Structural evidence for distinct IgG subclass-specific Fc receptors on mouse peritoneal macrophages.

Membrane proteins which selectively bind to the Fc portion of IgG were identified in the Nonidet P-40 extracts of radiolabeled thioglycollate- elicited mouse peritoneal macrophages. Affinity columns of various IgG preparations coupled to Sepharose 4B were used to absorb the Fc-binding proteins. Analysis of the acetic acid or sodium dodecyl sulfate (SDS) eluates from aggregated human IgG or antigen-complexed rabbit IgG columns revealed two Fc(gamma)/-specific proteins with apparent 67,000 and 52,000 mol wt. These proteins were not detected in acid or SDS eluates from F(ab')(2) columns or in eluates from IgG column, over which were passed lysates of Fc receptor-negative cells. With the use of affinity columns that contained aggregated mouse myeloma proteins of different IgG subclasses, we found that the 67,000-dahon protein selectively binds to IgG2a, whereas the 52,000-dalton protein binds to IgG1 and IgG2b. Neither protein was found in SDS eluates from IgG3 columns. Trypsin treatment of the macrophages before detergent lysis removed the 67,000-dalton protein, although it leaves intact the 52,000-dalton protein. These results provide structural confirmation for the existence of separate Fc receptors on mouse macrophages and indicate that the two Fc-binding proteins identified in this study represent all or part of the trypsin- sensitive Fc receptor which binds IgG2a and the trypsin-resistant Fc receptor which binds IgG2b and IgG1.

Effect of chemotherapy and immunotherapy on tumor-specific immunity in melanoma.

The effects of chemotherapy, with nitrosoureas or dimethyl-triazeno-imidazole-carboxamide (DTIC), or immunotherapy with Bacillus Calmette-Guérin (BCG), on cell-mediated immunity (CMI), and serum blocking factor (BF) to melanoma cells were studied in 23 patients. Studies were performed with autologous or allogenic melanoma target cells obtained from recent biopsy, in 16 mm diameter plastic wells. Assays for lymphocyte-mediated cytotoxicity and BF were performed at weekly intervals over the course of 3-4 mo, with some studies extending beyond 3 yr. The specificity of cytotoxicity was good with these methods. Nine patients given nitrosoureas, predominantly methyl-chloroethyl-cyclohexyl-nitrosourea, showed a transient decline in CMI from 42.2 to 14% 3 wk after administration of a single dose of the agent, with a rapid recovery within 1 week. 10 patients given 5-day courses of DTIC at 3-wk intervals showed no decline in CMI after two courses, and 7 of the 10 had no decline even after three courses. Three of the four patients who achieved a remission lost BF previously present: BF reappeared in both patients studied during a subsequent relapse. BCG intradermally or intralesionally elevated CMI within 2 mo after initiation of therapy, but despite continuation of the injections CMI returned to base line in all but two of the nine patients studied. These results indicate that chemotherapy for melanoma with nitrosoureas or DTIC at these schedules is not profoundly immunosuppressive towards tumor-specific immunity, as measured by our procedures. Putative immunotherapy with BCG at these schedules was likewise only transiently stimulatory.

Immunological responses to L-asparaginase.

In a series of 40 patients treated with L-asparaginase for various neoplastic diseases, 6 patients had generalized anaphylactic reactions to L-asparaginase. Each of these reactors had antibodies detectable by passive hemagglutination, but precipitins were detectable in only one of this group of six patients. That patient had received two courses of the enzyme. 1 wk after the anaphylactic reaction, complement-fixing antibodies were present in all the patients that were studied. Specific reagin antibodies (IgE) were demonstrated in one patient by the release of histamine from his leukocytes after incubation in vitro with L-asparaginase. Binding of L-asparaginase to serum antibodies after incubation in vitro was detected by selective precipitation of the complexes with 30% ammonium sulfate or by ultracentrifugation. Total inactivation of the enzyme did not occur even at optimal proportions or at antibody excess. Passive hemagglutinating antibodies to L-asparaginase were present in all patients who had an allergic reaction at least 1 day before the reaction occurred, when that sample was available, and were absent in all patients who did not manifest clinical allergy. Titration of antibodies by passive hemagglutination may thus provide a means of predicting impending anaphylaxis in this system, particularly when coupled with a sudden decrease in circulating levels of L-asparaginase activity.

Combining chemotherapy with biological response modifiers in treatment of cancer.

Combinations of chemotherapeutic agents with various biological response modifiers ("biomodulators") in the treatment of cancer are now being tested. Since most (though not all) chemotherapy is immunosuppressive, the schedules by which the chemotherapeutic agents and the biomodulators are combined are of critical importance. This review summarizes the effects of chemotherapy on immunity and suggests the most logical ways in which these categories of drugs might be combined with biomodulators. Specific examples are also provided of combination therapies that have proved useful or that are of significant potential utility. An understanding of the types of action and the mechanisms of action of specific chemotherapeutic agents and specific biomodulators may help to achieve additive effects (possibly synergism) with these combinations. At the very least, that understanding will avoid the hazard of one modality's negating the effects of the other.

Modulation of cell-mediated alloimmunity by BCG. II. Induction of specific, nonadherent, non-T-killer cells by BCG and alloantigen.

The cellular basis of the augmentation of cell-mediated immunity (CMI) by systemically administered BCG was investigated in C57BL/6 mice. BCG pretreatment potentiated the CMI to alloantigens as measured by a 48-hour microcytotoxicity assay (MCA) against P815Y tumor cells. The effector cell was not a T-cell, as demonstrated by its lack of sensitivity to antithymocyte serum and complement and its failure to kill in a short-term 51Cr release assay. The effector cell also was not a classical macrophage, because it was not depleted by treatment with silica. CMI as measured by the MCA was consistently depleted by magnetic separation after incubation with carbonyl iron. The active cell(s) showed variable adherence properties. The augmentation of alloimmunity was alloantigen-specific. The specificity appeared to be due to the interaction between effector cells without inherent specificity and sensitized T-cells.

Modulation of cell-mediated alloimmunity by BCG. I. Antagonism and potentiation of immunosuppression caused by cytarabine.

The ability of iv administered BCG to antagonize immunosuppression caused by injection of the antimetabolite cytarabine (beta-cytosine arabinoside; ara-C) was investigated in C57BL/6 mice. Treatment with BCG 10 days before alloimmunization with killed L1210 tumor cells decreased spleen T-cell-mediated cytolysis against allogeneic P815Y tumor cells, as measured by a short-term 51Cr release assay, and potentiated immunosuppression due to ara-C. In contrast, spleen cell-mediated immunity (CMI) that was assayed by a 48-hour microcytotoxicity assay (MCA) was augmented by systemic BCG administered before alloimmunization. Pretreatment with BCG resulted in a complete and long-lasting protection against the immunosuppressive effects of ara-C on this CMI as measured by the MCA. Treatment with BCG after cytoreductive therapy resulted in a significant, although transient, reversal of immunosuppression. Depending on the type of response and thus the type of effector cell measured, BCG acts as a moderate immunosuppressive agent or a strong immunopotentiator of CMI.

Stimulation of lymphoid cells by components of BCG.

BCG was fractionated into a delipidated mycobacterial cell fraction (DMC) and lipid by exhaustive chloroform-methanol extraction. The effects of these fractions were tested on mouse spleen cells, nonadherent spleen cells (lymphocytes), thymus cells, and adherent spleen cells (macrophages) in vitro and were compared with effects of the whole bacilli and a methanol-extraction residue (MER). Tritiated thymidine incorporation into spleen cells, purified spleen lymphocytes, and thymus cells was measured as an indicator of activity on these cells; lymphocyte-activating factor (LAF) production was used to measure activation of macrophages. DMC and MER were at least equivalent to, and often exceeded, whole BCG in their stimulation of spleen cells and spleen lymphocytes. DMC was a poor thymic mitogen in contrast to MER, which was as strong as BCG in this regard. Lipid was far less effective a mitogen for all cells tested, and failed to augment the effectiveness of DMC on thymus cells when both were present in the incubation mixture. LAF production was significantly increased by whole BCG (18-fold above controls), whereas each fraction increased production threefold to sixfold. These in vitro results seemed to reflect the known in vivo activity of BCG and its components and suggest further antitumor applications.

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.

Immunological enhancement of leukemia L1210 by Corynebacterium parvum in allogeneic mice.

The effect of Corynebacterium parvum on the immune response of C57BL/6 mice (H-2b) to the allogeneic leukemia L1210 (H-2d) was investigated. Mice were either left untreated or given C. parvum i.v. or i.p. in various dosages. Seven days later they were challenged with 2.5 to 10 X 10(6) live L1210 cells i.p. Control animals almost always rejected the challenge. In contrast, most mice pretreated with either 1.0, 0.5, or 0.25 mg of C. parvum i.v. and 1.0 or 0.5 mg i.p. exhibited enhanced growth of leukemia L1210 as indicated by gross ascites and significantly greater weight gain. This sometimes progressed to the death of the animal, but more often regressed after several days. Spleen cell-mediated cytotoxicity to alloantigens, evaluated in vitro by release of 51Cr from P815Y (H-2d) target cells, was significantly decreased in the mice pretreated with either 1.0 or 0.5 mg of C. parvum i.v. or 0.5 mg of C. parvum i.p. This suppression could not be reversed by reduction of the concentration of macrophages in the spleen cell suspensions. Complement-dependent cytotoxic antibody, measured by release of 51Cr from L1210 cells, was profoundly suppressed in mice pretreated with C. parvum i.v. in dosages ranging from 1.0 to 0.1 mg. These data suggest an immunological basis for the enhanced growth of leukemia L1210 caused by C. parvum at these schedules.

Tumor-reactive human immunoglobulin G monoclonal antibody from a melanoma patient.

To study tumor-associated antigens that are immunogenic to humans, we have generated human monoclonal antibodies by fusing lymph node lymphocytes of a melanoma patient with a mouse myeloma cell line. We examined in detail the reactivity of one IgG antibody, termed 2-139-1. Immunostaining was performed with purified antibody conjugated to biotin. Binding was visualized by the avidin-biotin-peroxidase complex. With cultured cells, 2-139-1 stained 12 of 12 melanomas and 12 of 16 carcinomas. Reactivity was not detectable in seven neural crest tumors, six sarcomas, and 45 lymphomas and leukemias. This spectrum of reactivity was confirmed with sections of human tissues. The human monoclonal antibody 2-139-1 reacted against melanomas and not banal nevi. While the antibody reacted strongly to adenocarcinomas of the colon, prostate, rectum, and pancreas, it did not stain all the carcinomas tested. Furthermore, reactivity was not seen against sarcomas. Interestingly, 2-139-1 did not bind to the majority of the cells in normal tissues, including fetal tissues. The reactivity of 2-139-1 may be representative of the humoral immune response found in the regional lymph nodes of cancer patients. The distribution of this epitope in various tumors was fairly limited and appeared to be associated with malignant transformation.

Analysis and reversal of the inhibition of cytophilic antibody receptors produced by antibody.

Administration of hyperimmune antibody to leukemia L1210 to allogeneic mice inhibited the development of macrophage-mediated immunity to L1210 in those hosts. In contrast to immunized mice, animals pretreated with antibody showed rapid activation of their peritoneal macrophages, followed by their disappearance and the inability of the residual peritoneal monocytic cells to attach L1210 cells even in the presence of proved cytophilic antibody to L1210. The inhibitory activity of the antibody, which resided entirely in its IgG2 fraction, was manifested only when the specific antigen (L1210 cells) was also injected within 2 days. Pretreatment with antibody to a different leukemia, EL4, failed to inhibit the monocytic uptake of L1210, but it did inhibit uptake of EL4 by monocytes if injected with its homologous antigen. Restoration of the functional capacity of macrophages was accomplished by injecting 1 X 10-7 bone marrow cells i.v. into "suppressed" mice, but 1.5 X 10-7 thymocytes failed to correct the defect. Significantly, thymocytes antagonized the restorative capability of bone marrow cells when they were injected concomitantly. These results indicate that specific inhibition of cytophilic antibody receptors on monocytes could be accomplished through a direct mechanism involving activation and exhaustion of macrophages and an indirect mechanism, perhaps mediated through "suppressor" thymus-derived cells. Although enhancement of the growth of leukemia cells did not occur, several parallels exist in mice with enhanced growth of different tumors. This inhibiotry phenomenon may thus represent another instance of "blocking" in tumor immunity, where the target of suppressive antibody-antigen is the macrophage as well as the lymphocyte.

Protection against experimental cerebral metastases of murine melanoma B16 by active immunization.

Melanoma patients often develop brain metastases despite effective systemic immunotherapy against melanoma. We have attempted to establish a mouse model to develop strategies to combat this problem. Immunization of C57BL/6 (H-2b) mice with a combination of the syngeneic G3.12/BM2 melanoma (a B16 subclone) and the allogeneic Cloudman-S91 melanoma was effective in preventing the growth of 10,000 viable, s.c. injected G3.12 cells in 93% of the mice. Irradiated whole tumor cells pretreated with gamma-interferon for 2 days were most effective. A nonspecific adjuvant (DETOX) was injected routinely together with the tumor cells. Active immunization with 2 different doses of irradiated melanoma cells (1 x 10(5) or 2.5 x 10(6) cells/injection x 5 injections) protected against intracerebral challenge with 200 live G3.12 cells in 69% of the mice. This challenge caused the death of all control mice within 30 days. T-cell-mediated, tumor-specific cytotoxicity against G3.12 melanoma was demonstrated in the spleen of immunized mice. Histological observations in the brain, 80 days after tumor challenge, indicated complete eradication of the melanoma, but although CD4+ and CD8+ T-cells and macrophages were present, their number was low. Gliosis was present in both immunized and control animals. Thus, in this murine melanoma model syngeneic mice were protected from death by s.c. and intracerebrally inoculated tumor cells if pretreated with a sufficient number of irradiated syngeneic and allogeneic melanoma cells and an immunological adjuvant. Whether this regimen can treat established tumors of the brain, alone or in combination, is uncertain. Yet its success suggests that the "blood-brain barrier" impeding immunity to tumors may not be absolute.

Comparative analysis of the immunosuppressive properties of two antiviral, iodinated thymidine analogs, 5-iodo-2'-deoxyuridine and 5-iodo-5'-amino-2',5'-dideoxyuridine.

5-Iodo-5'-amino-2',5'-dideoxyuridine (AldUrd), given as five single i.p. injections on Days 0 to 4 after antigenic challenge with sheep erythrocytes, had no demonstrable effect on serum hemagglutinin titers in doses as high as 2000 mg/kg/day. This was the maximum feasible single dose, but no 10% lethal dose was determinable. Similarly, 5-iodo-2'-deoxyuridine (IdUrd), 50 mg/kg/day (10% lethal dose), on Days 0 to 4 did not significantly affect this humoral response. However, with a more sensitive assay, immunocytoadherence, reductions in the number of hemagglutinin-forming cells in the spleen were found at several levels of AldUrd and IdUrd, but the same level of inhibition was attained by a course of AldUrd, 2000 mg/kg, or IdUrd, 50 mg/kg. Spleen cell-mediated immunity against lethally irradiated L1210 was measured by 4-hr 51Cr release and 48-hr growth inhibition assays. Both drugs showed dose-related immunosuppression. With AldUrd, 2000 mg/kg/day, and IdUrd, 100 mg/kg/day, on Days 0 to 4, cytotoxicity was inhibited by 35 to 68% and 73 to 90%, respectively. In comparison, a similar course of 1-beta-D arabinofuranosylcytosine, 40 mg/kg/day, completely abrogated both humoral and cell-mediated immunity. When AldUrd and IdUrd were administered on Days -5 to -1, little effect on either type of immunity was found, while pretreatment with the alkylating agent, cyclophosphamide, abolished all T-cell-mediated killing as measured on Day 7. Thus, AldUrd appears to be a very mild and IdUrd a moderate to strong cell cycle-dependent immunosuppressive.

Phase I study of recombinant beta-interferon given by four-hour infusion.

Twelve patients with advanced malignant disease were entered onto a Phase I study of escalating doses of beta-interferon serine given by 4-h i.v. infusion twice a wk. Three patients each were entered at starting doses of 0.01, 1, 10, and 30 million units (MU)/m2. Doses escalation within individual patients was allowed to a maximum dose of 400 MU/m2. Fever, chills, fatigue, and acral cyanosis were commonly seen and increased in frequency at higher doses. Myalgia, nausea, diarrhea, headache, and confusion were seen at lesser frequencies. Mild leukopenia, paresthesia, infusion site erythema, and hypotension were each seen in one patient. No conventional maximal tolerated dose could be defined, since several patients underwent escalation to the highest allowable dose and seemed to develop tolerance to acute toxicities. However, a maximal starting dose of 10 MU/m2 was identified, such that those begun at this level or below tolerated semiweekly dose escalation, while those begun at 30 MU/m2 could not tolerate continued therapy. Detectable serum interferon levels were noted during treatment at 10 and 30 MU/m2, the levels at which significant toxicity also first appeared. A maximal starting dose of 10 MU/m2, with gradual escalation as tolerance to side effects develops, is suggested if therapy with high-dose beta-interferon serine is given by 4-h infusion.

Novel gene sequences expressed by human melanoma cells identified by molecular subtraction.

Despite the variety of approaches used, only a limited number of tumor-associated antigens have been described for each histological type of tumor. In this report, we present a new strategy involving molecular subtraction to identify novel melanoma-associated gene sequences. Toward this end, 156 complementary DNA clones were isolated with a subtracted melanoma complementary DNA probe (melanoma minus lung carcinoma) after screening 2 x 10(4) independent recombinants of a melanoma expression library by in situ plaque hybridization. These clones were then polymerase chain reaction amplified, screened for duplication, and categorized into 53 discrete genes. By applying poissonian distribution to the numbers of duplicate isolates, we found most of the genes to be rare messages, present at less than 1 copy/200 molecules of mRNA in a typical somatic cell. Messages specific for a type of tissue are usually expressed in this range. The expression of the 53 genes was further studied in human tumor cell lines and normal tissues. Partial sequence data obtained for 20 complementary DNA clones revealed 8 novel human genes. The mRNA transcripts for 5 of the novel genes were identified by Northern blot analysis. Thus, molecular subtraction appears to be applicable for the identification of novel tumor-associated sequences. Some of the potential advantages and limitations of this technology are discussed, including its application to the molecular characterization of immunogenic melanoma-associated antigens.

Active specific immunotherapy for melanoma: phase I trial of allogeneic lysates and a novel adjuvant.

A Phase I trial of active specific immunotherapy for melanoma was performed to measure the toxicity and immunological effects of the therapy. A mixture of mechanical lysates (homogenates) of two melanoma cell lines was injected together with a novel adjuvant, DETOX, into 22 patients. Several types of cell-mediated and humoral immunity to melanoma-associated antigens were measured serially. In the 17 patients with measurable disease, the sizes of lesions were also noted serially. At least six patients per group were injected s.c. with either 100, 200, or 400 antigenic units (approximately 10, 20, and 40 million tumor cell-equivalents) of the lysates mixed with 0.25 ml of DETOX s.c. on weeks 1, 2, 3, 4, and 6. Three patients at each dose level also received 300 mg/m2 of cyclophosphamide i.v. 4 days before the start of immunization. Evidence for successful immunization was obtained in 13 of the 22 patients. An increase in the frequency of peripheral blood cytolytic lymphocyte precursors reacting against melanoma cells occurred in 12 patients, as measured by a limiting dilution assay involving in vitro re-exposure to irradiated melanoma cells for 9-10 days. Eight of the 12 patients had received cyclophosphamide. By cold-target competition assays, these cytolytic lymphocytes appeared to be atypical T-cells, which recognized melanoma-associated antigens on several allogeneic lines without apparent major histocompatibility complex restriction. An increase in antibody titers against melanoma-associated antigens, measured by enzyme immunoassay, was found in five of 22 patients, and a change in delayed hypersensitivity against the melanoma lysate, in three patients. Responses were found at all three dosage levels of lysate, without an obvious dose optimum. No toxicity except minor local soreness was noted. Therefore, no maximum tolerable dose was defined. Five of 17 patients with measurable lesions had a remission of their melanoma, two complete and three partial, with three additional minor responses. A patient whose complete remission lasted 5.5 months, has no evidence of disease 22+ months after entry onto the study, with the aid of surgical resection of small s.c. recurrences on two separate occasions. Sites of regression included s.c. nodules, lymph nodes, and pulmonary nodules, with no responses in liver, adrenal gland, or bone. The patients who had an increase in cytolytic lymphocyte precursors comprised all eight with a clinical remission (five major, three minor). In contrast, none of the seven patients lacking an increase in cytotoxic lymphocytes had a clinical response.(ABSTRACT TRUNCATED AT 250 WORDS)

Human monoclonal antibodies directed against melanoma tumor-associated antigens.

To analyze the humoral immune response to melanoma, human-mouse hybridomas were generated by the fusions of regional lymph node lymphocytes of patients with the mouse myeloma cell line M5. Six stable hybridomas were cloned from six separate lymphocyte parents obtained from three patients. Ascites were obtained from nude mice after i.p. injection with cultured hybridoma cells. The monoclonal antibodies, four immunoglobulin Gs and two pentameric immunoglobulin Ms, were partially purified to remove mouse immunoglobulin and then conjugated to biotin for immunocytochemical and immunohistochemical studies. With the avidin:biotin:peroxidase complex method to detect and amplify binding by the biotin-conjugated human monoclonal antibodies, we found the six antibodies to be reactive against cytoplasmic determinants in five short-term melanoma cultures and formalin-fixed paraffin-embedded melanoma tumors from four patients. The antigenic target of the antibodies identified was not carcinoembryonic antigen. Two antibodies, 2-139-1 and 6-26-3, were studied in more detail. Each stained 25 of 25 specimens of melanomas. Little or no reactivity was detected against fixed sections of normal skin, which included tissues such as epidermis, dermis, monocytes, lymphocytes, and vascular endothelium. More striking was the absence of binding to melanocytes in the basal layer of the skin or to pigmented nevus cells. Both antibodies showed cross-reactivity against other tumors, in particular colonic and prostatic carcinomas. In the normal colon, reactivity was restricted to the surface of the columnar epithelium; no reactivity was detected against normal prostatic epithelium. Reactivity was also not observed against liver and lung. However, the epithelia of the renal tubules, pancreatic ducts, and salivary ducts were all reactive. These human monoclonal antibodies identify cytoplasmic melanoma-associated tumor antigens that appear different from the membrane antigens defined by serological approaches and by most mouse monoclonal antibodies.

A novel melanoma gene (MG50) encoding the interleukin 1 receptor antagonist and six epitopes recognized by human cytolytic T lymphocytes.

We identified a novel 8.1-kb human melanoma gene, MG50, derived from subtractive hybridization with a squamous lung carcinoma cell line, LU-1. 6.8 kb containing an open reading frame were sequenced, and the structure of the encoded 1496 amino acid protein was deduced. With HLA-A2.1-transduced Drosophila cells as antigen-presenting cells, we identified six epitopes restricted by HLA-A2.1 that elicited CTLs in vitro. Reactivity of the CTLs to melanoma cells containing MG50 indicated that the epitopes were displayed naturally. Significant cross-reactivity of CTLs immunized against a melanoma cell line that lacked HLA-A2.1 indicated that at least four of the epitopes were also recognized in a different HLA class I context, most likely HLA-A*6802. By quantitative reverse transcription, MG50 message was found in one of two skin melanoma cell lines, an ocular melanoma cell line, two of four metastatic skin melanomas, two of three mammary carcinomas, one of two colon carcinomas, and an ovarian carcinoma. Of six normal tissues, MG50 was found only in a specimen of normal skin and was absent from a congenital nevus. It is likely that MG50 is the gene for the interleukin 1 receptor antagonist because a reported sequence of cDNA from the latter had a sequence of 528 bases in the 3' region, a long contiguous base sequence, and 176 encoded amino acids identical with those of MG50. MG50 is one of the few melanoma-associated antigens that is not a differentiation antigen or a mutated protein. Because of its nature, it may prove to be important in the pathogenesis of the tumors in which it is found, as well as an immunogen and target for immunotherapy.

Increased lysis of melanoma by in vivo-elicited human lymphokine-activated killer cells after addition of antiganglioside antibodies in vitro.

Thirty-nine melanoma patients were treated with cyclophosphamide (350 mg/m2) followed 3 days later by 5 daily doses of interleukin 2 (3.6 million units/m2 i.v.) weekly for 2 weeks. This cycle was repeated at least twice with a 1-week interval between cycles. Natural killer and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells were measured before treatment and on the last day of each cycle by chromium release assays. Development of LAK activity of greater than 10 lytic units was correlated with a clinical response. There was no correlation between natural killer activity and clinical response. Antibody-dependent cell-mediated cytotoxicity of in vivo-induced LAK cells after the addition of mouse monoclonal antibodies (MAbs) in vitro was measured in 30 cases on the last day of each interleukin 2 cycle. Anti-GD3 MAbs MB3.6, 11C64, 6H4, and R24 increased LAK cell cytotoxicity against GD3-positive GD2 melanoma cells while anti-GD2 MAb 14.18 increased LAK cell cytotoxicity against GD3-negative GD2-positive melanoma cells. MAb 9.2.27 (IgG2a) directed against a chondroitin sulfate proteoglycan and its core protein with a molecular weight of 250,000 (p250) on human melanoma cells did not mediate antibody-dependent cell-mediated cytotoxicity. The effector cells in these antibody-dependent cell-mediated cytotoxicity. The effector cells in these antibody-dependent cell-mediated cytotoxicity reactions were Leu-19 positive. In preincubation experiments the MAbs showed superior binding to the melanoma target cells than to effector cells. Our results show that low dose interleukin 2 preceded by low dose cyclophosphamide effectively induces LAK cells in vivo. The cytotoxicity of these in vivo-activated LAK cells can be augmented in vitro by mouse MAbs against glycolipid antigens on the tumor.

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.