A vaccine of Bacillus Calmette-Gu~rin (BCG) admixed with tumor cells induced systemic immunity and had a therapeutic effect on subclinical, disseminated mierometastasis. Inbred strain-2 guinea-pigs given I V injections of 5 x 103 to 106 syngeneic LIO hepatocarcinoma cells were vaccinated after metastatic foei were established in the lung parenchyma. The purpose of this study was to establish the variables that can be manipulated to assure optimal immunotherapy while minimizing deleterious side effects of the BCG. In the present study we examined the variables of source, dose, and ratio of BCG to tumor cells. Four BCG sources (lyophilized Tiee and Connaught; fresh-frozen Phipps and Tice) were compared. No significant differences among these BCG preparations couM be detected with respect to adjuvant potential when they were admixed with attenuated tumor cells in a vaccine. The dose study clearly demonstrated that a BCG dose dependency exists with relation to induction of effective cell-mediated immunity or survival from disseminated mierometastatic disease. Furthermore, evaluations of dose versus ratio of BCG to tumor cells also supported a BCG dose dependency, with the lowest effective BCG dose being directly influenced by tumor burden of the host. Cutaneous reactivity and hypersensitivity of the primary and secondary immunization sites of tumor-bearing animals treated with effective and ineffective vaccines supported the direct association of reaction to BCG and specific tumor immunity. However, when an in vitro leukocyte migration inhibition assay was used, the degree of reactivity to BCG could not be exploited as a quantitative, diagnostic monitor of effective systemic tumor immunity.
Agarose microdroplet leukocyte migration inhibition (LMI) assays were performed to measure reactivity against line 10 hepatocarcinoma antigens and purified protein derivative (PPD) with the use of peripheral blood leukocytes from line 10 and/or BCG-sensitized syngeneic guinea pigs. The assay was quite sensitive and detected leukocyte migration inhibition with concentrations as low as 12.6 ng protein/ml of the crude sonicate of the line 10 tumor and 0.1 pg PPD. Specificity was shown by lack of reactivity in leukocytes of line 10 and/or BCG-sensitized animals with antigen preparations of L2C leukemia cells or normal syngeneic liver. Furthermore, leukocytes from normal control guinea pigs failed to react with any antigen. The results also suggested antigen cross-reactivity between line 10 tumor and BCG. Leukocytes from guinea pigs sensitized to only BCG became LMI reactive to the line 10 sonicate as well as PPD. No reactivity was observed with leukocytes of the animals in simultaneous tests with a sonicate of guinea pig L2C leukemia cells. The results demonstrated the usefulness of this microassay in detection of LMI reactivity with low antigen concentrations and small volumes of whole blood.
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