytes (CTL). In the present study attempts were made to amplify the cytotoxic response by various approaches, either at the induction phase or at the effector phase. CTL were generated in one-way mixed lymphocyte-tumor cell cultures (MLTC) comprising of splenocytes from unsensitized mice and mitomycin C-treated syngeneic leukemia cells (EL4, RBL-5, YAC). The cytotoxic activity was measured on day 6 by a 3 h ⁵¹Cr release assay. A 2-10-fold augmentation in cytotoxic capacity was obtained with each of the following treatments: (1) sensitization culture medium supplemented with 2% polyethylene glycol (PEG) or 0.5-2 μg/ml MER-BCG; (2) responder lymphocytes derived from mice previously treated with 50-100 mg/kg cyclophosphamide or 2.5 mg hydrocortisone; (3) responder lymphocytes depleted of nylon-adherent or Fc receptor-bearing cells; (4) responder lymphocytes treated with trypsin or papain (0.01-0.1 mg/ml); (5) stimulator leukemia cells treated with either trypsin (0.1-1 mg/ml), bromelain (0.1-1 mg/ml), or trinitrobenzenesulfonic acid (TNBS, 10^-4-10^-5 M); (6) 6 day sensitized effector cells treated with trypsin, papain, or bromelain (0.01-0.1 mg/ml); (7) 6 day sensitized effector cells separated on a density gradient of bovine serum albumin. Compared to ordinary sensitization a 100-fold increase in cytotoxic activity (on a per cell basis) was achieved by a combination of several such techniques (e.g. Nos. 1, 5 and 7). The various possible mechanisms responsible for the augmentation of cytotoxic reactivity are discussed.