James T. Hunter

Mechanisms of immunological eradication of a syngeneic guinea pig tumor. II. Effect of methotrexate treatment and T cell depletion of the recipient on adoptive immunity.

The influence of methotrexate on the development of immunity to the line 10 hepatoma was studied in guinea pigs. Chronic methotrexate treatment had no apparent effect on the ability of immune guinea pigs to suppress the growth of inoculated tumor cells. In contrast, the same methotrexate regimen inhibited the development of tumor immunity if started before the 8th day after immunization with a vaccine containing viable line 10 cells admixed with Bacillus Calmette-Guérin (BCG) cell walls. Thus, methotrexate selectively inhibited the afferent limb of the immune response. In adoptive transfer experiments, methotrexate-treated recipient guinea pigs were capable of being passively sensitized with immune spleen cells, indicating that the primary cell-mediated immune response of the recipient was not required for adoptive immunity. The contribution of recipient T cells in adoptive immunity was further investigated in guinea pigs depleted of T cells by thymectomy, irradiation, and bone marrow reconstitution. Despite demonstrable deficiency in T lymphocyte reactions, “B” animals were fully capable of rejecting tumors after transfer of immune cells. These results suggest that the expression of adoptive immunity was independent of recipient T cell participation. In addition, sublethal irradiation of immune spleen cells prior to adoptive transfer abolished their efficacy. Proliferation of transferred immune cells in the recipient may be essential for expression of adoptive immunity.

Eradication of microscopic hepatic metastases by active specific immunization

SummaryStrain-2 guinea pigs, each with microscopic deposits of line 10 hepatocarcinoma in the liver, were treated by ID immunization with a mixture of irradiated tumor cells and an oil-in-water emulsion containing cell walls of Mycobacterium bovis strain Bacillus Calmette-Guérin (BCG CWE). Injection of line 10 hepatoma cells into the hepatic portal vein led to the development of tumor foci in the liver, metastasis in the hepatic lymph node, malignant ascites, and death. Active immunization using irradiated line 10 cells and BCG CWE was effective therapy when administered 1, 7, and 14 days after intraportal injection of line 10 cells. Effective immunization required both irradiated line 10 tumor cells and the BCG cell wall emulsion. Immunization with BCG CWE admixed with irradiated line 1 tumor cells, a hepatoma antigenically distinct from line 10, did not prevent outgrowth of line 10 deposits in the liver. Animals rendered free of disease could reject a challenge of line 10 tumor cells but not of line 1 tumor cells.

Pulmonary metastases in guinea pigs as a consequence of dermal implantation of line-10 tumor cells

SummaryMetastases to the lungs of guinea pigs occurred at high frequency as a consequence of intradermal implantation of tumor cells derived from the syngeneic hepatocellular carcinoma line-10. Surgery had a major influence on the proportion of guinea pigs found to have pulmonary metastases at necropsy. Without surgery all guinea pigs died with extensive lymph node metastases; macroscopic pulmonary metastases were present in a minority of the animals. Animals treated by excision of dermal tumors survived longer than untreated animals, and macroscopic pulmonary metastases were present in the majority of the animals. Animals treated by excision of dermal tumor and regional lymph nodes were rendered tumor-free. The data suggest that lymph node metastases were the most likely source of the tumor cells that spread to the lungs in animals from whom the dermal tumor transplant had been removed.

Safety and efficacy of living BCG or BCG cell walls (CW) in the treatment of a guinea pig hepatoma

Guinea pigs with established intradermal tumors and microscopic lymph node metastases were treated by intralesional injection of graded doses of living BCG or BCG CW. The lowest dose of living BCG used produced a significant cure rate and no grossly evident toxicity. An intermediate and the highest dose of living BCG used cured some animals but others lost weight and a significant number died as a result of the treatment. Histologic examination of animals with significant weight loss showed fatty degeneration of the liver, granulomatous hepatitis and histiocytic infiltration of the spleen. None of the doses of BCG CW used was toxic and they were at least as effective as living BCG in intralesional treatment. In some experiments in which treatment was delayed it was found that the extent of disease required to render treatment in‐effective was about the same for living BCG and for BCG CW. Cancer 43:484–491, 1979.

Efficacy of mycobacterial components in the immunotherapy of mice with pulmonary tumor deposits

SummaryThe effectiveness of each of two mycobacterial components and a synthetic analog of one of them in the eradication of pulmonary deposits of intravenously injected syngeneic fibrosarcoma 1023 in C3H mice was studied. BCG cell walls (BCG CW), trehalose 6,6′-dimycolate (TDM) or 6,6′-di-0-2-tetradecyl, 3-hydroxyoctadecanoyl-α,α-trehalose (C76), a synthetic analog of TDM, was administered in emulsified form by three different routes: intraperitoneal, intradermal, or intravenous, 24 h after intravenous injection of 1023 tumor cells. The most effective form of therapy was TDM given by the intraperitoneal route; about 50% of treated animals were cured. Higher doses of BCG CW or C76 also led to a significant number of cures. Each agent caused a significant prolongation of survival time of the treated mice at two or more of the dosages tested; however, their routes of optimal activity varied.

Immunotherapy of a guinea pig hepatoma with vaccines containing mycobacterial fractions

SummaryBCG cell walls (CW) were compared to trehalose-6,6′-dimycolate (TDM) in admixture with endotoxin (ET) for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basis, CW were at least as active as ET-TDM. Toxicity of ET and of ET-TDM were greater than that of CW as judged by weight loss after intravenous administration to normal guinea pigs.

Active specific immunotherapy of guinea pigs with visceral tumor implants

SummaryGuinea pigs, each with established, 7-day-old, syngeneic visceral micrometastases of line 10 tumor implanted intravenously, were immunized by intradermal inoculations into several sites of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG or Mycobacterium phlei bacilli. This treatment led to survival of 72 of 80 treated animals (90%). Therapeutic effectiveness depended on the dose of mycobacteria and on that of irradiated tumor cells. Animals treated by intradermal injection of mycobacteria attached to oil droplets alone or with irradiated tumor cells alone, all died with multiple foci of pulmonary tumor.

Treatment of mice with lung metastasis from a dermally implanted fibrosarcoma: comparison of intratumoral trehalose-6,6'-dimycolate (cord factor) and surgery.

Abstract Intralesional administration of emulsified trehalose- 6,6′ -dimycolate was compared to surgery in the treatment of mice with growing intradermal implants of a syngeneic fibrosarcoma. Treatments were given about one month after tumor implantation when a majority of animals so treated had metastatic foci of tumor in the lungs. Mice treated with trehalose- 6,6′ -dimycolate or by surgery survived significantly longer than untreated mice. Some treated mice were alive and tumor-free six months after tumor inoculation. The differences in cure rates and prolongation of survival between the trehalose- 6,6′ -dimycolate treated group and the surgically treated group were not statistically significant. Several mice died soon after surgery; no deaths could be attributed to the acute effects of immunotherapy.

A specific vaccine effective against stage I and stage II malignant disease in guinea pigs effect of variations in preparations and storage

SummaryGuinea pigs, each with an established, syngeneic dermal line 10 tumor and lymph node metastases, were immunized by intradermal injection of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG. Immunization eradicated 7- or 10-day-old dermal tumors (about 10 or 12 mm in diameter, respectively) and prevented growth of microscopic lymph node metastases. Fourteen-day-old dermal tumors (about 15 mm in diameter) were not rejected by immunization.Guinea pigs with stage II disease (21-day-old dermal tumors and palpable metastases in the first draining lymph node) were treated by excision of the dermal tumor and the first draining lymph node, and by specific immunization. This treatment eliminated tumor cells remaining in the second draining lymph nodes. The surgical treatment alone was not curative, palpable metastases in the second draining lymph nodes progressed and the animals died (some with visible lung metastases).Emulsions containing killed BCG were good adjuvants even after prolonged storage at 4° C, but lost most of their adjuvant activity after autoclaving or freezing.