Antitumor immunity in strain 2 guinea pigs immunized with potassium chloride extracts of L2C tumor cells.

Extracts of L2C tumor cells stimulated in vitro production of macrophage migration inhibitory factor (MIF) in peritoneal exudate cells from guinea pigs immunized with L2C tumor cells. Guinea pigs immunized with extracts of L2C tumor cells that were active in vitro (in the MIF assay) were completely resistant to challenge with viable tumor cells given 2 weeks later. Furthermore, guinea pigs immunized with extracts of L2C tumor cells within 1 hour after challenge with viable L2C tumor cells survived substantially longer than did nonimmunized controls. The immunoprotective and immunotherapeutic effects seen in guinea pigs given injections of viable L2C tumor cells were obtained with extracts of L2C tumor cells but not with extracts of another guinea pig tumor (line 10 hepatoma) or with extracts of normal guinea pig lymphoid cells.

Opposite effects of different strains or batches of same strain of BCG on in vitro generation of syngeneic and allogeneic antitumor cytotoxicity.

Pretreatment of mice with three batches of BCG Phipps strain 10 days before in vitro immunization of their spleen cells with syngeneic or allogeneic tumor cells augmented the levels of antitumor cytotoxicity (compared to the levels exhibited by in vitro immunized spleen cells from normal mice), whereas pretreatment with another batch of BCG Phipps strain or with a batch of BCG Tice strain suppressed antitumor cytotoxicity. The suppressive effects of these BCG vaccines could not be attributed to route of administration, dose of BCG, or percentage of colony-forming units in an inoculum. The effect of the interval between BCG pretreatment and in vitro immunization on the generation of antitumor cytotoxicity was evaluated; one BCG batch was of special interest, inasmuch as augmented cytotoxicity was obtained when the interval was short and suppressed cytotoxicity was obtained when the interval was long.

Role of antitumor immunity in cyclophosphamide-induced rejection of subcutaneous nonpalpable MOPC-315 tumors.

Previously, we had reported that a single i.p. injection of 15 mg cyclophosphamide (CY) per kg cured most mice bearing large MOPC-315 tumors (20 to 25 mm; Day 12 to Day 16 tumors) but rarely cured mice bearing nonpalpable tumors (Day 4 tumors). Also, mice that were not cured if treated with CY, 15 mg/kg, when they had nonpalpable tumors could not be cured if treated again with CY, 15 mg/kg, when they had large tumors (14). Here, we show that CY therapy with 15 mg/kg at early stages of tumor growth did not lead to alteration in the biology of the tumor so as to cause an increased resistance to CY-tumoricidal effects, increased resistance to immune lysis, and/or decreased immunogenicity. Treatment of nonpalpable tumor bearers with CY, 15 mg/kg, prior to in vitro immunization of their spleen cells did not reduce the ability of the spleen cells to generate antitumor cytotoxicity in vitro. However, the level of antitumor cytotoxicity generated was lower than that exhibited by in vitro-immunized spleen cells from mice treated with CY, 15 mg/kg, when they had large tumors. With CY, 15 mg/kg, mice bearing nonpalpable tumors could be cured in two ways: (a) by treating a mouse bearing a nonpalpable tumor in the presence of a contralateral large tumor; (b) by adoptive transfer of immune spleen cells given 1 day post-CY therapy. Both procedures resulted in higher levels of antitumor immunity which was apparently responsible for the cure of the mice in cooperation with CY. Thus, the ineffectiveness of CY therapy with 15 mg/kg at early stages of tumor growth correlated with the presence of relatively low levels of host antitumor immunity.

Cooperation between cyclophosphamide tumoricidal activity and host antitumor immunity in the cure of mice bearing large MOPC-315 tumors.

A single i.p. injection of cyclophosphamide (CY), 15 mg/kg, was shown previously to be curative if administered to BALB/c mice 10 to 16 days post-MOPC-315 tumor inoculation when the tumors reached 20 to 25 mm (large tumors) but not if administered to mice four days post-tumor inoculation when their tumors were nonpalpable. Here we show that the curative effect of CY, 15 mg/kg, for mice bearing large tumors was not due solely to the tumoricidal activity of the drug, because three or four days after therapy, when the CY had been cleared from the circulation, viable proliferative tumor cells were present in the primary s.c. tumor site. During tumor regression, the tumors became heavily infiltrated by mononuclear cells. Following therapy, mice bearing large tumors exhibited an active antitumor response, as illustrated by their ability to reject a tumor challenge with 350-fold the minimum lethal tumor dose given as early as 24 hr posttherapy. That the curative effect of CY, 15 mg/kg, for mice bearing large tumors required the presence of T-cell-dependent antitumor immunity (cellular and/or humoral), was indicated by the fact that tumor regression was abrogated by treatment of the tumor bearers with anti-thymocyte serum. Thus, CY drug tumoricidal activity and host antitumor immunity cooperated in the eradication of large MOPC-315 tumors.

Cytotoxic activity of human pulmonary alveolar macrophages.

The functions of human pulmonary alveolar macrophages (PAMs) have been relatively little studied compared with those of their circulating counterparts, blood monocytes. This study examined the ability of human PAMs to kill primary human tumor cell cultures and control normal fibroblasts in vitro. PAMs were derived by bronchial lavage from patients with lung cancer of various histological types and stages, patients with acute or chronic noncancerous pulmonary disorders, and subjects with a presumed illness who proved to be normal. After extensive washing, the PAMs were cocultured with [3H]proline-labeled tumor cells, principally lung cancers and melanomas, at various effector:target ratios for 60 hr. Cytotoxicity was measured by comparing radioactivity associated with the remaining adherent tumor cells cultured in the presence or absence of PAMs. Twenty-eight of 42 preparations of PAMs from 42 individuals were cytotoxic to one or more short-term primary tumor cultures. All 28 specimens from patients with lung cancer or chronic pulmonary disease were cytotoxic; all of the 14 PAM preparations lacking cytotoxicity were from individuals with acute pulmonary disorders or who were proved free of pulmonary disease. PAMs were cytotoxic even at effector:target ratios of 2.5:1 or 1.25:1. Fibroblasts were unaffected at any ratio. Sarcoidosis patients in remission had noncytotoxic PAMs, whereas the disease in relapse was characterized by cytotoxic PAMs. Serial study of 2 patients confirmed a loss of reactivity during remission. Smoking did not correlate with the presence or absence of spontaneous cytotoxicity and did not influence the degree of cytotoxicity in "reactors." Partially purified alpha-interferon enhanced the killing of cytotoxic PAMs in 10 of 21 instances but did not induce cytotoxicity in 9 tests on nonreactive PAMs. We conclude that human PAMs from patients with lung cancer or chronic pulmonary diseases, including active sarcoidosis, were cytotoxic to several recently explanted tumor cell cultures. PAMs from acute pulmonary dysfunctions and those from patients with inactive sarcoidosis were not spontaneously cytotoxic.

Correlation between cytotoxic and suppressor activities of human pulmonary alveolar macrophages.

We have reported previously that pulmonary alveolar macrophages (PAMs) from individuals with lung cancer and active chronic pulmonary diseases were cytotoxic to tumor cells in vitro, whereas PAMs from normal individuals or patients with acute pulmonary disorders were noncytotoxic. In the present study, we evaluated 20 PAM preparations for both suppressor and cytotoxic functions to determine if PAMs could function as suppressor cells and, if so, whether a correlation between the two functions exists. Cytotoxicity was assessed in a 60-hr cytotoxicity assay against [3H]proline-prelabeled human melanoma target cells. More than 20% cytotoxicity was considered to be significant. Suppressor activity was measured by determining whether admixing PAMs at various ratios with autologous or allogeneic mononuclear cells could suppress concanavalin A-induced blastogenesis by T-lymphocytes. At least 50% suppression was considered to be significant. Of the 20 specimens evaluated, 13 were cytotoxic and 5 of these exhibited suppressor activity. None of the 7 noncytotoxic PAM preparations had suppressor activity. Suppression was nonspecific and not HLA restricted, since autologous and allogeneic mononuclear cells were inhibited to a similar extent. Suppression was probably not due to prostaglandin production by the PAMs since assays were performed under optimal conditions and required extremely high concentrations of prostaglandins. A significant correlation between suppressor and cytotoxic activity was found. Suppression was observed only with PAM specimens that were also highly cytotoxic to tumors, but not all cytotoxic PAM specimens were suppressive. Whether these actions reflect different levels of activation of PAMs or are the properties of different macrophage subsets remains to be clarified.

Staphylococcal Protein A column: correlation of mitogenicity of perfused plasma with clinical response.

Eleven patients with advanced breast cancer and four with astrocytoma were treated with plasma perfused over columns containing staphylococcal Protein A (SPA). Doses of 5 to 20 mg of SPA were bound to collodion charcoal particles, and this treatment resulted in partial remissions in one patient with astrocytoma and in two patients with breast cancer. Remission duration was 6 wk to 6 mo. Resolution of lymphadenopathy and a decrease in carcinoembryonic antigen were noted in an additional two breast cancer patients. Systemic reactions to infused plasma consisted of fever, chills, and rigors. In brain cancer patients, increased intracranial pressure was also noted. A mitogenic substance was generated in plasma of 11 patients after it was perfused over the SPA charcoal matrix. The mitogenic material induced lymphoproliferation comparable to concanavalin A and required the presence of SPA on the collodion charcoal but was not due to leakage of SPA from the column during plasma perfusion. Of considerable significance was that only patients whose column perfused plasma contained this mitogenic activity exhibited systemic reactions, and five of these patients obtained antitumor responses. This striking correlation implies that the mitogenic factor is an active component of SPA therapy. The ability to demonstrate mitogenicity in column perfused plasma might also be useful for selecting patients amenable to SPA therapy. These findings attest to the therapeutic value of this mode of treatment and provide an initial definition of a mediator of SPA antitumor activity.

Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction.

Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.

Staphylococcal protein A immunoadsorptive column induces mitogenicity in perfused plasma.

A Phase I trial of therapy with the staphylococcal protein A immunoadsorptive column was conducted in six patients with advanced breast or brain cancer. Four of the patients reacted strongly to the therapy with high fever, chills, and rigors. The plasma of these four patients after perfusion over the column was strongly mitogenic to normal lymphocytes. This mitogenicity apparently was dependent on the amount of protein A on the matrix; small attached doses caused mitogenicity, while higher doses also induced clinical symptoms. Mitogenicity was not due to protein A leached from the column, as determined by heat-inactivation experiments. From these data it appears likely that the mitogenic plasma component generated by perfusion of the plasma over a protein A matrix is responsible, at least in part, for the immunomodulatory activity of the staphylococcal protein A immunoadsorptive column.

Immunological effects of recombinant interferon-alpha 2 in cancer patients.

Fifteen patients with various types of cancer, resistant to conventional therapy, were entered into a phase I trial of pure interferon-alpha 2 (IFN-alpha 2) produced by recombinant DNA technology. Groups of patients received either 3, 10, 30, or 50 x 10(6) units (U) of IFN-alpha 2 subcutaneously daily for 4 weeks and were closely followed for possible toxicity. At the higher doses, toxicity was encountered, which led to reduction in the frequency of administration. For immunological testing, peripheral blood mononuclear cells were obtained before treatment on day 1, on day 2, and during the 2nd, 3rd, and 7th weeks of the study. The cells were tested for natural killer (NK) activity, antibody-dependent cellular cytotoxicity (ADCC), monocyte-mediated antibody-dependent cellular cytotoxicity (MMADCC), and spontaneous monocyte-mediated cytotoxicity (SMC). ADCC was augmented at all doses in 9 of 15 patients, 6 of whom exhibited elevated levels by day 2. In direct contrast, MMADCC was decreased in 12 of 14 patients 7-20 days after beginning treatment. SMC was increased on day 2 in 1 of 2 patients given 3 x 10(6) U and in 3 of 4 patients given 10 x 10(6) U. SMC in the other patients given these doses was unchanged on day 2, with no decreases noted. In contrast, SMC was decreased in 2 of 4 patients given 30 x 10(6) U. SMC in the other patients given these doses was unchanged on day 2, with no decreases noted. In contrast, SMC was decreased in 2 of 4 patients given 30 x 10(6) U and in 2 of 3 patients given 50 x 10(6) U. NK cell activity was increased in 2 of 3 patients given 3 x 10(6) U, but was either unchanged or decreased in patients given higher doses. The only exception was an increase found in a patient given 50 x 10(6) U, whose renal cell carcinoma responded significantly to treatment. Data on NK and SMC from a phase II study of breast cancer treated daily with 3 x 10(6) U IFN-alpha 2 support our phase I observations. NK-cell activity was increased on day 2 in 3 of 8 patients, and SMC increased in 2 of 8 patients. As in the phase I study, no decreases in these functions occurred. In summary, for those responses that were dose dependent, such as NK and SMC, lower doses of recombinant IFN-alpha 2 (3 x 10(6) and possibly 10 x 10(6) U) may be more effective in increasing these antitumor activities than are higher doses.