Increased or decreased immunogenicity of tumor-associated antigen according to the amount of virus-associated antigen in rat tumor cells infected with friend virus

Summary The immunogenicity of tumor-associated antigen (TAA) in 3-methylcholanthrene-induced rat fibrosarcoma KMT-17 cells was investigated with particular reference to the amount of virus-associated antigen (VAA) produced on the cell surface after artificial infection of tumor cells with Friend murine leukemia virus (FV). To compare the TAA immunogenicity of FV-infected KMT-17 tumor cells (FV-KMT-17) with non-infected KMT-17 cells, rats were immunized with the above two tumor-line cells, and the growth of original non-infected KMT-17 cells was observed. The result showed that TAA immunogenicity was not always paralleled by the amount of VAA newly produced. The amount of VAA increased in proportion to the number of passage generations of FV-KMT-17 cells through FV-tolerant rats that had been neonatally injected with Friend virus. The TAA immunogenicity of FV-KMT-17 cells after two passages was lower than that of non-infected KMT-17 cells. High TAA immunogenicity was observed in FV-KMT-17 tumor cells of the fourth passage generation; it then weakened gradually after subsequent passages of FV-KMT-17 tumor cells, and finally dropped to a level lower than the immunogenicity of the original non-infected tumor cells. However, such variations of immunogenicity were never observed in FV-tolerant rats. These observations suggest that TAA immunogenicity definitely increases when an appropriate amount of VAA is expressed on the tumor cell surface and decreases when a relatively low or excessive amount of VAA is expressed. We discuss the mechanisms leading to the above findings. Abbreviations used in this paper are: FV, Friend murine leukemia virus; TAA, KMT-17 tumor-associated antigen; VAA, FV-associated antigen     

Development of highly immunogenic variants of a rat fibrosarcoma line during in vitro cultivation

Summary Rat firosarcoma KMT-17 cells descreased in tumorigenicity when cultured in vitro. Eight clones derived from cultured KMT-17 cell lines (c-KMT-17) were examined for their tumorigenicity, immunosensitivity, and immunogenicity. All the clones were less or nontumorigenic in normal syngeneic rats than KMT-17 cells maintained in vivo. All eight clones produced tumors in rats immuno-suppressed with 600 rad ⁶⁰Co; differences in degree of tumorigenicity were seen among clones in rats irradiated with 250 rad ⁶⁰Co. Although immunosensitivity of the eight clones to complement-dependent and cell-mediated cytotoxicity was the same or less than that of KMT-17 cells, al leight clones induced greater transplantation resistance to KMT-17 than KMT-17 itself. Cold target inhibition tests demonstrated new antigens in a highly immunogenic variant in addition to the original tumor associated antigen (TAA). New glycolipids, not observed in KMT-17 cells, were demonstrated in the clones by thin layer chromatography. These results suggest that new antigens appearing during culture of KMT-17 may act as helper antigens for TAA, increasing the immunogenicity and decreasing the tumorigenicity of the cultured cells.This work was partially supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science und Culture, Japan.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

Nonspecific inhibition of tumor growth in vivo by admixed allogeneic tumor-sensitized lymphoid cells and identical inactivated allogeneic tumor cells.

With the in vivo tumor neutralization test (Winn test), growth of a transplanted (KMT-17) from Wistar-King-Aptekman rats was inhibited by allogeneic tumor (AH-66 from Donryu rats)-sensitized syngeneic lymphoid cells admixed with mitomycin C (MMC)-treated AH-66 cells. The observed tumor inhibition may be immunologically nonspecific, since no cross-antigens were detected by membrane immunofluorescence on the surfaces of KMT-17 and AH-66 cells. Close contact among KMT-17, AH-66-sensitized lymphoid cells and MMC-treated AH-66 cells was required for the inhibition of KMT-17 growth. AH-66 cells pretreated with formalin or ultrasonication lost tumor inhibitory activity when they were admixed with AH-66-sensitized lymphoid cells, and only MMC-treatment effectively preserved the tumor inhibitory activity of AH-66 cells. The sensitized spleen cells, draining lymph node, or peripheral blood cells inhibited tumor growth when they were admixed with MMC-treated AH-66 cells, whereas nucleated cells from bone marrow, thymus, or distal lymph node did not. Growths of KMT-17 were inhibited by admixed sensitized spleen cells and MMC-treated AH-66 even when pre-irradiated rats were used as recipients.     

Leukotriene D4-induced Ca2+ Mobilization in Ehrlich Ascites Tumor Cells

Stimulation of Ehrlich ascites tumor cells with leukotriene D₄ (LTD₄) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca²⁺ concentration, [Ca²⁺] _i . The Ca²⁺ peak time, i.e., the time between addition of LTD₄ and the highest measured [Ca²⁺] _i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD₄ (100 nm), the highest measured increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium is 260 ± 14 nm and the EC₅₀ value for LTD₄-induced Ca²⁺ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC₄ and LTE₄ nor LTB₄ are able to mimic or block the LTD₄-induced Ca²⁺ mobilization, hence the receptor is specific for LTD₄. Removal of Ca²⁺ from the experimental buffer significantly reduces the size of the LTD₄-induced increase in [Ca²⁺] _i . Furthermore, depletion of the intracellular Ins(1,4,5)P₃-sensitive Ca²⁺ stores by addition of the ER-Ca²⁺-ATPase inhibitor thapsigargin also reduces the size of the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium, and completely abolishes the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-free medium containing EGTA. Thus, the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells involves an influx of Ca²⁺ from the extracellular compartment as well as a release of Ca²⁺ from intracellular Ins(1,4,5)P₃-sensitive stores. The Ca²⁺ peak times for the LTD₄-induced Ca²⁺ influx and for the LTD₄-induced Ca²⁺ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD₄ also induces a transient increase in Ins(1,4,5)P₃ generation in the Ehrlich cells, and the Ins(1,4,5)P₃ peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P₃ content seems to increase before the LTD₄-induced Ca²⁺ release from the intracellular stores but after the LTD₄-induced Ca²⁺ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD₄-induced increase in Ins(1,4,5)P₃ as well as the LTD₄-induced increase in [Ca²⁺] _i , indicating that a U73122-sensitive phospholipase C is involved in the LTD₄-induced Ca²⁺ mobilization in Ehrlich cells. The LTD₄-induced Ca²⁺ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD₄-activated Ca²⁺ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD₄ acts in the Ehrlich cells via a specific receptor for LTD₄, which upon stimulation initiates an influx of Ca²⁺, through yet unidentified Ca²⁺ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P₃ formation and finally release of Ca²⁺ from the intracellular Ins(1,4,5)P₃-sensitive stores. Received: 9 February 1996/Revised: 15 August 1996     

Leukotriene-D4 induced cell shrinkage in Ehrlich ascites tumor cells

Summary The nature of the leukotriene-D₄ (LTD₄) induced cell shrinkage in Ehrlich ascites tumor cells has been investigated. LTD₄ treatment of Ehrlich cells induces net loss of cellular KCl and cell shrinkage independent of the initial cell volume. LTD₄ also produces water loss and reduction in cell volume when all extracellular and all intracellular Cl has been replaced by NO₃. On the other hand, LTD₄ fails to produce any significant changes in cell volume in the presence of the K-channel blocker quinine, suggesting that LTD₄ in Ehrlich cells induces Cl-independent K loss through the Ca²⁺-dependent K channels. However, the effect of physiological doses of LTD₄ on cell volume seems not to be as potent in Cl-free, NO₃ cells when compared to Cl-containing cells, indicating that LTD₄ in Ehrlich cells also provokes Cl-dependent K loss. LTD₄ seems not to produce K loss through an electroneutral K⁺/H⁺ exchange system. LTD₄ still produces Cl-independent K loss and cell shrinkage in the presence of the anticalmodulin drug pimozide but not in the presence of the LTD₄ receptor antagonist L-649,923 or the 5-lipoxygenase inhibitor NDGA. Pretreatment of the cells with pertussis toxin, which inactivates inhibitory guanine nucleotide binding proteins (G-proteins), leads to partial inhibition of the LTD₄-induced shrinkage. It is suggested that the LTD₄-induced activation of K and Cl transporting systems in Ehrlich ascites tumor cells is mediated via a G-protein coupled receptor and that LTD₄ might exert its effect through another lipoxygenase product. The Ca²⁺-calmodulin complex is not involved in the LTD₄-induced activation of K and Cl transporting systems.     

Leukotriene D4 induced calcium changes in U937 cells may utilize mechanisms additional to inositol phosphate production that are pertussis toxin insensitive but are blocked by phorbol myristate acetate

DMSO differentiated U937 cells responded to 10⁻⁶ M LTD₄, LTB₄ and FMLP with an increase in both InsP formation and [Ca²⁺]_i. FMLP caused a greater rise in InsPs than either LTD₄ or LTB₄, which were equivalent. LTD₄, however, caused a greater increase in [Ca²⁺]_i than LTB₄ (4-fold) or FMLP. The FMLP [Ca²⁺]_i and InsP responses were abolished by pertussis toxin (100 ng/ml for 4 h) but were unaffected by PMA (10⁻⁷ M for 3 min). In contrast, the LTD₄ [Ca²⁺]_i and InsP responses were reduced by only 50% by pertussis toxin, whilst PMA reduced the [Ca²⁺]_i and InsP responses to LTD₄ by 75 and 30%, respectively. These results suggest that mechanisms additional to InsP formation exist for mediating LTD₄ evoked increases in [Ca²⁺]_i.     

Enhanced susceptibility of target KMT-17 cells to activated macrophages by treatment with the antitumor agent bleomycin

Summary We observed that after KMT-17 cells had been treated with bleomycin (BLM), even with a dose as high as 160 g/ml, they were still able to form colonies in soft agar. We then studied the susceptibility of KMT-17 cells treated with BLM to activated macrophages. During a colony inhibition assay, BLM-treated KMT-17 cells were found to be much more susceptile to activated macrophages than nontreated KMT-17 cells, moreover, a tumor neutralizing assay showed that the growth of BLM-treated KMT-17 cells was also significantly inhibited by activated macrophages as compared with nontreated KMT-17 cells. Macrophages activated by both BLM and the Nocardia rubra cell wall skeleton were able to mediate such tumor inhibition activity in BLM-treated KMT-17 cells. Activated macrophages did not seem to have strong antitumor activity against nontreated KMT-17 cells in vivo, however, the life span of the rats which were inoculated i. p. with KMT-17 cells was significantly expanded after the tumorbearing rats were given BLM i.p. The data presented here suggest that not only does BLM have a direct tumoricidal effect on KMT-17 cells, it also regulates immunosensitivity of targets to immune effectors. We also discuss the mechanism for enhancing the susceptibility of KMT-17 cells to activated macrophages brought about by treatment with BLM.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education, Science, and Culture     

The improved effects of specific active immunotherapy on a rat fibrosarcoma by antitumor drugs

Summary We have tried to find out if the combination of a xenogenized tumor cell vaccine and antitumor drugs is able to induce a synergistic increase in the antitumor therapeutic effect. The degree of increase in the LTD₅₀ (50% lethal tumor dose) is expressed numerically, as a quantitative index designed to compare degrees of transplantation resistance to tumor cell challenge. A LTD₅₀ was achieved by an intradermal (i. d.) immunization with xenogenized tumor cells when challenged with tumor cells implanted intraperitoneally 2 weeks after the immunization: this LTD₅₀ value was 527 000 times higher than that of the non-immunized group. When we combined this type of immunization with appropriate doses of bleomycin (BLM) or cyclophosphamide (CY), which are able to augment antitumor immunity, the LTD₅₀ was 723 000–1190 000 times higher than that of the non-immunized group. This increase in the LTD₅₀ is definitely higher than that achieved by a single immunization with irradiated tumor cells (× 33 000) and combined with either BLM (× 93 000) or CY (×140 000). We also studied the therapeutic effect of a tumor cell vaccine combined with antitumor drugs BLM or CY in tumor-bearing rats. We observed a synergistic effect caused by BLM or CY after i. d. immunization with xenogenized tumor cells: this showed a significant increase when compared with the therapeutic effects obtained by chemotherapy alone (P <0.01). Nevertheless, there was no evidence that the above antitumor effects is superior to the effect achieved by irradiated tumor cells.     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

Feasibility of UV-inactivated vaccinia virus in the modification of tumor cells for augmentation of their immunogenicity

Summary We compared the immunity induced by tumor cells modified with UV-inactivated purified vaccinia virus (UV-VV) and with live purified vaccinia virus (L-VV). C3H/HeN mice were inoculated i.p. with UV-VV or L-VV after whole-body irradiation with 150 rads of X-rays (priming). After 3 weeks the mice were immunized i.p. 3 times at weekly intervals with syngeneic X5563 or MH134 cells that had been adsorbed in vitro with UV-VV or infected with L-VV and subsequently irradiated with 10⁴ rads of X-rays. Then 1 week after the last immunization, the mice were challenged s.c. with X5563 viable tumor cells or challenged i.p. with MH134 viable tumor cells. The 50% lethal dose (TLD₅₀) of X5563 in mice primed and immunized with UV-VV (UV-VV group) on s.c. challenge (10^6.06) was the same as for mice treated with L-VV (L-VV group), whereas the TLD₅₀ of unprimed or nonimmunized mice (control group) was 10^2.61. The TLD₅₀ of MH134 in the UV-VV treated group on i.p. challenge (10^6.48) was similar to that of the L-VV treated group (10^6.54), while the TLD₅₀ of the control group was 10^1.00. The difference between the TLD₅₀ values of X5563 on s.c. challenge of mice primed and immunized with UV-VV or L-VV and control mice was 10^3.4. The difference between the TLD₅₀ values of MH134 on i.p. challenge of primed and immunized mice and control mice was 10^5.5. These results indicate that the in vivo helper function of UV-VV is similar to that of L-VV and that the augmenting effect of this protocol depends on the kind of tumor.     

Augmentation of immune response to syngeneic fibrosarcoma T241 with in vivo protection

Summary Lewis T241 fibrosarcoma, a syngeneic tumor in C57 BL/6J mice, was found to be poorly immunogenic. When tumor-bearing animals (TBA) were challenged with tumor cells either concomitantly or after excision of a growing tumor no protection was observed. In vivo (Winn) neutralization assays also showed a lack of tumor immunogenicity. However, in vitro studies showed that a significant proliferative response could be elicited from the spleen cells of TBA when these cells were cultured with either mitomycin-C-treated tumor cells or KCl tumor extract. Similarly, macrophage migration inhibition factor (MIF) was produced by TBA spleen cells upon incubation with KCl tumor extract, but no cell-mediated cytotoxicity to T241 target cells was observed with various lymphoid cell populations at any stage of tumor growth. Immunization of syngeneic animals with Vibrio cholerae neuraminidase(VCN)-treated, irradiated tumor cells alone or admixed with Freund's complete adjuvant (FCA) resulted in decreased tumor growth and fewer pulmonary metastases following challenge with 10⁶ tumor cells. No complete tumor rejection was observed. In contrast, 13 of 16 animals immunized with irradiated tumor cells admixed with FCA rejected 10⁵ tumor cells. Animals that grew tumors had significantly reduced tumor growths and pulmonary metastases. Lymph node and peritoneal exudate cells (PEC) of immunized animals showed significant cytotoxicity to T241 cells.     

Cellular immune response against tumor cells. I. In vitro immunization of allogeneic and syngeneic mouse spleen cell suspensions against DBA mastocytoma cells

Spleen cell populations stimulated in vitro with as few as 1000 tumor cells produce cytotoxic effector cells. Syngeneic as well as allogeneic spleen cells respond to DBA mastocytoma tumor cells. There is a significant cellular immune response to allogeneic tumor cells 72 hr after exposure to antigen. By contrast, the response of DBA spleen cells to DBA mastocytoma tumor cells is first detectable at 120 hr following exposure to antigen. C57BL/6 spleen cells immunized against DBA mastocytoma antigen kill both DBA mastocytoma tumor cells and normal cells from DBA animals. DBA spleen cells immunized against DBA mastocytoma antigen kill only the DBA mastocytoma tumor cells, and not normal cells from DBA animals.     

Species difference in increased vascular permeability by synthetic leukotriene C4 and D4

The activity of synthetic LTC₄ was tested in guinea-pig ileum and was 200 times more potent than histamine in contraction of the ileum (3 × 10^?11 M- 3 × 10^?9 M). The activities of LTC₄ and LTD₄ in increased vascular permeability in guinea pigs, rats and rabbits were compared with those histamine, bradykinin and prostaglandin (PG) E₂. LTC₄ was approximately equipotent to bradykinin on a molar basis in guinea pigs and rats and 5–100 times more potent than histamin. LTD₄ was about 10 times more potent than LTC₄ in guinea pigs and as equipotent to LTC₄ in rats. On the contrary, in rabbits, neither LTC₄ (upto 30 nmole/site) nor LTD₄ (1 nmole/site) induced the dye exduation. These results show that species difference is present in activity of LTC₄ and LTD₄ in vascular permeability. Furthermore, in guinea pigs, the vascular permeability increased by LTC₄ was not affected after pretreatment with pyrilamine (2.5 mg/kg, i.v.), and LTC₄ and LTD₄ did not potenciate the activity of bradykinin in vascular permeability.     

Augmentation of the therapeutic efficacy of adoptive tumor immunotherapy by in vivo administration of slowly released recombinant interleukin 2

Summary Immunization of C57BL/6 mice with MMC-treated syngeneic lymphoma cells, MBL-2, caused the generation of antitumor effector cells in vivo and the immunized mice permanently rejected viable MBL-2 lymphoma cells. Both plastic nonadherent T cells and plastic adherent MØ obtained from MBL-2 immunized mouse peritoneal exudate cells revealed strong cytotoxic activity against MBL-2 lymphoma cells, whereas immune spleen cells were not highly active against MBL-2 lymphoma cells in vitro. However, systemic adoptive transfer of immune spleen cells into the MBL-2-bearing mice by i.v. infusion in conjunction with i.p. cyclophosphamide (100 mg/kg) treatment cured the mice of tumor. This therapeutic efficacy of immune spleen cells was reflected by the number of transferred effector cells and over 5×10⁷ immune spleen cells were required to cure the mice completely. The cells mediating in vivo rejection of MBL-2 lymphoma cells were Thy 1.2⁺ T cells. This ACIT was specific against MBL-2 lymphoma cells and had no effect on the growth of other syngeneic tumors, B16 melanoma or BMC6A fibrosarcoma. In vivo administration of recombinant interleukin 2 (r-IL 2) combined with ACIT greatly modulated the cure rate of tumor-bearing mice. In addition, we found that slowly released r-IL 2 administratered from an ALZET miniosmotic pump was more effective in augmenting the therapeutic efficacy of immune spleen cells in ACIT than a single injection of the same total dose of r-IL 2.     

Biological response modifiers (BRM) as antigens

In order to investigate tumoricidal effector cells in therapy by biological response modifiers (BRM) such asPropionibacterium acnes, bacillus Calmette-Guérin (BCG),Streptococcus pyogenes and a protein-bound polysaccharide (PSK), we established T cell lines specific for each BRM from BALB/c mice immunized with the corresponding BRM. These T cell lines proliferated and produced interleukin-2-(IL-2) and/or IL-4, but only in the presence of the relevant BRM and BALB/c spleen cells as the antigen and antigen-presenting cells respectively. Cross-functional experiments indicated that each BRM acts as a nominal antigen, but not as a non-specific immunostimulator. In addition, the T cell lines killed Ia-positive syngeneic B lymphoma cells, but only in the presence of the relevant BRM. These experiments excluded the possibility of cytotoxic effects by each BRM. The T cell lines and clones also killed Ia-negative bystander target cells, but only in the presence of both a relevant antigen and antigen-presenting cells. The T cell clones specific forS. pyogenes orP. acnes tested were Thy1⁺, L3T4⁺ and Lyt2^–. These results indicate that some BRM exert tumoricidal activity by inducing T cells that recognize them as an antigen and kill tumor cells in an antigen-specific manner. The T cells killed tumor targets in either a tumor-necrosis-factor(TNF)-dependent or a TNF-independent manner. The mediator of the latter pathway remains to be elucidated.     

Non-specific immunosuppression in experimental cryptococcosis in rats

The delayed type hypersensitivity response to human serum albumin (HSA) of rats infected intraperitoneally with 10⁷ viable C. neoformans cells, and 7 days after, immunized with human serum albumin was significantly diminished (p<0.05) when compared with the response observed in rats immunized with human serum albumin and non infected. The spleen mononuclear cells from suppressed rats transferred to normal syngeneic recipients of the same sex suppress the afferent phase of the response (p<0.02) suggesting that cells present in the spleen might be one of the responsible mechanisms for the suppression to nonrelated antigens in infected animals.     

Adjuvant Activity of Mycobacterial Fractions

A quantitative assay and characterization of oil-attached cell wall of Mycobacterium bovis BCG (BCG-CWS) which stimulates cell-mediated immunity of spleen cells to alloantigens in mice were carried out by an in vitro cell-mediated cytotoxicity test using ⁵¹Cr-labeled target cells. C57BL/6J mice (H-2^b) were immunized intraperitoneally with mastocytoma cells (H-2^d) with or without oil-attached BCG-CWS. The cytotoxicity, comparable to that of spleen cells from mice immunized with mastocytoma cells (3 × 10⁷), could be induced in spleens of mice immunized with a mixture of mastocytoma cells (10⁴) and oil-attached BCG-CWS. The enhancing effect persisted for 55 days or more after the alloantigenic immunization. Oil-attached BCG-CWS enhanced cell-mediated cytotoxicity of T cells in the spleen and the mesenteric lymph node, but not in the thymus. The cytotoxicity showed specificity toward the alloantigen used for immunization. In addition to BCG-CWS, the cell walls of Nocardia rubra and Corynebacterium diphtheriae PW8 and the peptidoglycolipids of Mycobacterium tuberculosis Aoyama B were found to be potent stimulants of cell-mediated cytotoxicity in mice. Oil-attached BCG-CWS did not enhance humoral response to mastocytoma cells but enhanced cell-mediated cytotoxicity when viable mastocytoma cells were used as antigen. The above result was supported by the fact that anti-hapten antibody response induced by viable trinitrophenyl (TNP)-mastocytoma cells (10⁴) plus oil-attached BCG-CWS did not increase to the maximum level as was observed in mice immunized with a larger number of mastocytoma cells (3 × 10⁷) alone, while cell-mediated cytotoxicity induced by the same treatment increased to the maximum level obtained by immunization with mastocytoma cells (3 × 10⁷) alone.