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The in vivo effects of BCG were studied on antibody-dependent cellular cytotoxicity (ADCC) using an assay in which the effectors were spleen cells of BCG-treated mice, and the targets antibody-coated chicken red blood cells (CRBC). In the study of the time course of action, an increased ADCC-activity was observed starting at day 10 with a maximum (four-fold augmentation) at day 14 after BCG treatment. The nature of the effector cell was investigated: in normal mice the activity was found in two cell populations, i.e. macrophages and theta-negative (theta-) nylon-adherent spleen cells. A BCG treatment increased the ADCC activity of macrophages of theta- nylon-adherent spleen cells and induced a population of theta- nylon-non-adherent spleen cells. In old mice, the effects of BCG were compared after a weekly long-term (6 months) treatment and after administration of a single dose. It was concluded that ADCC is higher in old mice than in young mice and can be further increased by treatment with a single dose of BCG in contrast with the long-term treatment which impaired this immune reaction.

Because of the experimental and clinical studies which have been extensively conducted with bacillus Calmette-Guérin (BCG) as a systemic adjuvant in cancer immunotherapy, we have analyzed the main factors and conditions which determine its beneficial action and have underlined some of these (eg, the dose factor which controls the amplification of suppressor cells which is probably responsible for failures and even the possible tumor-enhancing effect of immunotherapy). Knowing those factors and conditions, we have been able to establish a systematic immunopharmacologic study of systemic immunity adjuvants, which has resulted in the discovery of agents whose actions are more rapid than that of BCG on one or a few populations of cells involved in immunity and which, unlike BCG, do not induce suppressor cell amplification. This amplification may explain the difference in the results obtained with this mycobacterium in various clinical immunotherapy trials in which it was applied differently. It is proposed to combine these mono- or pauc-functional adjuvants in order to try to obtain all of the beneficial effects of BCG without the amplification of suppressor cells.

The immunomodulating effects of bacillus Calmette-Guérin (BCG) and levamisole were tested in young adult mice after a single administration and in 12-month-old mice after continuous administration. In young mice, BCG was shown to activate macrophages, to potentiate antibody responses and delayed hypersensitivity reaction, to increase antibody-dependent cell-mediated cytotoxicity, and to induce nonspecific suppressor cells. Levamisole was not able to modify any of these immune responses in young mice. The action of these adjuvants differed markedly when tested in age-immunodepressed mice. BCG was found to be strongly immunosuppressive on antibody formation and induced suppressor cell activity in the spleen. Moreover, the survival of immunodepressed mice continuously treated with BCG was shortened in comparison to untreated aged mice. In contrast, levamisole acted as an immunorestoring agent because it strongly stimulated the antibody response compared to aged controls and did not induce suppressor cell population. The survival of levamisole-treated mice was prolonged when compared to untreated aged mice. When the surviving mice were killed and autopsied at the age of 24 months, the incidence of spontaneous tumors was significantly lower in the group of mice treated by levamisole.

Fresh living Bacillus Calmette-Guérin (BCG), injected i.v. into (C57Bl/6xDBA/2)Fl mice, activated peritoneal macrophages rendering them highly cytotoxic for tumor cells in vitro. This cytotoxic activity was already maximal 14 days after injection of 1 mg of BCG and remained stable when 3 or 5 mg of BCG were given. At the same time spleen cells of the BCG-treated mice showed strongly depressed responses to the T-cell mitogens, PHA and Con A, irrespective of the dose of BCG injected. The inhibitory effect was shown to be mediated by suppressor cells which had characteristics of macrophages since they could be removed by carbonyl iron and magnet treatment and were adherent to plastic. In contrast to it was observed after injection of 1 mg of BCG, these suppressor cells alone did not account for the depression of T-cell responses induced by higher doses of BCG. Nylon-nonadherent cell populations obtained from spleen cells treated with 3 or 5 mg BCG partially retained the inhibitory activity suggesting that suppressor T cells were also induced after injection of high doses of BCG.

A combined treatment modality imcorporating surgery plus BCG immunotherapy was administered to (C57Bl/6 X DBA/2)F1 mice grafted with EAkR lymphosarcoma. A single preoperative local or postoperative systemic BCG administration cured 20-30% of the animals, but did not prolong the survival time of these groups. Repeated s.c. injections of BCG resulted in a significant increase in survival time compared to the group submitted to surgery alone. In contrast, multiple i.v. injections of BCG before and after surgery were no more effective than surgery alone and were less effective than a single postsurgical i.v. injection in producing cures. We have concluded that for local BCG therapy, multiple injections before and after surgery are more effective than a single injection. However, for systemic therapy, multiple injections are less effective than a single injection applied postoperatively.

A combined treatment modality incorporating surgery plus BCG immunotherapy was administered to (DBA/2 X C57B/6) F1 mice grafted with EAkR lymphosarcoma. 30% of mice survived free of disease when BCG was injected intravenously two days after operation consisting of tumorectomy plus excision of the regional lymph node performed on the 8th day after tumor inoculation. Similar results were obtained when BCG was injected subcutaneously 6 days before surgery. Pre-operative intravenous and post-operative subcutanous BCG administration failed to cure the animals just as BCG or surgery alone.