Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/ fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p<0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.Abbreviations RLN regional lymph node(s) - OK-432/fbg OK-432/fibrinogen solution - PHA phytohemagglutinin - NK natural killer - LAK lymphocyte activated killer     

Augumentation of splenic antitumor immunity by local immunotherapy in gastric cancer patients

We previously reported that the antitumor effect of OK-432, a streptococcal preparation, was markedly augmented when this agent was injected into tumors together with fibrinogen. In order to elucidate the effect of this treatment on the spleen, we assessed splenic function in gastric cancer patients receiving preoperative local immunotherapy with OK-432 and fibrinogen. Immunohistochemical studies of the spleen at 7 days after intratumoral injection therapy revealed numerous macrophages phagocytizing OK-432 in the splenic sinuses. Phenotypic analysis of splenocytes by flow cytometry revealed an increase in the CD4/CD8 ratio and in the expression of HLA-DR, CD25, and Leu M3 by splenic T cells of the patients treated with OK-432 plus fibrinogen when compared to patients treated with OK-432 alone or untreated patients. Splenic T cells from patients treated with OK-432 plus fibrinogen showed significantly higher cytotoxicity against Daudi and K562 cells than T cells from control patients (p<0.05), and culture of these splenic T cells with recombinant IL-2 induced the expansion of lymphokine-activated killer cells. These results demonstrate that local immunotherapy with a mixture of OK-432 and fibrinogen effectively augumented splenic antitumor immunity in gastric cancer patients.     

Combination immunotherapy with OK-432, recombinant granulocyte-colony-stimulating factor and recombinant interleukin-2 for human hepatocellular carcinoma

 The antitumor effects of immunotherapy using streptococcal preparations (OK-432), recombinant granulocyte-colony-stimulating factor (rG-CSF) and recombinant interleukin-2 (rIL-2) were examined for human hepatocellular carcinoma (HCC). Following subcutaneous injection of OK-432 (2 KE) and rG-CSF (50 – 60 μg), low-dose intratumoral administration of OK-432 (3 – 12 KE) was performed. Thereafter, 2×10⁵ JRU of rIL-2 was subcutaneously injected. This therapeutic regimen was repeated twice. Serum α-fetoprotein levels were markedly decreased in three of seven patients with HCC by this treatment. Post-therapeutic histological examination revealed that trabecular cords or pseudoglandular arrangements of tumor cells were completely disordered in all cases and that extensive infiltration of lymphocytes into the tumor stroma was present in five cases. The number of CD4- and CD57-positive cells among tumor-infiltrating lymphocytes after immunotherapy was significantly higher than that in patients without immunotherapy (P <0.01). These findings suggest that even a small intratumoral injection of OK-432 can induce extensive infiltration of helper/inducer and natural killer cells into the tumor stroma when combined with subcutaneous injection of OK-432, rG-CSF and rIL-2 and that these cells might play important roles in tumor cytotoxicity. Received: 30 December 1994 / Accepted: 6 November 1995     

Streptococcal preparation OK-432 promotes fusion efficiency and enhances induction of antigen-specific CTL by fusions of dendritic cells and colorectal cancer cells

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4(+) and CD8(+) T cells capable of high levels of IFN-gamma production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.     

Establishment of cytotoxic CD4+ T cell clones from cancer patients treated by local immunotherapy

We have previously reported that the antitumor effect of OK-432, aStreptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (Cancer, 69: 636–642, 1992). In order to elucidate the mechanism of the antitumor effects, we established T cell clones from regional lymph nodes of colorectal cancer patients who received this local immunotherapy. By culture of lymph node lymphocytes, in the presence of IL-2 and OK-432, 4 clones of T cells were established from 4 patients treated by local immunotherapy. These clones had a helper T cell phenotype (CD3⁺, CD4⁺, CD8^–, CD56^–, WT31⁺) and were successfully maintained for several months. The cells strongly expressed CD25 when stimulated with OK-432 and exhibited a high level of cytotoxic activity in part explained by the increased expression of ICAM-1 and LFA-1, and the release of TNF. These results suggest that the CD4⁺ T cells play a role in the antitumor mechanism of local immunotherapy.     

Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.     

Effector molecules from antitumor macrophages induced with OK-432 and cyclophosphamide

Summary The present study was designed to determine whether antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on their ability to produce a soluble factor, that is,l-arginine-dependent nitric oxide as measured by nitrite concentration. Nitrite production by peritoneal macrophages from NIH Swiss mice pretreated with OK-432 (125 KE/kg) i.p. twice at 1-week intervals and with cyclophosphamide (200 mg/kg) i.p. 2 days before the second OK-432 treatment, increased with time for 24 h, and proportionally depended on macrophage numbers. Nitrite production was inhibited by actinomycin D and puromycin but not by mitomycin C.N ^G-Monomethyl-l-arginine, a specific competitive inhibitor ofl-arginine-dependent nitric oxide synthesis, also inhibited production. There was a close correlation between nitrite production and antitumor activity in macrophages from mice pretreated with either OK-432 and cyclophosphamide, OK-432, or thioglycolate broth. OK-432 increased both nitrite production and antitumor activities when added to the macrophage from mice pretreated with OK-432 but not with thioglycolate broth. Both activities of macrophages from mice pretreated with OK-432 and cyclophosphamide were enhanced with increasing concentrations ofl-arginine (0.125–1 mM) in the culture medium.d-Arginine, however, did not substitute forl-arginine. Neither activity was affected by contact between the macrophage and the EL4 cell. The macrophage showed antitumor activity through a membrane filter though the activity was greatly reduced. This antitumor activity of macrophages through a membrane was also inhibited byN ^G-Monomethyl-l-arginine, and increased by OK-432. However, conditioned media, obtained by culturing macrophages induced with OK-432 and cyclophosphamide, inhibited growth of EL4 cells. This activity was carried out by dialysable and non-dialysable factors. One of the dialysable factors was nitrite, an oxidized product of nitric oxide. The antitumor activity of non-dialysable factors was heat-stable and production of factors was increased byN ^G-Monomethyl-l-arginine and OK-432. Also, non-dialysable factors increased both antitumor and nitrite production activities of OK-432-elicited macrophages, when incubated with factors. Such activity of factors was also heat-stable. The production of factors increased with incubation time of macrophages, and was not inhibited byN ^G-Monomethyl-l-arginine. These results indicate that in vitro antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent onl-arginine-dependent nitric oxide, and that macrophageassociated soluble factors other than nitric oxide were also needed to inhibit fully tumor growth in vitro.     

Stimulation of arachidonic acid metabolism by a streptococcal preparation (OK-432) in rat peritoneal macrophages.

A streptococcal preparation OK-432 is reported to be an immunopotentiator and a potent antitumor agent. In order to elucidate the mechanism of biologic action, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages were investigated. Prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were found to be stimulated by OK-432 in a concentration-dependent manner (5 to 80 micrograms/ml). Heat-treatment of OK-432 further stimulated its effects. These stimulative effects on arachidonic acid metabolism by OK-432 were not observed in MDCK cells that have no phagocytotic activity. Furthermore, cytochalasin B treatment completely suppressed the stimulative effects induced by OK-432 in macrophages. These results strongly indicate that the stimulative effects by OK-432 on arachidonic acid metabolism are dependent on phagocytosis of OK-432 particles. Significance of stimulation of arachidonic acid metabolism in macrophages by OK-432 for its biological effects is discussed.     

The concurrent administration of OK432 augments the antitumor vaccination effect with tumor cells by sustaining locally infiltrating natural killer cells

 The effect of a local injection with a streptococcal preparation OK432 on the antitumor vaccination with tumor cells was investigated. Natural killer (NK) cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells after an intraperitoneal (i.p.) injection with syngeneic B16 melanoma cells. Furthermore, a concurrent i.p. injection with OK432 efficiently sustained the locally infiltrating NK cells. The OK432 treatment also sustained the augmented NK and lymphokine-activated killer activities in the peritoneal exudate cells. This treatment also increased the ability of the locally infiltrating NK cells to produce interferon γ in response to the tumor cells. In addition, the concurrent i.p. injection with OK432 in combination with the tumor cells enhanced the capacity of the spleen cells to turn into anti-(B16 melanoma) cytotoxic T lymphocytes after in vitro restimulation. This augmenting effect of OK432 was dependent on NK cells. Moreover, the concurrent injection with OK432 at the time of antitumor vaccination significantly enhanced the protective immunity against B16 melanoma at the rechallenge. Taken together, these findings indicate that a concurrent local injection with OK432 in combination with tumor cells efficiently augments the antitumor vaccination effect, in part, by sustaining the locally infiltrating activated NK cells. Received: 6 March 1996 / Accepted: 30 May 1996     

Adjuvant immunotherapy with intrapleuralStreptococcus pyogenes (OK-432) in lung cancer patients after resection

A prospective randomized study to evaluate the effect of adjuvant intrapleural OK-432 immunotherapy after resection of lung tumor was conducted in 93 patients with primary lung cancer. Among them, 46 patients had had intrapleural OK-432 injection, 47 had not. In the meantime, serial measurements of serum immunosuppressive acidic protein, of serum interleukin-2 receptor and of the sub-population of the peripheral blood cells and lymphocytes were performed in all these patients. Patient characteristics in these two groups (sex, age, histological type, pathological stage, type of operation, and performance status) were compatible. The results showed that adjuvant intrapleural OK-432 injection after resection had no beneficial effect on a patient's survival time. Patients who received intrapleural OK-432, had an increase in blood leukocytes, granulocytes and monocytes and serum immunosuppressive acidic protein level. But the cell numbers of total T cells, suppressor/cytoxic cells, helper/inducer cells and natural killer cells of peripheral blood were decreased in the OK-432 positive group. Over half of the patients had transient 1- or 2-day febrile reactions after intrapleural OK-432 injection. It was concluded that neither clinical observation nor immunological monitoring of peripheral blood could demonstrate a beneficial effect from intrapleural OK-432 immunotherapy after complete resection of the tumor.     

Enhancement of rat hepatic macrophages by treatment with interleukin-2 and streptococcal preparation OK432, with reference to antitumor activity,soluble factor production and Ia expression

Summary The effect of biological response modifiers, such as interleukin-2 (IL-2) and streptococcal preparation OK432, on the functions of hepatic macrophages was investigated. The macrophages, even with no exogenous stimulation, produced superoxide anion (O ₂ ^- ) and tumor necrosis factor (TNF), displayed cytotoxicity against K562 cells and cytostasis against P815 cells and expressed immune-region-associated antigen (Ia). IL-2 administered in vitro or in vivo enhanced O ₂ ^- production by hepatic macrophages and the intravenous injection of OK432 also enhanced O ₂ ^- production. Furthermore, IL-2 added to the culture medium of hepatic macrophages isolated from OK432-injected rats augmented O ₂ ^- production even more. The TNF production and Ia expression of the macrophages were also increased by the intravenous injection of OK432. As with O ₂ ^- production, the cytotoxicity of the cells was enhanced by OK432 injection or by IL-2 added to the culture medium and the combination of OK432 and IL-2 augmented their cytotoxicity even more. Thus, the present study suggested that IL-2 and OK432 induce the augmentation of the antitumor activity of hepatic macrophages, partly as a result of the increase in production of O ₂ ^- and TNF and Ia expression.     

The neutrophil as an information transmitter in tumor inhibition by a streptococcal biological response modifier,OK-432

 Effective treatment of a rat transplanted ascites tumor by i. p. injection of a streptococcal biological response modifier, OK-432, was abrogated by selective in vivo depletion of neutrophils by a monoclonal antibody, RP-3. The mechanisms by which neutrophils participate in the therapeutic action of OK-432 were studied with Winn’s assay using peritoneal exudate cells periodically obtained from rats i. p. injected with this biological response modifier. Intraperitoneal resident macrophages were first activated with OK-432, and within 3 h, tumor-inhibitory activity had moved to the early exuded neutrophils. However, 6 h after injection, exuded macrophages were the only cells involved in tumor inhibition. Considered together with other findings, it is likely that, in this system, neutrophils may transmit information from resident macrophages to exuded inflammatory macrophages in a series of responses induced by i. p. injection of OK-432. Received: 29 April 1996 / Accepted: 27 July 1996     

The augmentative effect of a protein-bound polysaccharide (PSK) on tumoricidal activity of the soluble factor produced by a streptococcal preparation (OK-432)-activated macrophages

The enhancement of antitumor activities of the tumoricidal soluble factor (SF) from a streptococcal preparation (OK-432)-activated macrophages by the pretreatment with a protein-bound polysaccharide (PSK) was investigated in tumor-bearing mice.Two-step stimulations with OK-432 atin vivo priming andin vitro eliciting were required for the production of the tumoricidal SF by macrophages, and the tumoricidal activity of the SF apparently correlated with the uptake of OK-432 by macrophages at priming phase.Tumoricidal activity of the SF from OK-432-activated macrophages in proteose-peptone (P-P)-pretreated mice significantly decreased with the development of the tumor, whereas in PSK-pretreated mice did not. Pretreatment of tumor-bearing mice with PSK saved a decrease in the macrophages carrying Ia^k or asialo GM1 antigens and an increase in wheat germ agglutinin (WGA) receptors. Furthermore, the uptake of OK-432 by macrophages at priming phase was enhanced. The tumoricidal activity of the SF from OK-432-activated macrophages was augmented.Thus, PSK may restore the depressed functions of macrophages, and the combination therapy with PSK and OK-432 may be effective to enhance the production of tumoricidal SF in tumor-bearing mice.     

Augmentation of therapeutic effect of adoptive immunotherapy through a synergy between transferred killer cells and host's fresh lymphocytes]

Among several approaches to augment the therapeutic effect of adoptive immunotherapy, we focused the antitumor synergy between transferred killer cells and host's fresh lymphocytes. Immunotherapy models using murine tumors or clinical experiments revealed that preadministration of immunostimulator such as OK-432, followed by chemotherapeutic agents such as cyclophosphamide, can induce host's non-cytotoxic fresh lymphocytes that act synergistically with cultured killer cells against autologous tumor cells. Immuno-chemo-lymphocytotherapy (a sequential treatment with OK-432, chemotherapy and adoptive immunotherapy) is useful to treat the patients with advanced cancer even if the number of transferred lymphocytes is limited.     

Search for immunobiological parameters predictive of clinical effects of OK-432 in patients with malignant ascites

Although OK-432, a potent BRM, has been known to induce the remarkable improvement of clinical conditions in cancer patients through its strong effects on their immune capabilities, no specific immune parameters have been identified to best predict the clinical outcome after the OK-432 treatment. In an attempt to identify early parameters indicative of the clinical effects, we have administered 0.1 mg of OK-432 intraperitoneally to a total of 12 patients with malignant ascites and examined peritoneal fluid and peripheral blood obtained on 4 days before, 1, 3, and 7 days after the OK-432 injection using various immunobiological assays. Four weeks later, clinical improvements were evaluated by the disappearance of malignant cells from and/or substantial decrease in ascites. Four patients (responders) showed the improvements while 8 patients (nonresponders) showed no clinical evidence for improvement. In a few parameters among the many examined, significantly different patterns of changes were noted between responders and nonresponders. Thus, in nonresponder patients MØ and T cell population returned to an initial low level after early increases (on days 1 and/or 3), while they remained increased day 1 through 7 in responders. In responder patients, the cytotoxicity of peritoneal mononuclear cells against K562 and Daudi cells were augmented on day 7, but not in nonresponder patients. Thein vitro stimulation of the mononuclear cells with OK-432 enhanced the cytotoxic activity and induced the interferon (IFN) production in the responders but not in nonresponders. These parameters will be useful for the early prediction of the expected clinical effects of OK-432.     

Purification and characterization of a lectin-like molecule specific for galactose/N-acetyl-galactosamine from tumoricidal macrophages

A lectin-like molecule (macrophage lectin) was purified from murine peritoneal exudate macrophages which had been induced with an antitumor streptococcal preparation, OK-432. The purified macrophage lectin from both 3H-labeled and unlabeled macrophages after rechromatography on a beta-D-galactose-Bio-Gel P-100 column gave a broad single band corresponding to 45-60 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The broadness of this band was due to high N-glycosylation of the lectin, because the lectin gave a compact band corresponding to 35 kDa on SDS-PAGE after deglycosylation. The lectin required Ca2+ for binding and showed an optimum pH of around 6. The sugar specificity of the lectin was examined by means of an inhibition assay using simple sugars and neoglycoproteins. The lectin was found to be specific for D-galactose/N-acetyl-D-galactosamine, and not inhibited with D-mannose or N-acetyl-D-glucosamine at all. The lectin was detected on the surface of OK-432-elicited and thioglycolate-elicited macrophages, but it was not detected on resident macrophages. Moreover, the binding of tumor cells to macrophages was inhibited by the addition of the purified lectin to the binding mixture. These results suggest that this lectin is expressed on the surface of activated macrophages, and that it participates in the interaction between tumoricidal macrophages and tumor cells.     

Studies of the properties of a streptococcal preparation, OK-432 (NSC-B116209), as an immunopotentiator

Summary The present study was designed to examine the mechanism by which OK-432 triggers the cytotoxic activity of peritoneal exudate cells (PEC). When OK-432 was incubated with freshly harvested mouse serum, the formation of complexes of OK-432 with the third component of complement (C3) was demonstrated by using ¹³¹I-labeled mouse C3. The formation of C3-OK-432 complexes was totally abolished by a chelating compound, EDTA, which had been shown to inhibit the OK-432 induced activation of the alternative complement pathway. The C3-OK-432 complexes thus obtained bound to the resident PEC, which were subsequently shown to be activated. These activated PEC had augmented cytostatic activity against MM2 cells, a mouse mammary carcinoma.Further, the PEC from mice which had received an IP injection of OK-432 4–5 days previously were cytostatic against MM2 cells and also inhibited the growth of MM2 cells in culture. In contrast, resident PEC stimulated rather than inhibited the ³H-thymidine uptake by MM2 cells and the growth of MM2 cells. The mechanism of PEC (presumably macrophages) activation by OK-432 is discussed.     

Recruitment of T lymphocytes and induction of tumor necrosis factor in thyroid cancer by a local immunotherapy

Summary To elucidate the mechanism of action for intratumoral injection of immunopotentiators, infiltrating mononuclear cells and tumor necrosis factor (TNF) were assayed by immunostaining tissue samples of differentiated thyroid cancer resected with or without presurgical local application of OK-432, a streptococcal preparation. Frozen sections of resected specimens were stained with monoclonal antibodies using either a conventional or a modified immunoperoxidase method. The tumors injected with OK-432 showed increased T lymphocyte infiltration and HLA-DR expression on cancer cells as compared to the non-injected controls. Among these T cells, the CD4⁺ subset was more numerous than the CD8⁺ population. In four out of the seven cases constituting the injected group, numerous TNF-positive cells were seen in clusters or lines as well as scattered, while none of the seven cases in the control group was associated with a considerable amount of these cells. In their morphology and distribution pattern, these TNF-positive cells appeared to be of macrophage lineage. Thus local injection of OK-432 in thyroid cancer was shown to recruit T lymphocytes of predominantly the CD4⁺ subset and to induce in situ production of TNF, a known potent tumoricidal cytokine. The present data warrant further studies in this direction besides wider clinical intratumoral application of the reagent.     

Locoregional immunotherapy of head and neck cancers utilizing allogeneic spleen cells — a report of 2 cases

Frozen-stored human spleen cells (SC) cultured with streptococcus preparation OK-432 acquired direct cytotoxicity to autologous as well as allogeneic tumor cells. The activated cells started to produce cytocidal cytokine TGIF, which is distinct from previously known cytokines.We examined the possibility of allogeneic adoptive immunotherapy (AIT) using these OK-432-stimulated spleen cells (OK-SC) in two cancer patients. Rapid necrosis of cancer tissue and remarkable decreases of tumor markers in tumor effusion were observed. There were no severe side effects.     

Cyclophosphamide modifies the induction kinetics but not cell types and cytotoxic mechanisms of antitumor cells elicited with OK-432 plus attenuated tumor cells

Summary The present study was designed to examine whether the antitumor cells induced by treatment with mitomycin-C-treated EL4 cells (EL4_MMC) plus OK-432 plus cyclophosphamide differed from those induced by treatment with EL4_MMC plus OK-432 in terms of their cell types and antitumor mechanisms. Antitumor activity of peritoneal exudate cells (PEC) from mice receiving either treatment was nonspecific, and inhibition of their target cell growth increased for up to 24 h. Macrophage toxin, silica and trypan blue abrogated the activity in vitro and in vivo, respectively. The activity of the PEC was inhibited with inhibitors of the arachidonic acid cascade, such as dexamethasone, 4-bromophenacyl bromide and nordihydroguaiaretic acid but not esculetin, ibuprofen, indomethacin and BW755C.N ^G-monomethyl-l-arginine, a specific competitive inhibitor of thel-arginine-dependent nitric oxide synthesis, also inhibited the activity. These results and morphological observations indicated that antitumor cells in the PEC from mice receiving either treatment were macrophages, and that their activity was closely related to the arachidonic acid cascade and to nitric oxide. Antitumor activity of the PEC spontaneously decayed in vitro and this decay was inhibited by the addition of OK-432 or lipopolysaccharide. On the other hand, cyclophosphamide sustained the appearance of antitumor cells in mice treated with EL4_MMC plus OK-432. Therefore, cyclophosphamide treatment did not modify cell types and cytotoxic mechanisms of antitumor cells elicited with EL4_MMC plus OK-432, but did modify the induction kinetics of such antitumor macrophages.