Antitumor effect of intrapleural administration of Lactobacillus casei in mice

Summary The antitumor effect of intrapleural (i.pl.) administration of Lactobacillus casei YIT 9018 (LC 9018) on Meth A sarcoma in BALB/c mice was examined. Inoculation of Meth A cells into the thoracic cavity of BALB/c mice caused growth of the cells and the mice died from the tumor with an increased amount of pleural fluid. LC 9018 was given i.pl. to BALB/c mice before or after i.pl. inoculation of Meth A cells and the survival of the mice was determined. The i.pl. administration of LC 9018 was effective in prolonging the survival of the mice after i.pl. inoculation of Meth A tumor, and pretreatment with LC 9018 i.pl. also prolonged survival. Moreover, i.pl. administration of LC 9018 not only increased the number of thoracic exudate cells (TEC) but also augmented both cytolytic activity of thoracic macrophages and natural killer cell activity of TEC. Furthermore, phagocytic activity of thoracic macrophages against sheep red blood cells was enhanced and Ia antigen-positive cells in TEC were increased by the i.pl. treatment with LC 9018. These results showed that TEC induced by i.pl. administration of LC 9018 had antitumor activity against Meth A tumor inoculated i.pl. into BALB/c mice.     

Macrophage activation by Lactobacillus casei in mice

Effects of Lactobacillus casei YIT 9018 (LC 9018), which has antitumor activities against allogeneic and syngeneic murine tumors, on macrophage functions were examined. By intraperitoneal (i.p.) injection of LC 9018, acid phosphatase activity and phagocytic activity of peritoneal macrophages were enhanced significantly compared with those of normal peritoneal macrophages. The phagocytic activities showed peaks 2-3 days after the LC 9018-injection. LC 9018 accelerated the phagocytic function of the reticuloendotherial system in ICR mice tested by the carbon clearance test. The cytostatic activity of peritoneal exudate cells (PEC) induced by i.p. injection of LC 9018 into C57BL/6 mice against EL4 cells was also enhanced. On the other hand, PEC induced by L. fermentum YIT 0159, which has no antitumor activity, did not have cytostatic activity. These observations showed that LC 9018 was able to activate macrophages in mice.     

Augmentation of Mouse Natural Killer Cell Activity by Lactobacillus casei and Its Surface Antigens

Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate-polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250 μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A-bearing mice. In vitro treatment with anti-asialo GM1 antibody plus complement completely-abrogated the LC 9018-augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti-Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018-treated mice. One population was asialo GM1-positive and Thy 1-negative, the other was asialo GM1-positive and Thy 1-positive.     

Antimetastatic effect of Lactobacillus casei YIT9018 (LC 9018) on a highly metastatic variant of B16 melanoma in C57BL/6J mice

Summary The effect of Lactobacillus casei YIT9018 (LC 9018) on a highly metastatic variant of B16 melanoma, B16-BL6, was determined in C57BL/6 mice. Intralesional (i.l.) injection of LC 9018 inhibited tumor growth and prolonged the survival after s.c. inoculation of B16-BL6 into C57BL/6 mice. Injection of LC 9018 i.v. protected the mice against pulmonary metastasis after i.v. inoculation of B16-BL6. Injection of LC 9018 i.l. before surgical excision of the primary tumor inhibited axillary lymph node metastasis and i.v. injection of LC 9018 after surgical excision of the primary tumor inhibited both axillary lymph node and lung metastases. On the other hand, the combination of i.l. and i.v. injections of LC 9018 markedly inhibited both lymph node and lung metastases. Natural killer cell activity of axillary lymph node cells was augmented by the injection of LC 9018 into a front footpad, while the cytolytic activity of axillary lymph node cells was significantly enhanced. However, the cytolytic activity was diminished by depleting whole lymph node cells of the plastic adherent cells. Furthermore, alveolar macrophage-mediated cytotoxic activity was augmented by the i.v. injection of LC 9018.     

Protective effect of lipoteichoic acid from Lactobacillus casei and Lactobacillus fermentum against Pseudomonas aeruginosa in mice

Lipoteichoic acid (LTA) from Lactobacillus casei YIT 9018 or Lactobacillus fermentum YIT 0159 augmented the resistance of C57BL/6 mice to infection with Pseudomonas aeruginosa, but conferred no resistance to Listeria monocytogenes. It is suggested that LTA was unable to activate macrophages.     

Anti-tumour effect of humoral and cellular immunities mediated by a bacterial immunopotentiator,Lactobacillus casei,in mice

Summary Administration of a mixture containing Lactobacillus casei YIT 9018 (LC9018) and methylcholanthrene-induced fibrosarcoma (Meth A) cells into the peritoneum of syngeneic BALB/c mice suppressed the tumour growth and protected the mice from tumour death. With the appearance of the anti-tumour activity, serum complement-dependent tumour cytotoxic (CDC) antibody was induced on the 5th day after the administration as a result of the adjuvant effect. The cytotoxic antibody was not found in serum on the 5th day after inoculation of Meth A cells alone, but it was induced before the mice died of the tumours. Adjuvant induction of the cytotoxic serum antibody at an early time was also observed using Kirsten murine sarcoma virus-transformed tumour (K234) cells. Both of these cytotoxic antibodies in sera from Meth A-suppressed and the tumour-bearing mice were specific for the tumour cells and were IgM class, since they were absorbed with rabbit anti-mouse IgM antibody. However, the cytotoxic antibody was not found in the peritoneal cavity which was the tumour inoculation site, but binding antibody against the tumour cells was faintly detected in the region using an enzyme-linked immunoabsorbent assay (ELISA). In neutralization tests, the cytotoxic antibody did not exert anti-tumour activity in recipient mice when it was administered to the mice along with the tumour cells or when it was administered i. v. at the time of tumour inoculation. Moreover, the cytotoxic antibody was not available for the antibody-dependent cell-mediated cytotoxicity (ADCC). These results suggest that the cytotoxic antibody did not exert anti-tumour activity in the tumour-suppressed mice. In contrast, peritoneal exudate cells (PEC) on the 5th day, and PEC and spleen cells on the 15th day after i. p. administration of the mixture exerted strong anti-tumour activity as measured by the Winn test.In conclusion, the adjuvant effect of LC9018 induced tumour-specific humoral and cellular immunities but the anti-tumour activity was dependent only on the cellular effectors of the host. The possible use of LC9018 in tumour immunotherapy is discussed.     

Anti-tumour activity of Lactobacillus casei on lewis lung carcinoma and line-10 hepatoma in syngeneic mice and guinea pigs

Summary The anti-tumour activity of Lactobacillus casei YIT 9018 (LC 9018) on Lewis lung carcinoma (3LL) in C57BL/6 mice and line-10 hepatoma in strain-2 guinea pigs was examined. Intravenous injection of LC 9018 was effective for inhibition of pulmonary metastases in C57BL/6 mice after s.c. inoculation with 3LL tumours. Intralesional (i.l.) injection of LC 9018 was also effective for both prolongation of the survival period and inhibition of pulmonary metastases in 3LL tumour-bearing mice. The combination treatment of i.l. and i.v. injections of LC 9018 before or after surgical excision of the primary tumour remarkably inhibited the pulmonary metastases after inoculation with 3LL tumour. Intralesional injection of LC 9018 was effective for regression of the established tumours of line-10 hepatoma inoculated i.d. and for induction of systemic tumour immunity in strain-2 guinea pigs.     

Cytotoxic factor production by kupffer cells elicited with Lactobacillus casei and Corynebacterium parvum

Summary The ability of Kupffer cells, spleen macrophages, pulmonary macrophages, and peritoneal macrophages (PM) to produce cytotoxic factor (CTF) was investigated in vitro. The production of CTF by Kupffer cells elicited with Corynebacterium parvum (CP) or Lactobacillus casei YIT9018 (LC9018) was higher than that of spleen, pulmonary macrophages, or PM. In addition, oxygen radical (OR) production by Kupffer cells or PM was measured. The production of OR by Kupffer cells or PM was significantly augmented by i.v. or i.p. injection of LC9018 or CP. No significant correlation was observed between the increase in OR production by Kupffer cells or PM and CTF production by Kupffer cells or PM elicited with either organism. It was suggested that activated Kupffer cells may be one important source of CTF production in serum and that the CTF-producing macrophages may be different from the OR-producing macrophages.     

Inhibition of tumor metastasis of lewis lung carcinoma in C57BL/6 mice by intrapleural administration of Lactobacillus casei

Summary The antimetastatic effect of Lactobacillus casei YIT9018 (LC 9018) against Lewis lung carcinoma (3LL) in C57BL/6 mice was determined. Intrapleural (i.pl.) administration of LC 9018 was effective in inhibiting pulmonary metastasis after s.c. inoculation of 3LL tumors into C57BL/6 mice. The combination of i.pl. and intralesional or i.v. injections of LC 9018 also markedly inhibited pulmonary metastasis in 3LL-bearing mice. The i.pl. administration of LC 9018 into mice induced an increase in the number of thoracic exudate cells (TEC) and the cell population in the TEC was mainly polymorphonuclear leukocytes in the early stage, while macrophages were dominant in the late stage. In addition, in vitro cytolytic activity against 3LL cells and natural killer cell activity of TEC were augmented by the i.pl. administration of LC 9018. Furthermore, i.pl. administration of LC 9018 into the mice rendered their lung macrophages tumoricidal for 3LL cells in vitro. These results show that TEC induced by i.pl. administration of LC 9018 played a key role in the inhibtion of metastasis in 3LL-bearing mice.     

Protective and therapeutic efficacy of Lactobacillus casei against experimental murine infections due to Mycobacterium fortuitum complex

A bacterial immunopotentiator, LC 9018 (heat-killed Lactobacillus casei), was studied for its protective and therapeutic efficacies against Mycobacterium fortuitum and M. chelonae infections in mice. This agent reduced the incidence of spinning disease and gross renal lesions and enhanced the elimination of organisms at the site of infection in the host mice, when administered intramuscularly six times a week (0.1 mg dry weight per injection, one injection on each day of treatment) from 1 week before to 2 weeks after infection. The LC 9018 injections in this protocol caused a marked increase in the phagocytic function, O2- -producing ability and chemiluminescence of host peritoneal macrophages. Moreover, LC 9018 injections using the same schedule resulted in an enhancement of interleukin-1-producing function of the macrophages, particularly in the infected mice. These findings indicate that LC 9018 administration with the present protocol can activate macrophage functions, in particular those related to microbicidal activity. This would partly explain the protective and therapeutic efficacy of LC 9018 against infection due to M. fortuitum complex.     

Protection of mice against herpes simplex virus infection by a Lactobacillus casei preparation (LC 9018) in combination with inactivated viral antigen

Heat-killed Lactobacillus casei YIT 9018 (LC 9018) cells enhanced the resistance to herpes simplex virus type 1 (HSV-1) in adult mice, but not significantly. The protection of mice against HSV-1 infection and the production of neutralizing antibodies were significantly enhanced by the administration of LC 9018 in combination with inactivated HSV-1 antigen. The optimal enhancement of resistance was seen in mice 14 days after the simultaneous administration of these substances. The resistance to HSV-1 infection in mice could be transferred with peritoneal exudate cells from syngeneic mice previously treated with LC 9018 alone and LC 9018 in combination with inactivated HSV-1 antigen or with thioglycollate broth, whereas the transfer of peritoneal exudate cells induced by thioglycollate broth alone and of spleen cells induced by LC 9018 in combination with thioglycollate broth or by thioglycollate broth alone was not effective. These results suggest that mouse peritoneal macrophages induced by the administration of LC 9018 in combination with inactivated HSV-1 antigen may play an important role in host defense mechanisms against HSV-1 infection.     

Isolation of Cycloclavine from the Culture Broth of Aspergillus japonicus SAITO

Changes of protein components (LA, LB, LC, and X) in mouse serum after administration of an antitumor agent, LC 9018 (lyophilized preparation of Lactobacillus casei, YIT 9018), were investigated. The intraperitoneal injection of LC 9018 caused an increase in these proteins one or two days after administration. The LC component was purified from mouse serum and was identified as haptoglobin. In addition, LA was identified as an intermediate of a haptoglobin-hemoglobin complex [Hp-Hb(α₁β₁)]and X as a haptoglobin-hemoglobin complex [Hp-Hb(α₂β₂)] because addition of increasing amount of hemoglobin to purified haptoglobin formed X via LA and all of these components were immunoprecipitated with anti-haptoglobin IgG.     

Prevention of 5-fluorouracil-induced infection with indigenous Escherichia coli in tumor-bearing mice by nonspecific immunostimulation.

We have previously reported that the lethal toxicity of 5-fluorouracil (5-FU) in specific-pathogen-free mice is due to an indigenous infection with Escherichia coli (K. Nomoto, T. Yokokura, Y. Yoshikai, et al. Can. J. Microbiol. 37:244-247, 1991). In the present study, we demonstrate that nonspecific immunostimulation augments host resistance against the lethal toxicity of 5-FU in tumor-bearing mice. Intravenous administration of a preparation of heat-killed Lactobacillus casei (LC 9018), a nonspecific immunostimulant, at a dose of 20 mg/kg to BALB/c mice augmented their resistance against the lethal toxicity of 5-FU if the preparation was injected into the mice 10-40 days before administration of 5-FU. Injection of LC 9018 into BALB/c mice bearing Meth A fibrosarcoma also enhanced their resistance against the lethality of 5-FU. Systemic infection with E. coli was induced in all of the 5-FU-treated tumor-bearing mice 10 days or more after administration of the drug at a lethal dose of 500 mg/kg, and it was accompanied by an overgrowth of the bacteria in the intestine. Treatment of tumor-bearing mice with LC 9018 resulted in decreased rates of occurrence of systemic infection with E. coli and inhibition of overgrowth of the bacteria in the intestine after administration of 5-FU. A single administration of either LC 9018 or 5-FU significantly inhibited the growth of Meth A cells in vivo, and a combined antitumor effect was shown in the mice treated with both 5-FU and LC 9018.     

In vitro and in vivo release of cytostatic factors from Lactobacillus casei-elicited peritoneal macrophages after stimulation with tumor cells and immunostimulants

Summary The effect of tumor cells and immunostimulants on the release of cytostatic factors (CF) from Lactobacillus casei YIT 9018 (LC)-, Corynebacterium parvum (CP)- or peptone-elicited peritoneal macrophages (PM) was investigated in vitro and in vivo. Significant release of CF into the culture medium from PM elicited with LC was induced by seven of eight mitomycin C-pretreated tumor cell lines and not by normal spleen cells, while no CF was released extracellularly from peptone-elicited PM given the same stimulus. CF were released from LC-elicited PM (LCEPM) after stimulation with LC, bacille Calmette-Guérin, streptococcal preparation OK-432, fucoidan or lipopolysaccharide, and LC but not CP induced CF production in the peritoneal cavities of LC- or CP-primed mice. The release of CF from LCEPM after stimulation with mitomycin C-pretreated 3T12-3 cells was inhibited by D-mannose and not by L-fucose. L-Rhamnose and mannose 6-phosphate, but not D-mannose or L-fucose, caused the release of CF from the PM.It was suggested that the release of CF from activated PM is caused by stimulation by some tumor cells, sugars, or bacterial immunostimulants, D-Mannose and L-rhamnose on the surface of tumor cells or bacteria, respectively, may play an important role in the release of CF from activated macrophages.     

Mechanism(s) of in vitro macrophage activation with Nocardia rubra cell wall skeleton: the effects on macrophage activating factor production by lymphocytes

Summary BALB/c mouse peritoneal macrophages prepared from WPC which had been treated with N. CWS demonstrated potent cytostatic activity against syngeneic Meth A fibrosarcoma cells. The maximum cytostatic activity developed in the macrophages when WPC were incubated with 25 g/ml N. CWS for 3 days. NAPC from BALB/c mice given an i. p. injection with 100 g N. CWS 7 days previously (N. CWS-NAPC) or supernatants from N. CWS-NAPC also activated peritoneal macrophages in vitro. However, when peritoneal macrophages were incubated with N. CWS in the absence of NAPC, or when T cells were depleted from WPC by treatment with anti-Thy 1.2 antibody and complement, N. CWS failed to enhance the cytostatic activity of the macrophages. Furthermore, thioglycollate-elicited peritoneal macrophages from C3H/HeN mice increased their cytolytic properties by incubation with supernatant fluids from N. CWS-treated spleen cells. These findings suggest that in vitro macrophage activation with N. CWS depends on MAF secreted from T lymphocytes. Abbreviations used: N. CWS, Nocardia rubra cell-wall skeleton; BRM, biological response modifier; MAF, macrophage activating factor; IL-1, interleukin 1; IL-2, interleukin 2; IFN-, interferon gamma; PCCM, peritoneal cell culture medium; SCCM, spleen cell culture medium; TCM, tumor culture medium; HI-FCS, heat-inactivated fetal calf serum; Con A, concanavalin A; PC, peritoneal cells; PEC, peritoneal exudate cells; WPC, whole peritoneal cells; APC, adherent peritoneal cells; TGC-APC, thioglycollateelicited adherent peritoneal cells; NAPC, nonadherent peritoneal cells; SN, supernatants; NK cells, natural killer cells; LAK cells, lymphokine activated killer cells E:T ratio, effector: target cell ratio; WSA, water soluble adjuvant; LPS, lipopolysaccharide; MDP, N-acetyl-muramyl-L-alanyl-D-isoglutamine     

Role of culture supernatant of cytotoxic/cytostatic macrophages in activation of murine resident peritoneal macrophages

Summary Peritoneal macrophages elicited by Lactobacillus casei YIT9018 (LCEPM) were incubated in culture for 18 h with L. casei; the culture supernatant (LCM) was then harvested and tested for its ability to increase the cytostatic activity of resident peritoneal macrophages (RPM) and LCEPM. Treatment of RPM with LCM induced activation of macrophages to a cytostatic state against L929, Colon 26, P815, P388D1 and L1210 cells. A combination of recombinant human tumor necrosis factor (rhTNF), recombinant mouse TNF (rmTNF), recombinant human interleukin-1 (rhIL-1) or bacterial lipopolysaccharide with recombinant mouse interferon (rmIFN-) resulted in the synergistic induction of cytostatic activity in RPM. Recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) plus rhTNF increased the cytostatic activity of RPM a little but rmGM-CSF or rhTNF combined with rhIL-1 or alone had no effect. The effect of LCM on RPM was not inhibited by polymyxin B, anti-mTNF antiserum or below 20 U/ml monoclonal anti-rmIFN- antibody (anti-rmIFN-) but was inhibited by more than 40 U/ml anti-rmIFN-, and LCM did not have any interferon antiviral activity. These results suggest that the cytostatic activity of RPM was augmented by the LCM, and that the effect of the LCM may be not due to IFN-, TNF, GM-CSF, IL-1 or a small amount of contaminating lipopolysaccharide.     

An extract of seeds fromAeginetia indica L., a parasitic plant,induces potent antigen-specific antitumor immunity in Meth A-bearing BALB/c mice

Summary The antitumor activity of an extract of seeds fromAeginetia indica L., a parasitic plant, was investigated. BALB/c mice, inoculated i.p. 1 × 10⁵ syngeneic Meth A tumor cells, were administered 2.5 mg/kgA. indica extract i.p. every 2 days from day 0. The untreated mice died of an ascitic form of tumor growth within 21 days, whereas all the treated mice completely recovered from tumor challenge without any side-effects. The extract did not exert direct cytotoxic activity against Meth A in vitro. Mice that survived after the first challenge as a result ofA. indica treatment overcame the rechallenge with homologous Meth A without additional administration of the extract. On the other hand, those mice could not survive after rechallenge with Meth 1 tumor cells, which were also established in BALB/c mice but were different in antigenicity from Meth A, suggesting the development of antigen-specific concomitant immunity in theA. indica-cured mice. In the induction phase of antitumor resistance in this system, CD4⁺ T cells appeared to be the main contributors, since in vivo administration of anti-CD4 mAb completely abolished such resistance. In contrast, anti-CD8 mAb administration did not influence the effect ofA. indica. The importance of CD4⁺ T cells in antitumor immunity was again clarified by Winn assay; that is, spleen and lymph node cells depleted of CD4⁺ T cells in vitro prior to assay abolished antitumor activity on co-grafted Meth A tumor cells in vivo.     

Optimizing conditions for the growth of Lactobacillus casei YIT 9018 in tryptone-yeast extract-glucose medium by using response surface methodology.

This study was undertaken to find optimum conditions of tryptone, yeast extract, glucose, Tween 80, and incubation temperature for the growth of Lactobacillus casei YIT 9018 and to assess the effects of these factors by use of response surface methodology. A central composite design was used as an experimental design for allocation of treatment combinations. A second-order polynomial regression model, which was used at first for analysis of the experiment, had a significant lack of fit. Therefore, cubic and quartic terms were incorporated into the regression model through variable selection procedures. Effects involving incubation temperature, yeast extract, glucose, and tryptone were significant, whereas the only significant effect involving Tween 80 was the interaction effect between temperature and Tween 80. It turned out that growth of L. casei YIT 9018 was most strongly affected by the incubation temperature. Estimated optimum conditions of the factors for growth of L. casei YIT 9018 are as follows: tryptone, 3.04%; yeast extract, 0.892%; glucose, 1.58%; Tween 80, 0%; incubation temperature, 35 degrees C.     

Augmentation of antitumor effect on syngeneic murine solid tumors by an interleukin 2 slow delivery system,the IL-2 mini-pellet

We evaluated the antitumor effect of an interleukin 2 (IL-2) slow delivery system, the IL-2 mini-pellet, in two murine solid tumor models, and also investigated the enhancement of its therapeutic effect by serial administration. The IL-2 mini-pellet contains 1 × 106 units of IL-2 and releases it slowlyin vivo. In our experiment, the IL-2 mini-pellet was administered subcutaneously near the tumor site in combination with the intravenous injection of lymphokine-activated killer (LAK) cells. When this regimen was given on days 8 and 11 after the subcutaneous inoculation of Meth A fibrosarcoma into BALB/c mice, tumor growth was significantly inhibited (p < 0.05) compared to tumor growth in untreated controls. Moreover, the IL-2 mini-pellet alone was also effective in inhibiting tumor growth. In another experiment, MH134 hepatoma was inoculated into C3H/He mice. Both administration of the IL-2 minipellet alone and in combination with LAK cells resulted in complete tumor regression in four of five mice. In a third experiment, serial administration of the IL-2 mini-pellet at 3- or 5-day intervals prolonged the suppression of Meth A fibrosarcoma growth in BALB/c mice. These results suggested that the IL-2 mini-pellet could be applied to cancer immunotherapy and that its antitumor effect could be prolonged by serial administration.     

Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells

The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurred between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the "effector phase" as well as in the "induction phase". The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the "bystander effect."