Immunological characteristics of leukemia and lymphoma in allogeneic cell immunity: growth of syngeneic tumors in rats immunized with allogeneic cells

The growth of transplanted tumors was strongly inhibited in syngeneic Wistar King Aptekman (WKA) rats immunized with allogeneic tumor cells from Donryu rats. This phenomenon of non-specific immunity against tumors is referred to as "allogeneic cell immunity". However, an exception to the "allogeneic cell immunity" was observed in leukemias and lymphomas. Four transplanted leukemia or lymphoma lines were not inhibited in syngeneic rats immunized with allogenic tumor cells. Furthermore, immunization with allogeneic leukemic cells had only a relatively weak inhibitory effect upon a syngeneic fibrosarcoma and no inhibitory effect upon leukemias. In WKA rats immunized with allogeneic lymphoid cells from Donryu rats, the growth of fibrosarcoma, but not of lymphoma, was inhibited. Using transplantation experiments, both fibrosarcoma and lymphoma were defined as antigenic tumors in WKA rats. Transplantation of mixtures of syngeneic tumor cells and allogeneic tumor cells in WKA rats confirmed the above findings. These results revealed that leukemia and lymphoma differ from non-leukemic tumors in regard to "allogeneic cell immunity".     

Establishment and characterization of a continuous in vitro line from a rat choriocarcinoma

A continuous in vitro cell line of rat choriocarcinoma has been established. It is composed of pure trophoblast cells which multiply and differentiate. The morphology of the cells is very similar to normal rat cytotrophoblasts and giant cells. The cultured cells contain cytokeratin, alkaline phosphatase and express the receptors for Bandeira simplificifolia Agglutinin-I (BSA-I). They are hormonally active as demonstrated by the presence of lactogen and progesterone in the supernatant of the culture. The injected cells develop into choriocarcinoma in syngeneic as well as allogeneic rats. The morphological, biological and immunohistochemical features of these tumors are identical to those described in the transplantable neoplasm from which the in vitro line was established. The presence of Y chromosome in cultured cells proves the paternal origin of the primary tumor developed from extra-embryonic membranes in fetectomized rat and makes this neoplasm similar to human post-gestation choriocarcinoma.     

Inhibition of syngeneic tumor growth in rats immunized with allogeneic skin grafts

The growth of a transplantable tumor (KMT-17) in syngeneic Wistar King Aptekman/Mk (WKA) rats was inhibited by preimmunization with allogeneic normal cells from Donryu strain rats. The phenomenon is referred to as allogeneic cell immunity. A slight inhibition was observed in rats immunized with normal liver, spleen, kidney, embryonal cells, whole blood and white blood cells from allogeneic Donryu rats. A strong inhibition was observed in animals which had rejected allogeneic skin grafts, particularly from the Donryu and Kyoto rats. The inhibition was comparatively weak after immunization with skin grafts from the Long Evans, ACI, Buffalo, Fischer, Sprague Dawley or Tokyo allogeneic rat strains. This immunizing effect was dependent on the viability of the grafts and was easily abrogated by low-dose irradiation. The mechanism of allogeneic cell immunity is considered to be a non-specific stimulation of the immunity against antigenic tumors in the syngeneic host.     

Reduced transplantability of syngeneic tumors in rats immunized with allogeneic tumors

The growth of transplanted syngeneic tumors in Wistar/KA (WKA) rats was inhibited by three immunizations with tumors from allogeneic Donryu rats which possibly are unrelated antigenically. Inhibition of syngeneic tumor growth was not associated with the source or type of the tumors (carcinoma and sarcoma) used. A slight inhibition was also observed in rats immunized with a large amount of xenogeneic tumor or allogeneic normal spleen, liver and kidney cells. Inhibition was also observed after immunization with syngeneic tumors artificially infected with murine leukemia virus. However, the inhibition was strongest in rats immunized with allogeneic tumors. The mechanism of inhibition may be immunological, and it may be associated with an increase in the non-specific immunity of the host.     

Potent antitumor activity of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine in choriocarcinoma-bearing rats

The acyclic nucleoside phosphonate 9-(2-phosphonyl-methoxyethyl)adenine (PMEA) is a potent and selective antiretroviral agent which is currently evaluated in its oral prodrug form, bis(POM)PMEA (adefovir dipivoxil), in phase II and III clinical trials in human hepatitis B virus (HBV)- and human immunodeficiency virus (HIV)-infected individuals, respectively. We have now found that PMEA is also a potent inhibitor of growth of the highly aggressive choriocarcinoma tumor arising from rat choriocarcinoma RCHO cells grafted under the kidney capsule of syngeneic WKA/H rats. In untreated rats, massive invasive RCHO tumors, covering the whole surface of the kidney and resulting in a marked enlargement of the kidney, were observed at day 10 after tumor cell grafting. Daily treatment with PMEA at 25 mg/kg/day afforded a marked reduction in tumor size (i.e., smaller tumors and slight, if any, enlargement of the kidney). Increasing the PMEA dose to 50, 100 or 250 mg/kg/day resulted in a gradual increase of the antitumor effect of the compound. At the highest dose tested, i.e., 250 mg/kg/day, PMEA completely suppressed tumor growth. The antitumor activity of PMEA persisted for at least 10 days after termination of drug treatment. In addition, delayed treatment with PMEA at a dose of 200 mg/kg/day, started at a time point where choriocarcinoma tumors had already developed, stopped further growth and even induced regression of the tumors. PMPA, a closely related structural analogue of PMEA, failed to inhibit choriocarcinoma tumor growth. This observation points to the specificity of PMEA as an antitumor agent. In view of our findings, the therapeutic potential of PMEA for the treatment of neoplastic diseases appears to merit further investigation. Int. J. Cancer 76:595–600, 1998.© 1998 Wiley-Liss, Inc.     

Identification of the transformation-associated cell surface antigen expressed on the rat fetus-derived fibroblast

A WKA rat fetus-derived fibroblast cell line WFB showed strict nontransformant phenotypes in vitro such as anchorage dependency of cell growth in soft agar, contact inhibition, and serum dependency on the monolayer cell culture. Transfection of 6.6-kilobase EJras oncogene into WFB resulted in the acquisition of tumorigenicity in vitro and in vivo. The cell surface antigen that is moderately or highly expressed on these WFB transformants, designated as W14 and W31, was analyzed using monoclonal antibody 109 that was produced after the immunization of BALB/c mice with W31. Moab 109 recognized a glycoprotein with a molecular weight of 36,000 composed of a single polypeptide chain with 5.4 isoelectric point value. This antigen was highly expressed on WFB EJras and polyoma middle T-DNA transformants, but was undetectable or at the best only faintly recognized on WFB parental cells, transfectants of WFB with c-myc, and normal thymus, liver and kidney of WKA adult rats. It was also clearly expressed on the EJras transformants of Fisher rat fetus-derived 3Y1 fibroblast, but very faintly on parental 3Y1. Furthermore, this antigen was detected on some rat T-lymphoma and gliosarcoma lines. However, it was undetectable on EJras transformants on NRK-49F rat kidney cells and NIH3T3 and BALB3T3 mouse cells. In addition, this antigen appeared on the cell surface of concanavalin A-activated WKA rat lymphocytes and WKA rat on the 16th day of embryo but not on the 8th. These results suggested that the cell surface antigen detected by Moab 109 was clearly unrelated to the ras oncogene product p21 that was highly expressed on EJras-transformants of WFB or 3Y1 cells. Furthermore, it was shown that W14 and W31 cells but not parental WFB cells were susceptible to rat splenic NK cells that were induced by poly(I-C) treatment. Pretreatment of these W14 or W31 cells with Moab 109 could block the NK cell activity against W14 and W31. These data suggest that this antigen may act as one of the NK target structures, and plays an important role as a tumor antigen on the host tumor surveillance, since the antigen was expressed (a) on the cell surface after the cell transformation or enhanced DNA synthesis of some particular cells, and (b) in the W31 tumor developing progressively in the syngeneic rats.     

9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine: a novel prodrug of 9-(2-phosphonylmethoxyethyl)guanine with improved antitumor efficacy and selectivity in choriocarcinoma-bearing rats

The novel acyclic nucleoside phosphonate analogue 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPr-PMEDAP) was shown in vitro to act as an intracellular prodrug of 9-(2-phosphonylmethoxyethyl)guanine (PMEG). We compared the in vivo antitumor efficacy and selectivity of cPr-PMEDAP, its progenitor PMEDAP, and PMEG in a rat choriocarcinoma tumor model. The rats, inoculated with rat choriocarcinoma (RCHO) cells under the renal capsule, were treated IP during 10 days. Macroscopical and histological examination of the RCHO-inoculated kidneys was performed at two time points (i.e., immediately after the end of treatment or after an additional drug-free period of 2 weeks). Complete inhibition of choriocarcinoma tumor development was achieved upon treatment with cPr-PMEDAP, PMEG, and PMEDAP at a daily dose of 10, 1, and 50 mg/kg, respectively. At these doses, all three compounds produced moderate to strong toxicity (evidenced by atrophy of lymphoid organs and reduced body weight gain). When compared at the maximum tolerated (sublethal) doses (i.e., 0.5, 10, and 50 mg/kg for PMEG, cPr-PMEDAP, and PMEDAP, respectively), cPr-PMEDAP proved superior to PMEG and PMEDAP in achieving a complete inhibition of tumor development. Also, whereas PMEG was unable to produce a prolonged antitumor effect, the animals treated with cPr-PMEDAP still showed prominent inhibition of tumor development when tumor size was evaluated at 2 weeks after end of treatment. Based on its efficacy and therapeutic safety, cPr-PMEDAP can be regarded as a promising antitumor agent, which merits further in vivo evaluation in additional tumor models for human neoplasms.     

Increased sensitivity of murine leukemia virus-infected tumor cells to lymphocyte-mediated cytotoxicity

The cytotoxic sensitivity of murine leukemia virus (MuLV)-infected and noninfected fibrosarcoma cells in syngeneic inbred WKA/Hok rats was compared by in vitro cell-mediated 51Cr release cytotoxicity assay. A highly significant increase in cytotoxic sensitivity of target cells was observe in MuLV-infected tumor cells as compared with noninfected cells when spleen cells from syngeneic tumor-bearing hosts (TBH) were used as a source of effector lymphocytes. The cytotoxicity of spleen cells against MuLV-infected tumor cells was specifically directed to the tumor-associated antigen (TAA), but not to the virus-associated antigen. However, there was no quantitative difference in the amount of TAA on the cell membranes between virus-infected and noninfected tumor cells as measured by a quantitative absorption test of anti-TAA serum. The cytotoxic activity of spleen cells from TBH against MuLV-infected tumor cells was abrogated by the treatment of anti-T-serum plus complement and significantly decreased after trypsin treatment. Spleen cells from normal rats given injections of immune sera from TBH acquired the cytotoxic activity against MuLV-infected tumor cells.     

大鼠树突状细胞融合瘤苗的特异性抗骨肉瘤作用

背景与目的:树突状细胞(dendriticcells,DCs)是目前已知的功能最为强大的抗原呈递细胞,但以DC为基础的针对骨肉瘤的免疫治疗未见报道。本研究旨在探讨大鼠树突状细胞融合瘤苗诱导的特异性抗骨肉瘤作用。方法:应用重组大鼠rGM-CSF、rIL-4和rTNF-α培养大鼠骨髓前体细胞获得大量DCs,用标记抗大鼠OX62单抗免疫磁珠分离纯化DCs,经形态学观察、表型检测、功能学实验鉴定,然后通过电融合的方法将细胞系UMR-106与异基因或同基因大鼠DCs相融合制备肿瘤疫苗,观察其抗肿瘤的效果。结果:预防接种异基因肿瘤疫苗组和同基因肿瘤疫苗组大鼠的存活率分别为70%和50%,存活的大鼠经历了第2次大剂量骨肉瘤细胞的免疫攻击,7周之后继续得以存活。在针对骨肉瘤动物模型进行主动免疫治疗的研究中,60%的荷瘤大鼠瘤体萎缩、消失而得以长期存活。结论:大鼠骨肉瘤细胞系UMR-106与同种异基因树突状细胞的融合瘤苗有较强的特异性抗骨肉瘤作用。     

Effects of doxorubicin on cancer cells after two-thirds hepatectomy in rats

A rat model of liver metastases generated by intraportal injection of syngeneic tumor cells after two-thirds hepatectomy was used to determine the optimal regional chemotherapeutic modality for early hepatic metastases. WKA rats had viable tumor cells injected directly into the portal vein after two-thirds hepatectomy. Ten rats were used as a control; the remaining groups were given doxorubicin (4/3 mg/kg) injected directly into the hepatic artery at 24 hr, 72 hr, and 7 days (after liver regeneration) postoperatively. The mean survival period in each group was 21.0, 20.0, 20.5, and 20.7 days, respectively, compared with those treated with doxorubicin (4 mg/kg) injection at 24 hr, 72 hr, and 7 days postoperatively, with a mean survival period in each group of 20.0, 21.6, and 25.6 days, respectively. When a comparison was made with regard to the doses of doxorubicin administered, statistically significant differences in survival rates were recognized between the rats that had doxorubicin (4 mg/kg) injection 7 days postoperatively and the others (P < 0.01). Based on these findings, we believe that appropriate adjuvant chemotherapy should be given after the liver regeneration phase. © 1995 Wiley-Liss, Inc.     

Modulation of the immune response to transplanted tumors in rats by selective depletion of neutrophils in vivo using a monoclonal antibody: abrogation of specific transplantation resistance to chemical carcinogen-induced syngeneic tumors by selective depletion of neutrophils in vivo

The role of neutrophils in transplantation immunity to syngeneic rat tumors was examined using a monoclonal antibody (RP-3) that depletes rat neutrophils selectively in vivo. We used 2 chemical carcinogen-induced transplanted tumors of different antigenic specificity (KMT-17 and KDH-8 of WKA rat origin). When neutrophils were selectively depleted by i.p. injection of RP-3 at the time of in vivo priming with X-irradiated tumor cells, the growth of subsequently s.c. transplanted identical tumors was not inhibited, in contrast to the group of rats immunized without RP-3 treatment. Tumor growth was also not inhibited when the immune rats were treated with RP-3 at the time of identical viable tumor cell challenge. These results suggest that neutrophils play a role in both the priming and effector phases of specific transplantation resistance to syngeneic tumors.     

Establishment and characterization of a transplantable rat myelomonocytic leukemia

A transplantable myelomonocytic leukemia was established from a leukemia of a WKA/Hok rat which had been inoculated with Rauscher virus at birth. The tumor grew in ascites form in normal syngeneic rats and, after the middle stage of i.p. transplantation, leukemia cells consisting of a mixed population of monocytic and granulocytic cells were observed in the peripheral blood. A complement-dependent cytotoxicity test failed to demonstrate Rauscher virus-related antigen on the tumor cell surface. Membrane marker analysis revealed that most of the tumor cells possessed receptors for both complement and neuraminidase-treated sheep RBC. More than 90% of ascitic tumor cells displayed phagocytic activity and a positive nonspecific esterase reaction. Serum from rats bearing this tumor contained high levels of muramidase. Ultrastructurally, the tumor cells resembled both immature and mature cells of the monocyte-macrophage series. On serial transplantation into the peritoneal cavity, the tumor displayed consistent differentiation from undifferentiated blast cells to monocytes and cells indistinguishable from granulocytes. The karyotype analysis revealed that the modal number of chromosomes of the tumor cells was 81, and no structural abnormalities of chromosomes were observed after quinacrine mustard staining. This transplantable leukemia will provide a useful experimental model for the study of granulocyte-monocyte differentiation and for human myelomonocytic leukemia.     

Activation in vitro of mouse macrophages by syngeneic, allogeneic, or xenogeneic lymphocyte supernatants.

Macrophages from normal C57BL/6 mice, those with a subcutaneous B16 melanoma, and mice immunized against the tumor were examined for in vitro cytotoxicity to B16 tumor cells. Macrophages were treated by incubation with supernatants from B16 cells grown either in unmixed cultures or in cultures containing syngeneic, normal, or sensitized allogeneic (A mouse), or xenogeneic (rat) lymphocytes. The various treated and untreated macrophages were then cultured for 5 days with viable B16 cells prelabeled with 125I-5-iodo-2'-deoxyuridine; the cultures were terminated, and the extent of destruction of the B16 target cells was determined from the amounts of radioactivity remaining in adherent tumor cells. Of the untreated macrophages, only those from immunized mice were cytotoxic to the tumor cells; macrophages from normal and tumor-bearing mice became cytotoxic by incubation with supernatants from cultures containing lymphocytes from immunized syngeneic mice, sensitized allogeneic mice, or sensitized rats; and macrophages incubated with supernatants from cultures containing normal nonsensitized allogeneic or xenogeneic lymphocytes showed no cytotoxicity. Thes results suggested that macrophages from tumor-bearing animals are potentially cytotoxic to their syngeneic tumors and can be activated by mediators released from sensitized syngeneic, allogeneic, and/or xenogeneic lymphocytes in vitro.     

Allogeneic mRNA-based electrotransfection of autologous dendritic cells and specific antitumor effects against osteosarcoma in rats

Vaccination with dendritic cells (DCs) transfected with tumor-derived mRNA antigen has emerged as a promising strategy for generating protective immunity in mammals. However, the integration of allogeneic osteosarcoma mRNA and autologous DCs has not been fully examined. This study was designed to investigate the antitumor effects of tumor vaccine produced by autologous DCs transfected of allogeneic osteosarcoma mRNA through electroporation in tumor-bearing rats model. In the present study, extraction of Wistar rat tumor mRNA was performed as a two-step procedure. First, total RNA was extracted by use of Trizol; then, mRNA purification was performed by use of polyT-coated magnetic beads. Then, we transfected the allogeneic-derived tumor mRNA to Sprague?CDawley (SD) rat bone marrow-derived DCs through electroporation. The tumor vaccine was applied to tumor-bearing rats model, and the specific antitumor effects of the tumor vaccine were observed. The immunization using autologous DCs electrotransfected with allogeneic osteosarcoma total RNA induced specific CTL responses, which were statistically significant (P?<?0.05), and the cytotoxic activity was confirmed in cold target inhibition assays and using mAbs blocking MHC class I molecules. In in vivo experiments, 70?% of the rats immunized with allogeneic osteosarcoma RNA transfected to DCs were typically able to reject tumor challenge and remained tumor-free. Vaccinated survivors developed long immunological memory and were able to reject a subsequent rechallenge with the same tumor cells but not a syngeneic unrelated tumor line. In the present study, we demonstrated that allogeneic tumor mRNA isolated from rat osteosarcoma cell line could be applied to produce tumor vaccine inducing specific antitumor effects, especially in DC-based immunotherapy strategy. This study also provides the foundations for an effective and broadly applicable treatment to a wide range of cancer indications for which tumor-associated antigens have not been identified.     

HTLV-1-associated Myelopathy/Tropical Spastic Paraparesis-like Rats by Intravenous Injection of HTLV-1-producing Rabbit or Human T-Cell Line into Adult WKA Rats

We intravenously injected Ra-1 cells or MT-2 cells into female adult WKA rats. Spastic paraparesis mainly in the hind-limbs was observed in 1 out of 2 Ra-1 cell-injected WKA rats and in 3 out of 8 MT-2 cell-injected WKA rats 20 27 months after injection. The main neuropathological finding was symmetrical white matter degeneration with mononuclear cell infiltration of the spinal cord, similar to that of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, and degeneration of nerve roots and peripheral nerves. Antibodies against HTLV-1 antigens were detected in plasma and cerebrospinal fluid from these HAM/TSP-like rats. HTLV-1 provirus was detected from the peripheral blood mononuclear cells of one of these rats 20 months after injection. Interestingly, spastic paraparesis was not observed in F344 rats.     

Correlation between various cell-surface antigens induced by murine leukemia virus in the rat: serological analysis

Analysis based on the cytotoxicity test using antisera produced in rats indicated the presence of various cell-surface antigens on the tumors in WKA/Mk rats induced or artificially infected by Friend, Rauscher or Gross viruses. Each line of the tumor was found to have a common antigen, namely group-specific cell-surface antigen. A Gross type-specific and Friend and Rauscher type-specific cell-surface antigens were also detected, in keeping with the observations in the mouse system. In addition, the present experiments strongly suggested that a Friend strain-specific antigen was detected only on the cell-surface of the tumors in WKA/Mk rats induced or artificially infected by Friend virus. These results were in parallel with the data of previously reported transplantation studies.     

In vivo Retrovirus-mediated Herpes Simplex Virus Thymidine Kinase Gene Therapy Approach for Adult T Cell Leukemia in a Rat Model

We have previously demonstrated that human T-lymphotropic virus type I (HTLV-tax-expressing human T cell lines are selectively eliminated in the presence of aciclovir, using a retroviral vector carrying the herpes simplex virus thymidine kinasc (HSV TK) gene under the control of the long terminal repeat (LTR) of HTLV-I. Based on these findings in vitro , we investigated whether this system could also be effective in vivo , using a rat model. Following infection of the HTLV-I-trans-formed and tot-expressing rat T cell line TARS-1 with this retrovirus (LNLTK virus), high levels of HSV TK expression were observed and resulted in increased susceptibility to ganciclovir (GCV). Tumors were generated by subcutaneous injection of TARS-1 in newborn syngeneic WKA/H rats. While the tumors derived from infected TARS-1 cells with control virus, as well as uninfected cells, continued to grow in all the rats with or without administration of GCV, those derived from LNLTK-infected cells exhibited dramatic regression upon GCV treatment. These results indicate that the HTLV-I LTR-HSV TK system also causes selective elimination of HTLV-I-transformed, (ax-expressing T cells in vivo. Therefore, our present study may provide a rationale for clinical gene therapy against adult T cell leukemia.     

Tumor-cytotoxicity of nitric-oxide produced from alveolar macrophages directly stimulated with tumor-cells

Macrophages activated by lipopolysaccharide or interferon-gamma have been shown to be cytotoxic to tumor cells by releasing nitric oxide. Here, we report that unstimulated rat alveolar macrophages cultured with certain tumor cells produce nitric oxide and are cytotoxic to these tumor cells. Alveolar macrophages were taken from BUF/Mna rats, which were known to produce spontaneous thymoma, and cultured with syngeneic BUF/Mna-derived thymoma cells. They were killed by syngeneic or allogeneic alveolar macrophages and this killing was partially abolished by addition of N(G)-monomethyl-L-arginine. X-ray irradiated, mitomycin C-treated or membranous fragments of BUF/Mna-derived thymoma cells directly stimulated rat alveolar macrophages to produce nitric oxide.     

Antimetastasis effect of lymphokine-activated killer (LAK) cells on fibrosarcoma in WKA rats

Treating WKA rats with fibrosarcoma with LAK cells, antimetastasis effect was studied. The results indicated that splenocytes incubated by IL-2 could produce lytic activity to WKA rats with fibrosarcoma in vitro. Passive infusion of LAK cells lead to a marked reduction in the number of metastatic nodules in the lung. After intravenous injection of LAK cells, cancer cells in the lung speedily disappeared. LAK cell administration for 3 times gave better result than once only (P less than 0.01). In this paper, the possibility of LAK cells used as an adoptive immunotherapy for human neoplasms is briefly discussed.     

Macrophage tumoricidal activity as a possible antitumor mechanism associated with the local injection of allogeneic spleen cells into rats

The in vivo antitumor effect of i.p. injection of allogeneic spleen cells was investigated. ACl rats were inoculated i.p. with 10(4) AMC-60 syngeneic fibrosarcoma cells and given injections i.p. of 4 X 10(7) Wistar spleen cells once a week for 3 wk from 1 day after tumor inoculation. This treatment significantly prolonged the survival period of the tumor-bearing rats. A similar effect was obtained by i.p. injections of Lewis spleen cells. Injection i.p. into ACl rats of spleen cells of these rat strains resulted in the apparent augmentation of cytolytic activity of peritoneal adherent but not of nonadherent cells against AMC-60 tumor cells. The cytotoxicity was exhibited nonspecifically to cells of a variety of tumor lines but not to concanavalin A blasts of ACl spleen cells and was inhibited by the addition of carrageenan. Irradiation (2000 R) of Lewis spleen cells or fractionation of the allogeneic spleen cells using nylon wool columns revealed that a radiosensitive and nylon wool-passed cell population, presumably a T-cell population, of the allogeneic spleen cells is responsible for the augmentation of peritoneal macrophage tumoricidal activity in ACl rats. Further, Lewis spleen cells irradiated at 2000 R neither augmented peritoneal macrophage cytotoxicity nor prolonged the survival period of ACl rats bearing AMC-60 tumor, suggesting that the augmentation of peritoneal macrophage cytotoxicity plays a major role in the in vivo antitumor effect of the allogeneic spleen cell transfer. ACl rats were given injections i.p. of 4 X 10(7) Lewis spleen cells. Two days after injection, cells including peritoneal cells of the ACl rats and Lewis spleen cells remaining in the peritoneal cavities were obtained by peritoneal lavages and then incubated for 5 days. Significant blastogenic proliferation was observed, and the supernatant of the culture was shown to be able to render thioglycollate-induced peritoneal macrophages of ACl rats cytotoxic to AMC-60 tumor cells, indicating that a certain cell population of the cell mixture produced a lymphokine(s) resembling macrophage activating factor (MAF) during the incubation. When ACl rats were given injections i.p. of irradiated Lewis spleen cells, neither the blastogenic proliferation nor the generation of MAF activity in the culture supernatant was observed. Indirect immunofluorescence analysis using rabbit anti-ACl and anti-Lewis antisera revealed that as many irradiated Lewis spleen cells were remaining in the peritoneal cavities as normal Lewis spleen cells 2 days after injection into ACl rats.(ABSTRACT TRUNCATED AT 400 WORDS)