Eradication of tumor cells after injection into immunized hosts compared with the eradication of tumor cells after transfer of immune peritoneal exudates into tumor-bearing recipients

Summary DBA/2 mice were immunized against the syngeneic SL2 lymphoma by two or five injections with irradiated lymphoma cells given IP or SC, respectively. The antitumor efficacy induced in immunized mice was tested by (a) IP injection of the immunized mice with nonirradiated tumor cells and (b) transfer of the total immune peritoneal exudate, the cellular fraction only, or the cell-free fraction only, IP into tumor-bearing recipients, or (c) tumor neutralization tests (Winn assay). It was shown that immunized mice were able to reject 5×10⁷ SL2 tumor cells (8 of 14 mice survived >100 days), while in most transfer experiments 2×10⁵ SL2 cells could be eradicated. In the tumor neutralization experiments a number of 10⁶ SL2 cells were eradicated. When the immune exudates were given before the inoculations of SL2 tumor cells the number of survivors increased significantly. Further, it was shown that the cellular fraction is the major contributor to the antitumor effect in the transfer experiments, since there was no significant difference in tumor eradication after injection of a complete immune exudate and after injection of the isolated cellular fraction. Injection of the noncellular fraction had no measurable antitumor effect. An increase in the number of injections with total peritoneal exudates from immunized mice did not result in an increase in tumor eradication in the tumor-bearing recipients.Extra stimulation (IP) of immunized mice with 10⁴ nonirradiated cells 6 days after the last immunization resulted in an increase of the antitumor efficacy of these peritoneal exudates of these mice when collected 4–24 h after this stimulation. Extra stimulation with 10⁶ irradiated cells had no measurable effect.     

Interleukin-5 induces tumor suppression by peritoneal exudate cells in mice

The antitumor activity of peritoneal exudate cells (PEC) induced by murine interleukin-5 (mIL-5) was examined using Meth-A sarcoma cells transplanted into the peritoneal cavity of mice. Although in vitro treatment of Meth-A sarcoma cells with mIL-5 did not result in inhibition of their growth, treatment of mice intraperitoneally with mIL-5 (1 g/day) from day –5 to +5 (tumor cells were inoculated on day 0) led to a significant increase in survival or even rejection of tumor cells. This antitumor effect depended on the dose of mIL-5. Interestingly, there was identical therapeutic activity when the protocol of days –10 to –1 was used as opposed to –5 to +5. In addition, post-treatment with mIL-5 from day +1 to +10 was ineffective. This suggests that the therapeutic activity of IL-5 is largely prophylactic. Under the former condition, the number of PEC was found to increase over 50-fold when compared to levels in control mice. Moreover, the antitumor effect of mIL-5 was completely abolished by subcutaneous injection of anti-mIL-5 monoclonal antibodies. The treatment of mice injected intraperitoneally with human IL-2 also resulted in an increase in survival. Winn assay experiments using PEC recovered from mIL-5-treated mice (1g/day, from day –10 to –1) revealed that these PEC could mediate antitumor activity against Meth-A sarcoma cells. Furthermore, when the cured mice were re-injected with Meth-A sarcoma cells or syngeneic MOPC 104E cells, they could reject Meth-A sarcoma cells but not MOPC 104E cells, indicating that immune memory had been generated. These results suggest that IL-5 augumented the PEC tumoricidal activity but we have no indication that the tumoricidal activity was mediated through a mIL-5-dependent mechanism.     

Activation of anti-tumor immune response and reduction of regulatory T cells with Mycobacterium indicus pranii (MIP) therapy in tumor bearing mice

Background

Role of immune system in protecting the host from cancer is well established. Growing cancer however subverts immune response towards Th2 type and escape from antitumor mechanism of the host. Activation of both innate and Th1 type response is crucial for host antitumor activity. In our previous study it was found, that Mycobacterium indicus pranii (MIP) also known as M. w induces Th1 type response and activates macrophages in animal model of tuberculosis. Hence, we studied the immunotherapeutic potential of MIP in mouse tumor model and the underlying mechanisms for its antitumor activity.

Methodology and Principal Findings

Tumors were implanted by injecting B16F10 melanoma cells subcutaneously into C57BL/6 mice. Using the optimized dose and treatment regimes, anti-tumor efficacy of heat killed MIP was evaluated. In MIP treated group, tumor appeared in only 50–60% of mice, tumor growth was delayed and tumor volume was less as compared to control. MIP mediated immune activation was analysed in the tumor microenvironment, tumor draining lymph node and spleen. Induction of Th1 response and higher infiltration of immune cells in the tumor microenvironment was observed in MIP treated mice. A large fraction of these immune cells were in activated state as confirmed by phenotypic and functional analysis. Interestingly, percentage of Treg cells in the tumor milieu of treated mice was less. We also evaluated efficacy of MIP along with chemotherapy and found a better response as compared to chemotherapy alone.

Conclusion

MIP therapy is effective in protecting mice from tumor. It activates the immune cells, increases their infiltration in tumor, and abrogates tumor mediated immune suppression.     

A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation

Background

Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation on tumor growth.

Methods

Three-month old CBA/Lac mice were used in the experiments. Mice were injected with CP and human dsDNA preparation. The percentage of mature dendritic cells (DCs) was estimated by staining of mononuclear cells isolated from spleen and bone marrow 3, 6, and 9 days later with monoclonal antibodies CD34, CD80, and CD86. In the next set of experiments, mice were given intramuscularly injections of 1-3 × 10⁵ tumor cells. Four days later, they were injected intravenously with 6-6.7 mg/kg Dox and intraperitoneally with 100-200 mg/kg CP; 200 mkg human DNA was injected intraperitoneally after CP administration. Differences in tumor size between groups were analyzed for statistical significance by Student's t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups.

Results

The conducted experiments showed that combined treatment with CP and dsDNA preparation produce an increase in the total amount of mature DCs in vivo. Treatment of tumor bearers with preparation of fragmented dsDNA on the background of pretreatment with Dox plus CP demonstrated a strong suppression of tumor growth in two models. RLS, a weakly immunogenic, resistant to alkalyting cytostatics tumor, grew 3.4-fold slower when compared with the control (p < 0.001). In experiment with Krebs-2 tumor, only 2 of the 10 mice in the Dox+CP+DNA group had a palpable tumor on day 16. The cytotoxic index of leucocytes was 86.5% in the Dox+CP+DNA group, but it was 0% in the Dox+CP group.

Conclusions

Thus, the set of experiments we performed showed that exogenous dsDNA, when administered on the background of pretreatment with Dox plus CP, has an antitumor effect possibly due to DC activation.     

Immunotherapy of tumor-bearing mice utilizing virus help

Summary Utilizing vaccinia virus (VV), a tumor-specific immunotherapy model was established in which a growing tumor regressed. C3H/HeN mice were primed with VV after low dose irradiation to generate amplified VV-reactive T cell activities. Then 4 weeks later, the mice were inoculated i. d. with syngeneic MH134 hepatoma cells, and 6 days after the tumor cell inoculation, live VV was injected into the tumor mass 3 times at 2-day intervals. Of 10 mice which had received VV priming and subsequent VV injection into the tumor mass, 8 exhibited complete tumor regression. On the contrary, mice which had received only intratumoral VV injection without VV priming failed to exhibit appreciable tumor regression. Mice whose tumor had completely regressed following the VV immunotherapy were shown to have acquired systemic antitumor immunity, which was confirmed by a challenge with syngeneic tumor cells after immunotherapy. In vitro analysis of these immune mice revealed that potent tumor-specific antibody responses were preferentially induced, but with no detectable antitumor cytotoxic T lymphocyte (CTL) responses. Such a potent tumor-specific immunity was not observed in mice which had received intratumoral VV injection in the absence of VV priming. Thus, the results clearly indicate that tumor regression was accompanied by the concurrent generation of a potent tumor-specific immunity, suggesting that cellular cooperation between VV-reactive T cells and tumor-specific effector cells might be functioning in this VV immunotherapy protocol. Therefore, the present model provides an effective maneuver for tumor-specific immunotherapy. This system is, in principle, applicable to the human situation.     

Augmentation of the antitumor effect of adoptive immunotherapy by in vivo sensitization of EL-4 lymphoma and pre-treatment with sizofiran

The antitumor effects of adoptive immunotherapy using LAKcells treated with sizofiran (SPG) following in vivoantigen sensitization with EL-4 lymphoma (EsLAK),comparing nonsensitized LAK cells (sLAK), were studied inmice with intraperitoneal implantation of EL-4 lymphoma.EL-4 cells treated with Mitomycin C (100 g /ml) wereintroduced by inoculation into the peritoneum of C57BL/6mice for antigen sensitization. Four days later, SPG (100g) was intramuscularly injected. Three days after SPG administration, mononuclear cells obtained from the spleen were prepared for LAK cells (EsLAK). The following resultswere obtained: 1) The survival period was significantlygreater in the sLAK and EsLAK groups than in the controlgroup. The survival period in the EsLAK group wassignificantly greater than that in the sLAK group. 2) Thenumber of EL-4 cells in the peritoneal exudate cells 11days postimplantation was lowest in the EsLAK group, andthe number of lymphocytes including LGL was largest in theEsLAK group, compared with the sLAK group and the controlgroup. 3 ) The EsLAK cells showed significantly moreenhanced cytotoxic activity against EL-4 than the sLAKcells. 4) Histopathological findingsof metastatic lesions of the liver and spleen stained by HE11 days postimplantation showed less infiltrating tumorcells and more lymphocytic infiltrations in the sLAK andEsLAK groups compared with the control group. Theseresults suggest that induction of LAK cells byadministration of SPG to lymphocytes treated by in vivosensitization with tumor antigen increasesthe efficacy of adoptive immunotherapy.     

Propionibacterium acnes Augments Antitumor,Anti-Angiogenesis and Immunomodulatory Effects of Melatonin on Breast Cancer Implanted in Mice

Breast cancer is one of the most invasive cancers with high mortality. The immune stimulating Propionibacterium acnes is a Gram positive bacterium that has the ability to cause inflammation and activate Th1-type cytokine immune response. Antitumor response was associated with the inflammation induced by P. acnes, but the antitumor effect of this bacterium was not evaluated in combination with other agents. The aim of this study was to test the antitumor potential of a combination of melatonin and P. acnes against breast cancer implanted in mice. Balb/C mice were transplanted with EMT6/P cell line and in vivo antitumor effect was assessed for P. acnes, melatonin, and a combination of melatonin and P. acnes. Tumor and organs sections were examined using hematoxylin/eosin staining protocol, and TUNEL colorimetric assay was used to detect apoptosis. The expression of vascular endothelial growth factor (VEGF) was measured in tumor sections and serum levels of INF-γ, and IL-4 were measured to evaluate the immune system function. To evaluate the toxicity of our combination, AST and ALT levels were measured in the serum of treated mice. The combination of melatonin and P. acnes has high efficiency in targeting breast cancer in mice. Forty percent of treated mice were completely cured using this combination and the combination inhibited metastasis of cancer cells to other organs. The combination therapy reduced angiogenesis, exhibited no toxicity, induced apoptosis, and stimulates strong Th1-type cytokine antitumor immune response. The combination of melatonin and P. acnes represents a promising option to treat breast cancer. However, carful preclinical and clinical evaluation is needed before considering this combination for human therapy.     

Protein A-induced apoptosis of cancer cells is effected by soluble immune mediators

Since Protein A (PA) of Staphylococcus aureus has been documented to have both antitumor and immunostimulatory properties, we attempted to determine whether PA-induced tumor cell death was effected through the immune system of the host, and analyze the mechanisms of such anti-tumor activity. For in vivo studies, Ehrlich's ascites carcinoma (EAC) cells were inoculated into the peritoneal cavity of Swiss albino mice. PA (1 micro g/20 g body weight) was injected biweekly for 2 weeks. To determine the role of immunomodulators in PA-induced tumor cell death, EAC were co-cultured with PA-primed splenic cells or with the spent medium of the same. Our results indicated a "two-step" mechanism of the induction of apoptosis in tumor cells, by PA, i.e. (1) activation of the immune system of the host to release different apoptogenic factors like tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO); and (2) induction of EAC apoptosis by these soluble immune mediators through the up-regulation of pro-apoptotic factors (p53 and Bax) and down-regulation of anti-apoptotic factor (Bcl-2), resulting in the activation of caspase-3. The present observations provide additional findings on an approach to cancer immunotherapy that causes apoptogenic insult to cancer cells.     

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus

Summary Two IgE-producing hybridomas were established from spleen cells of Balb/c mice, which had been immunized with mouse mammary tumor virus (MMTV). These IgE monoclonal antibodies (mAbs) reacted specifically with the major envelope glycoprotein (gp36) of MMTV, as established by the immunoblot assay and by passive cutaneous anaphylaxis. The effect of the IgE mAbs (produced by clone A8) on the growth of the MMTV-secreting mammary adenocarcinoma H2712 was investigated in syngeneic C3H/HeJ mice. The mice were inoculated s.c. with either 10⁵ (100 × LD₅₀) or 10⁶ (1000 × LD₅₀) tumor cells and received repeated i.p. injections of 25 µg anti-gp36 IgE mAbs at 4-day intervals for 8 weeks. This treatment prevented the development of subcutaneous tumors in 50% of the animals. Similar protection was observed when the tumor cells (10⁵/animal) were injected i.p. 4 days prior to the beginning of the i.p. treatment consisting of injections of 25 µg mAbs at 4-day intervals for 6 weeks. However, these mAbs did not protect C3H/HeJ mice against the MMTV-negative MA16/c carcinoma cells. Hence, these results support the view that IgE-mediated cytotoxic mechanisms may play an immunologically specific antitumor surveillance role and that laboratory-induced antitumor IgE mAbs have the potential of specific therapeutic agents for in vivo destruction of tumor cells.     

Analysis of effector cells in tumor-bearing mice pre-treated with active specific immunization followed by cyclophosphamide

In order to analyse the effector population in an immunization model, we treated BALB/c mice with intraperitoneal (i.p.) active specific immunization (ASI), which consists of interleukin (IL)-1- and sonicated tumor supernatant (SS) of a plasmacytoma MOPC-104E followed by i.p. injection of cyclophosphamide (CY). This ASI-CY treatment provoked a protective immunity against i.p. tumor inoculation more strongly than that of ASI alone. The main effector cells in tumor neutralizing assay were CD4+ T cells at this pont. The number of spleen cells of the ASI-CY treated mice were significantly lower than that of ASI alone treated mice but it increased significantly 6 days thereafter while this increase was not observed on the mice treated with ASI alone. The spleen cells of the ASI-CY treated mice responded to SS in vitro in the presence of IL-2, more profoundly in CD4 enriched population which produced high amount of TNF-. In vivo tumor-neutralizing activity at a later stage was dependent on CD8+ T cells in addition to CD4+ T cells. These results suggest that antitumor activity by ASI and CY is transduced by sequential population shift from CD4 alone to both of CD4 and CD8.     

In situ recruitment of antigen-presenting cells by intratumoral GM-CSF gene delivery

Proper antigen presentation is paramount to the induction of effective and persistent antitumor immune responses. In a murine model of hepatic metastasis of colon cancer, we found that the numbers of in situ mature dendritic cells (DCs) and macrophages in tumor-infiltrating leukocytes (TILs) were significantly increased in mice treated with the combination therapy of herpes simplex virus thymidine kinase, interleukin 2, and GM-CSF genes when compared with control groups without GM-CSF treatment. Significantly higher levels of IFN-, MIP-1, mIL-12, and GM-CSF were detected in the tumor after the combination therapy. T cells isolated from the combination therapy–treated mice exhibited higher ex vivo direct CTL activity than those from other treatment groups. Antigen-presenting cells (APCs) enriched from the TILs and liver of the combination therapy–treated mice induced higher levels of proliferation by the splenocytes from long-term surviving mice that had been cured of tumors at early time points (days 4 and 7) whereas significant APC activity was only observed in the spleen at the latter time point (day 7, 14) after the combination therapy. In contrast, APCs isolated from tk or tk + IL-2–treated mice did not induce any significant proliferation. Subcutaneous injection of fluorescence-labeled latex microspheres followed by the combination therapy showed a similar sequential trafficking of microspheres, day 4 after the combination therapy to tumor and day 14 to spleen. The results suggest that APCs recruited by intratumoral gene delivery of GM-CSF can capture antigens, mature to a stage suitable for antigen presentation, and subsequently migrate to the spleen where they can efficiently stimulate antigen-specific T cells.P.-Y. Pan and Y. Li contributed equally to this work.     

Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice

The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/ c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (106 cells) and left flank (2 × 105 cells), and were then injected with 5 mg of extracts of A. blazei in the right tumor on days 3, 4 and 5. Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibitition of growth of the left, non-injected tumor. The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments. The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3. When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 × 107 cells/mouse) into the Meth-A tumor, tumor growth was inhibited. The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity. Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue. Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3. These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.     

Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope

In this study, we developed a mouse model of adoptive immunotherapy reflecting immune recognition of syngeneic tumor cells naturally expressing an endogenous rejection Ag. Specifically, in a pulmonary metastases model, we examined the potency and maintenance of an antitumor CD8(+) CTL response in vivo, as well as its effectiveness against an "extensive" tumor burden. The approach taken was to first generate tumor-specific CTL from mice challenged with the CMS4 sarcoma coadministered with anti-CTLA4 mAb, which has been shown to facilitate the induction of Ag-specific T cell responses in vivo. An H-2L(d)-restricted nonamer peptide, derived from an endogenous murine leukemia provirus was identified as a CMS4-reactive CTL epitope based upon the following: CTL cross-recognition of another syngeneic tumor cell line (CT26 colon carcinoma) previously characterized to express that gene product; sensitization of Ag-negative lymphoblasts or P815 targets with the peptide; and by cold target inhibition assays. In vivo, the adoptive transfer of CMS4-reactive CTL (> or =1 x 10(6)) resulted in nearly the complete regression of 3-day established lung metastases. Furthermore, mice that rejected CMS4 following a single adoptive transfer of CTL displayed antitumor activity to a rechallenge 45 days later, not only in the lung, but also at a s.c. distal site. Lastly, the adoptive transfer of CTL to mice harboring extensive pulmonary metastases (> 150 nodules) led to a substantial reduction in tumor burden. Overall, these data suggest that the adoptive transfer of tumor-specific CTL may have therapeutic potential for malignancies that proliferate in or metastasize to the lung.     

Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor α combination treatment of EL4-lymphoma-bearing C57BL/6 mice

A combination treatment protocol initiated 12 days after tumor injection, when the tumor was large, by administering cyclophosphamide (CY, 150 or 250 mg/kg) intraperitoneally followed by intravenous tumor necrosis factor (TNF, 1000 units/injection) on days 13, 16, 18, 21, and 23, resulted in about 60% longterm survival (i.e., survival for at least 60 days) in the syngeneic C57BL/6 mouse/EL4 lymphoma model system. The establishment of a specific antitumor immune memory and its possible therapeutic relevance was verified by reinjecting 60-day survivors with EL4 cells; all 60-day survivors that had received the combination treatments rejected the implants and survived for a further 60 days. Thymic cellularity was reduced during treatment and its recovery appeared to correlate with long-term survival and immunity. Thymocytes from mice treated with the combination were found to express significant levels of specific anti-EL4 cytolytic activity following a 4-day stimulation culture with X-irradiated EL4 cells and low concentrations of interleukin-2. This response could not be generated with thymocytes from naive animals. In each case the effect seen with the combination of a moderate CY dose (150 mg/kg) with TNF was better than that seen with either dose of CY alone and equal to or better than that seen with the higher dose of CY combined with TNF. These results indicate that treatment with a single moderate dose of CY in combination with TNF is effective against a large, established tumor in this murine model. Furthermore, all the long-term survivors induced by this treatment developed protective immunity against reimplanted tumor and demonstrated a long-term specific immune memory in the thymus.This work was done in partial fulfillment of the M.S. degree requirements (C. M. K.) in the Program of Natural Sciences, Roswell Park Graduate Division, SUNY at Buffalo     

Transfer of antitumor immunity with ribonucleic acid-containing spleen extract of immunized animals

Summary Ribonucleic acid-containing spleen extract (i-extract) was prepared from the spleens of C57BL/6 mice immunized with mammary carcinoma Ca755. The i-extract contained a factor which could transfer antitumor immunity into the recipient mice, since the tumor growth was significantly retarded if mice received IP injections of i-extract at the same time as or at 6 days after tumor transplantation. Little or no inhibition of tumor growth was observed in mice which received injections of i-extract 6 days prior to tumor transplantation.Tumor growth was also inhibited in mice which had received live attenuated strain (SER) Salmonella enteritidis by IV injection 6 days prior to the tumor transplantation, whereas no growth inhibition was observed in mice which were treated by injection of live SER strain of S. enteritidis simultaneously with the tumor transplantation.Tumor growth was synergistically inhibited if mice received live SER by injection 6 days prior to and i-extract 6 days after tumor transplantation, and an extended survival was observed.     

Induction of antitumor immunity after cure of pulmonary metastases, using staphylococcal enterotoxin B and bispecific antibody

The combination of staphylococcal enterotoxin B (SEB) and anti-p97 x anti-CD3 bispecific antibody (bsAb) cures 60%-80% of mice with established pulmonary metastases of the syngeneic p97+ murine melanoma, CL62. We investigated the ability of cured mice to generate protective antitumor immunity. In tumor rechallenge experiments, CL62-cured mice developed protective immunity against rechallenge with CL62. The majority of mice also rejected the p97-negative parental cell line, K1735, indicating an immune response to tumor antigens common to both cell lines that were not bsAb-targeted. A significant humoral response developed against p97 antigen, but not against other antigens common to both CL62 and K1735. That the majority of cured mice nevertheless rejected K1735 suggests that tumor immunity is not antibody-dependent. Evidence of cellular immunity was obtained from the results of delayed-type hypersensitivity, proliferation and cytotoxicity assays, which revealed the presence of tumor-specific memory in bsAb-treated, CL62-cured mice. CD8+ T cells from cured, but not control mice were able to lyse tumor; however, memory CD4 cells had no cytolytic function. In vivo, however, both CD4 and CD8 T cells were required for effective protective immunity. These studies demonstrate that treatment with SEB and bsAb not only confers passive immune effects of tumor eradication, but also actively promotes the generation of a host antitumor immune response.     

Production of specific macrophage-arming factor precedes cytotoxic T lymphocyte activity in vivo during tumor rejection

Summary Recently we published a hypothesis on the immunological events occurring during tumor rejection. One of the implications of this hypothesis is that specific macrophage-arming factor (SMAF) is produced early during the initiation of the immune response, whereas the classical cell-mediated immune response components, such as cytotoxic T lymphocytes (CTL), are produced later, that is, during the amplifier-effector phase. In this paper we establish the kinetics of the induction of (a) lymphocytes producing SMAF and (b) CTL. Groups of DBA/2 mice were injected i.p. once, twice or three times with irradiated and/or non-irradiated syngeneic SL2 tumor cells, the injections being given at intervals of 10 days. After each of these injections the production of SMAF and the expression of CTL activity were established. The results showed that in the peritoneal cavity SMAF-producing lymphocytes appeared earlier than cytotoxic lymphocytes (CTL). In addition, it was shown (a) that SMAF does not interfere with the in vitro cytotoxicity expressed by CTL and (b) that in addition to CTL memory cells, SMAF-producing memory cells were also induced after injection of syngeneic tumor cells. These data support the hypothesis that SMAF is involved in the early phase of the cellular immune response against tumors, whereas CTL are induced later.     

The “double grafted tumor system”, proposed to find effector cells in the analyses of antitumor effect of BRMs

The antitumor effects of three biological response modifiers (BRMs; PSK, IFN A/D and OK432) and two chemotherapeutics (Mitomycin C and Neocarzinostatin) in a new experimental mouse model, the double grafted tumor system, were evaluated. BALB/c mice received simultaneous inoculations of Meth A fibrosarcoma cells on right flank (1 × 10⁶ cells) and left flank (2 × 10⁵ cells) on day 0, and drugs were given intratumorally into the right-flank tumor on day 3. The growth of the left-flank tumor was the real target for the evaluation of a given drug after 21 days. All tested five agents successfully cured the drug-injected right tumor with a pre-determined optimum dose. In addition, PSK, OK432, IFN A/D and MMC among the five, inhibited the left-flank tumor, whereas no inhibition was observed when treated with NCS. To understand the mechanism by which the antitumor effect of the above four agents is able to influence the growth of tumor on the other side, tumor cells (2 × 10⁵ cells) inoculated only into the left flank were treated with drugs given subcutaneously to the right flank (single tumor system). Among the four, MMC exhibited an effect similar to that obtained in the double tumor system, and IFN A/D showed a less pronounced but still definite antitumor effect. However, PSK and OK432 failed to express anti-tumor effect in the single tumor system. These results obtained with PSK, OK432 and IFNA/D suggest that the effect of the drug on the left-tumor may be mediated by certain effector cells, which are specifically induced by injection of the drug, in the right-tumor tissues. When effector cell analysis was conducted with spleen cells obtained after PSK treatment by means of intratumoral adoptive transfer into 3-day Meth A bearing recipients, these cells were shown to be Lyt-1⁺2^–-T and L3T4⁺-T cell.     

Modulation of the immune response to tumors by a novel synthetic compound,N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide (CL 259,763)

Summary CL 259,763, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide, is an orally active compound capable of modifying the reactivity of certain lymphoid cell populations affected by the growth of a tumor. The compound augmented the response of lymphocytes from tumor-primed animals to syngeneic tumor cells, resulting in a marked increase in tumor cell destruction. Likewise, it enhanced macrophage inhibitory effects on the growth of tumor cells in vitro. These activated macrophages were detectable in peritoneal exudates of treated mice 4 to 12 days after receiving a single oral dose of CL 259,763, with peak activity being demonstrable by day 7. The compound also restored the alloreactivity of lymphocytes from immunodepressed mice bearing the Lieberman plasma cell tumor, possibly by interferring with suppressor cells. Macrophages and lymphocytes from treated mice released significantly more IL-1 and IL-2-like factors in culture than did the control counterparts. Sera from treated mice also possessed more colony stimulating factor than those from normal mice. Immunoadjuvant effects were evident when the compound was administered with an inactivated L1210 leukemia vaccine and it enhanced the effectiveness of cytotoxic chemotherapy when given to mice challenged with P388 murine leukemia. These immunomodulating effects of CL 259,763 may hopefully be exploited in efforts to augment the immune response of the host to a progressively growing tumor.     

I-Ad antigen expression of pyran copolymer-induced peritoneal cells in tumor vaccine-primed mice and its association with the host antitumor response

Summary Mice inoculated IP with L1210 murine leukemia vaccine and subsequently with pyran copolymer-induced macrophages (pyran-M) lived for a prolonged time after live L1210 inoculation IP. Pyran-M as tentatively identified by anti-AcM.1 monoclonal antibody expressed I-A^d antigen in tumor vaccine-primed recipient mice and contributed to maintaining I-A^d antigen positive (I-A^d+) macrophages at high cell density in the peritoneal cavity of recipient mice. The relevance of these I-A^d+ cells to the host antitumor response was examined by experiments in which I-A^d+ cell density in the peritoneal cavity and host antitumor response behaved in a parallel fashion. Human interferon-A/D, an agent selectively inhibiting Ia antigen expression, and silica, a general antimacrophage agent, strongly suppressed I-A^d antigen expression of peritoneal mcrophages of tumor vaccine-primed and pyran-M-inoculated mice and, consistently with this, the antitumor response was nullified in these mice. Tumor vaccine-primed mice inoculated with sodium caseinate or thioglycollate-induced peritoneal cells did not survive L1210 inoculation and, in these mice, I-A^d+ peritoneal macrophages were suppressed in number as compared with those of tumor vaccine-primed and pyran-M-inoculated mice. These results warrant further study on the contribution of I-A^d+ macrophages to pyran copolymer-induced augmentation of the antitumor response in tumor vaccine-primed mice.