Co-expression of immunogenic determinants by the same cellular immunogen is required for the optimum immunotherapeutic benefit in mice with melanoma

 Tumor-associated T cell epitopes are recognized by T cells in the context of determinants specified by class I loci. Since the rejection of foreign histocompatibility antigens is known to enhance tumor immunity, immunization with a cellular vaccine that combined the expression of both syngeneic and allogeneic class I determinants could have important immunological advantages over a vaccine that expressed either syngeneic or allogeneic determinants alone. To investigate this question in a mouse melanoma model system, we tested the immunotherapeutic properties of B16 melanoma × LM fibroblast hybrid cells in C57BL/6J mice with melanoma. Like C57BL/6J mice, B16 cells expressed H-2K^b class I determinants and (antibody-defined) melanoma-associated antigens. LM cells, of C3H mouse origin, formed H-2K^k determinants along with B7.1, a co-stimulatory molecule that can activate T cells. The B16 × LM hybrid cells co-expressed H-2K^b and H-2K^k class I determinants, B7.1 and the melanoma-associated antigens. C57BL/6J mice with melanoma, immunized with the semi-allogeneic hybrid cells, developed CD8-mediated melanoma immunity and survived significantly (P<0.005) longer than mice with melanoma immunized with a mixture of the parental cell types. The failure of melanoma immunity to develop in mice injected with the mixture of parental cells indicated that co-expression of the immunogenic determinants by the same cellular immunogen was necessary for an optimum immunotherapeutic effect. Augmented immunity to melanoma in mice immunized with the semi-allogeneic hybrid cells points toward an analogous form of therapy for patients with melanoma. Received: 19 May 1997 / Accepted: 23 July 1997     

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.     

Rejection of B16 melanoma induced by expression of a transfected major histocompatibility complex class I gene.

Transfection of a functional major histocompatibility complex class I gene into certain tumor cells, induced by oncogenic viruses or chemical carcinogens, can effectively abrogate their tumorigenic activity. Since experimentally induced tumors possess strong tumor-specific transplantation antigens, expression of cell surface class I antigens may present the tumor cells to appropriate immune effector cells. Most spontaneously arising tumors do not possess tumor-specific transplantation antigens, and their tumorigenicity may not be affected by the expression of a transfected class I gene. We demonstrate that the poorly immunogenic B16-BL6 melanoma can be rendered nontumorigenic in syngeneic mice by the expression of the class I H-2K antigen but not the class II I-A antigen. Furthermore, the poorly tumorigenic, class I-expressing B16-BL6-transfected cells can effectively immunize syngeneic C57BL/6 mice against the highly tumorigenic, class I-deficient B16-BL6 parental cells. Our success in experimentally manipulating the tumorigenicity of a spontaneously derived neoplasm offers hope for a potential modality for the effective treatment of human cancer.     

A single heteroclitic epitope determines cancer immunity after xenogeneic DNA immunization against a tumor differentiation antigen

Successful active immunization against cancer requires induction of immunity against self or mutated self Ags. However, immunization against self Ags is difficult. Xenogeneic immunization with orthologous Ags induces cancer immunity. The present study evaluated the basis for immunity induced by active immunization against a melanoma differentiation Ag, gp100. Tumor rejection of melanoma was assessed after immunization with human gp100 (hgp100) DNA compared with mouse gp100 (mgp100). C57BL/6 mice immunized with xenogeneic full-length hgp100 DNA were protected against syngeneic melanoma challenge. In contrast, mice immunized with hgp100 DNA and given i.p. tolerizing doses of the hgp100 D(b)-restricted peptide, hgp100(25-33), were incapable of rejecting tumors. Furthermore, mice immunized with DNA constructs of hgp100 in which the hgp100(25-27) epitope was substituted with the weaker D(b)-binding epitope from mgp100 (mgp100(25-27)) or a mutated epitope unable to bind D(b) did not reject B16 melanoma. Mice immunized with a minigene construct of hgp100(25-33) rejected B16 melanoma, whereas mice immunized with the mgp100(25-33) minigene did not develop protective tumor immunity. In this model of xenogeneic DNA immunization, the presence of an hgp100 heteroclitic epitope with a higher affinity for MHC created by three amino acid (25 to 27) substitutions at predicted minor anchor residues was necessary and sufficient to induce protective tumor immunity in H-2(b) mice with melanoma.     

Isolated soluble fractions from the murine B16 melanoma induce primary in vitro syngeneic antitumor responses

Summary This paper extends our previous studies, which documented our ability to isolate immunogenic entities from nonimmunogenic or weakly immunogenic tumors.B16 melanoma cells failed, in our in vitro experimental system, to induce anti-B16 cytotoxic responses in spleen cells derived from normal syngeneic C57BL/6 mice. The B16 melanoma cellular homogenate was fractionated on an Ultrogel AcA 34 column, and the various fractions were tested for their ability to induce anti-B16 cytotoxic responses under the same conditions as those used for intact B16, the nonimmungenic tumor cells. Certain fractions, some of them with relatively low protein concentrations, induced anti-B16 cytotoxic responses in spleen cells of normal C57BL/6 mice, whereas others, some of them with relatively high protein concentrations, failed to induce such responses. One fraction (Fr.), designated Fr. 5/6, was examined in detail. It was found that in normal syngeneic spleen cells this fraction induced effector cells that efficiently killed (at various E : T ratios) the relevant B16 target cells and RBL5 syngeneic tumor cells, but not the YAC allogeneic tumor cells or C57BL/6 lymphoblasts. Furthermore, an excess of unlabeled B16 cells most efficiently blocked the ability of these anti-B16 effector cells to kill radiolabeled B16 target cells. RBL5 tumor cells, YAC tumor cells, or C57BL/6 lymphoblasts failed to block these effector cells efficiently. A significant fraction of the effector cells induced with Fr. 5/6 was characterized as thymus-derived cells (Thy-1⁺, Thy-2⁺3⁺ cells). It was suggested that another fraction of the cellular population was natural killer cells, which cytolyzed the RBL5 target cells. Various theoretical and practical aspects of these findings are discussed.     

Brucella spp. Lumazine Synthase Induces a TLR4-Mediated Protective Response against B16 Melanoma in Mice

Brucella Lumazine Synthase (BLS) is a highly immunogenic decameric protein which can accept the fusion of foreign proteins at its ten N-termini. These chimeras are very efficient to elicit systemic and oral immunity without adjuvants. BLS signaling via Toll-Like Receptor 4 (TLR4) regulates innate and adaptive immune responses, inducing dendritic cell maturation and CD8+ T-cell cytotoxicity. In this work we study the effect induced by BLS in TLR4-expressing B16 melanoma. In order to evaluate the effectiveness of BLS as a preventive vaccine, C57BL/6J mice were immunized with BLS or BLS-OVA, and 35 days later were subcutaneously inoculated with B16-OVA melanoma. BLS or BLS-OVA induced a significant inhibition of tumor growth, and 50% of mice immunized with the highest dose of BLS did not develop visible tumors. This effect was not observed in TLR4-deficient mice. For treatment experiments, mice were injected with BLS or BLS-OVA 2 days after the inoculation of B16 cells. Both treatments induced significant and equal tumor growth delay and increased survival. Moreover, BLS and BLS-OVA stimulation were also effective in TLR4-deficient mice. In order to study whether BLS has a direct effect on tumor cells, B16 cells were preincubated with BLS, and after 48h, cells were inoculated. Tumors induced by BLS-stimulated cells had inhibited growth and survival was increased. In the BLS group, 40% of mice did not develop tumors. This effect was abolished by the addition of TLR4/MD2 blocking antibody to cells before BLS stimulation. Our work demonstrates that BLS immunization induces a preventive antitumor response that depends on mice TLR4. We also show that BLS generates a therapeutic effect in mice inoculated with B16 cells. Our results show that BLS acts directly in cultured tumor cells via TLR4, highly suggesting that BLS elicits its therapeutic effects acting on the TLR4 from B16 melanoma cells.     

Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model

We characterized the metastatic ability and mortality of four different mouse melanoma cell lines, B16-F0, -F1, -F10 and -BL6. B16-F0 is the parent cell line. B16-F1 was obtained by a one-time selective procedure and B16-F10 by a ten-time selective procedure using Fidler's method. B16-BL6 derived from B16-F10 has much more invasive activity than B16-F10. To investigate the difference in mortal malignancy among B16-F0, -F1, -F10 and -BL6, we examined the survival time of syngeneic C57BL/6Cr mice intravenously inoculated with these cells. As a control, we used the C57BL/6J-embryo mouse fibroblast-like semi-normal cell line. The ability to form lung metastatic nodules in mice gradually increased in the order: B16-F0, -F1, and -F10 (=-BL6). C57BL/6J-embryo cell (1 x 10(5)/mouse)-inoculated mice survived for over 46 days. B16-F0, -F1, -F10 and -BL6 (1 x 10(5)/mouse)-inoculated mice survived 31.4+/-4.4 (7), 25.7+/-2.8 (7), 23.6+/-1.5 (7) and 25.3+/-2.3 (7) days [mean+/-S.D. (number of mice)], respectively. According to the Mann-Whitney test, the B16-F0 inoculated group versus -F1 inoculated group (P<0.05), -F0 inoculated group versus -BL6 inoculated group (P<0.05), and -F0 inoculated group versus -F10 inoculated group (P<0.01) were significantly different, but the B16-F1 group versus -F10 group, -F1 group versus -BL6 group, and -F10 group versus -BL6 group were not. These results suggest that mortal malignancy is not necessarily correlated with lung-colonizing potential and even only one-time selected B16-F0 mouse melanoma cells are useful as an experimental metastatic model in vivo.     

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A

Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naïve mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vβ and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vβ T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls.?These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.     

Inhibition of pulmonary metastases of B16 melanoma with irradiated tumor cells and BCG

Summary When the tumor-bearing leg of C57BL/6J mice was amputated 16 days after SC inoculation of 10⁶B16 melanoma cells, all the amputated mice died of pulmonary metastases. Transfer of lungs from the amputated to normal syngeneic mice revealed tumor cells in the lungs just after amputation. Repeated weekly injections of BCG and irradiated tumor cells, beginning 24 h after amputation of the tumor-bearing limb, prolonged the survival only of mice presensitized to BCG. Injections of BCG or irradiated melanoma cells alone, of neuraminidase- and mitomycin C-treated tumor cells or of Levamisole had no effect, but injections of ConA-coated tumor cells slightly prolonged the survival of the amputated mice. Both BCG and B16 cells induced humoral and cell-mediated immunity but there was no cross-reactivity between BCG and B16 cells. Abbreviations used: ConA, concanavalin A; SC, subcutaneous; IP, intraperitoneal; IV, intravenous; ID, intradermal; IT, intratumoral; PBS, phosphate-buffered saline (0.01 M sodium phosphate, pH 7.1); VCN, Vibrio cholerae neuraminidase; HBSS, Hank's balanced salt solution; RPMIM, Roswell Park Memorial Institute medium     

Induction of antitumor immunity with dendritic cells transduced with adenovirus vector-encoding endogenous tumor-associated antigens.

Dendritic cells (DCs) are professional Ag-presenting cells that are being considered as potential immunotherapeutic agents to promote host immune responses against tumor Ags. In this study, recombinant adenovirus (Ad) vectors encoding melanoma-associated Ags were used to transduce murine DCs, which were then tested for their ability to activate CTL and induce protective immunity against B16 melanoma tumor cells. Immunization of C57BL/6 mice with DCs transduced with Ad vector encoding the hugp100 melanoma Ag (Ad2/hugp100) elicited the development of gp100-specific CTLs capable of lysing syngeneic fibroblasts transduced with Ad2/hugp100, as well as B16 cells expressing endogenous murine gp100. The induction of gp100-specific CTLs was associated with long term protection against lethal s.c. challenge with B16 cells. It was also possible to induce effective immunity against a murine melanoma self Ag, tyrosinase-related protein-2, using DCs transduced with Ad vector encoding the Ag. The level of antitumor protection achieved was dependent on the dose of DCs and required CD4+ T cell activity. Importantly, immunization with Ad vector-transduced DCs was not impaired in mice that had been preimmunized against Ad to mimic the immune status of the general human population. Finally, DC-based immunization also afforded partial protection against established B16 tumor cells, and the inhibition of tumor growth was improved by simultaneous immunization against two melanoma-associated Ags as opposed to either one alone. Taken together, these results support the concept of cancer immunotherapy using DCs transduced with Ad vectors encoding tumor-associated Ags.     

Involvement of tumor necrosis factor α and very late activation antigen 4/vascular cell adhesion molecule 1 interaction in surgical-stress-enhanced experimental metastasis

 We examined the influence of surgical stress on hematogenous metastasis of malignant tumor cells. The study was performed by focusing on the involvement of inflammatory cytokines in the serum, raised acutely after surgery, and endothelial adhesion molecules in the metastatic process. Surgical stress, given to C57BL/6 mice before B16-BL6 melanoma inoculation, significantly enhanced the pulmonary metastasis. This enhancement was seen when the surgery lasted for more than 2 h. After the 2-h surgery, the enhancement of pulmonary metastasis was seen most remarkably when B16-BL6 was inoculated 24 h after surgery. The serum level of tumor necrosis factor α (TNFα) in the mice that underwent the 2-h surgery peaked 12 h after the surgery. In contrast, serum interferon γ was not detectable. Administration of an anti-TNFα mAb before the surgery inhibited the enhanced metastasis by inhibiting the increased expression of vascular cell adhesion molecule 1 (VCAM-1) on lung vascular endothelium after the surgery. Pretreatment of B16-BL6 cells with an anti-very late activation antigen 4 (anti-VLA-4) mAb completely inhibited the enhanced metastasis after surgery. Administration of an anti-VCAM-1 mAb before surgery also inhibited the enhancement. These results indicate that serum TNFα , raised by surgical stress, is critically involved in the enhanced pulmonary metastasis of mouse melanoma by inducing VCAM-1 expression on lung vascular endothelium. Received: 22 January 1996 / Accepted: 1 April 1996     

Effects of pulsed magnetic stimulation on tumor development and immune functions in mice

We investigated the effects of pulsed magnetic stimulation on tumor development processes and immune functions in mice. A circular coil (inner diameter = 15 mm, outer diameter = 75 mm) was used in the experiments. Stimulus conditions were pulse width = 238 micros, peak magnetic field = 0.25 T (at the center of the coil), frequency = 25 pulses/s, 1,000 pulses/sample/day and magnetically induced eddy currents in mice = 0.79-1.54 A/m(2). In an animal study, B16-BL6 melanoma model mice were exposed to the pulsed magnetic stimulation for 16 days from the day of injection of cancer cells. A tumor growth study revealed a significant tumor weight decrease in the stimulated group (54% of the sham group). In a cellular study, B16-BL6 cells were also exposed to the magnetic field (1,000 pulses/sample, and eddy currents at the bottom of the dish = 2.36-2.90 A/m(2)); however, the magnetically induced eddy currents had no effect on cell viabilities. Cytokine production in mouse spleens was measured to analyze the immunomodulatory effect after the pulsed magnetic stimulation. tumor necrosis factor (TNF-alpha) production in mouse spleens was significantly activated after the exposure of the stimulus condition described above. These results showed the first evidence of the anti-tumor effect and immunomodulatory effects brought about by the application of repetitive magnetic stimulation and also suggested the possible relationship between anti-tumor effects and the increase of TNF-alpha levels caused by pulsed magnetic stimulation.     

Enhanced pulmonary natural killer cell activity during murine encephalitozoonosis

Experimental infection with the protozoan parasite Encephalitozoon cuniculi induced an augmentation of pulmonary natural killer cell (NK) activity in C57BL/6 mice. Enhanced clearance of 51Cr-labelled YAC-1 lymphoma cells peaked on day 4 of infection and returned to normal levels by the tenth day of infection. Infected mice also demonstrated heightened resistance to pulmonary tumor formation compared to uninfected control mice following challenge with lung-homing B16F10 melanoma cells.     

Induction of antigen-specific tumor immunity by genetic and cellular vaccines against MAGE: enhanced tumor protection by coexpression of granulocyte-macrophage colony-stimulating factor and B7-1.

BACKGROUND: A number of tumors express antigens that are recognized by specific cytotoxic T cells. The normal host immune responses, however, are not usually sufficient to cause tumor rejection. Using appropriate immunization strategies, tumor-specific antigens may serve as targets against which tumor-destructive immune responses can be generated. MAGE-1 and MAGE-3 are two clinically relevant antigens expressed in many human melanomas and other tumors, but not in normal tissues, except testis. Here, we have investigated whether DNA and cellular vaccines against MAGE-1 and MAGE-3 can induce antigen-specific anti-tumor immunity and cause rejection of MAGE-expressing tumors. MATERIALS AND METHODS: Mice were immunized against MAGE-1 and MAGE-3 by subcutaneous injection of genetically modified embryonic fibroblasts or intramuscular injection of purified DNA. Mice were injected with lethal doses of B16 melanoma cells expressing the corresponding MAGE antigens or the unrelated protein SIV tat, and tumor development and survival were monitored. RESULTS: Intramuscular expression of MAGE-1 and MAGE-3 by plasmid DNA injection and subcutaneous immunization with syngeneic mouse embryonic fibroblasts transduced with recombinant retroviruses to express these antigens induced specific immunity against tumors expressing MAGE-1 and MAGE-3. Both CD4+ and CD8+ T cells were required for anti-tumor immunity. Coexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) or B7-1 significantly increased anti-tumor immunity in an antigen-specific manner and resulted in a considerable proportion of mice surviving lethal tumor challenge. CONCLUSIONS: Our results suggest that genetic and cellular vaccines against MAGE and other tumor antigens may be useful for the therapy of tumors expressing specific markers, and that GM-CSF and B7-1 are potent stimulators for the induction of antigen-specific tumor immunity.     

Down-regulation of ras and myc expression associated with mdr-1 overexpression in adriamycin-resistant tumor cells.

The murine melanoma tumor cells, B16-BL6, are a recognized model for experimental and spontaneous metastasis. B16-BL6 cells express a lower metastatic phenotype upon acquisition of resistance to adriamycin. Using this novel system, the role of ras, c-myc, and multidrug-resistant gene (mdr1) expression in the metastatic and drug-resistant phenotype was examined. The metastatic cells expressed a high level of c-Ki-ras and c-myc, whereas down-regulation of both proto-oncogenes was observed in the adriamycin-resistant cells. The mdr1 gene, which encodes P-glycoprotein of the drug-resistant superfamily gene, was overexpressed in drug-resistant melanoma cells. These results suggest that altered expression of genes that regulate cellular proliferation and growth may be a determinant of metastasis and drug sensitivity of tumor cells.     

Induction of tumor immunity by intact irradiated leukemic B cells (BCL1) bearing a tumor-associated cell-surface idiotype and the costimulatory B7 molecule

The idioptypic (Id) determinant of immunoglobulin expressed on the cell surface of malignant B cells represents a prototypical tumor-associated antigen (TAA), which has been used in a purified soluble form for active immunization in experimental tumor models and human hematological malignancies. Using a spontaneous transplantable murine model of B cell leukemia/lymphoma (BCL1), we have demonstrated the expression of the B7 costimulatory molecules in addition to the previously described Id determinant and class II major histocompatibility antigens. Intact irradiated BCL1 cells bearing these distinct determinants induced long lasting antitumor immunity in naive syngeneic mice. Induction was dose-dependent and most effective when three doses of 30×10⁶ intact irradiated BCL 1 cells were given at intervals of 7–10 days. The induced immunity protected 96% of 28 mice inoculated with a lethal dose of 10⁵–10⁶ nonirradiated BCL1 cells and 85% of 27 mice given a second challenge, whereas control mice died on day 20 after inoculation with 10⁶ BCL1 cells. Adoptive transfer of splenocytes derived from immune mice did not induce leukemia in syngeneic recipients. Such splenocytes, harvested more than 365 days following immunization and administered together with fresh BCL1 cells to adoptive recipients, were able to confer protection for 90 days, even following a second challenge given 104 days after the first one. BCL1 immune splenocytes transferred into BCL1-bearing mice exerted a therapeutic effect, preventing leukemia onset for at least 180 days. Our results demonstrate the ability of tumor cells to trigger effective anti-tumor immunity. These findings could ultimately be applied to the prevention of tumor relapse in treatment of hematological and other malignancies expressing TAA, class II MHC antigen and costimulatory molecules.These studies were supported by grant 942010-B from the Israel Cancer Association     

Studies of the mechanisms for the induction of in vivo tumor immunity. VI. Induction of specific and nonspecific cell-mediated immunity in tumor-bearing hosts and its correlation with transplantation tumor immunity

Studies were performed to determine the development of cell-mediated cytotoxic response at tumor site in C57BL/6 mice bearing progressively growing FBL-3 ascites leukemia. The effectors isolated from tumor ascites are found to be highly cytotoxic for leukemic target cells. The levels of cytotoxicity obtained with effectors isolated from tumor site are generally higher than those obtained with immune mice. This cytotoxicity is both specific and nonspecific. The specific cytotoxicity against tumor-associated antigen is mainly mediated by T cells and the nonspecific cytotoxicity against unrelated tumor cells is mediated largely by macrophages. The T-cell-enriched preparation did not give significant natural killer activity. When testing the ability of these effectors to produce in vivo immunity against the challenge of FBL-3, it was found that only T cells could confer the transplantation-type immunity, but the immunity was transient. The macrophage-enriched preparation isolated from tumor ascites failed to give in vivo protection. These findings indicate that in FBL-3 system, mice with progressively growing tumors are able to develop immune response against tumor cells. However, this immunity is probably interfered with by a suppressor factor(s) or suppressor cells which restrict their activity to eliminate the tumor cells effectively.     

Characterization of Effector Cells against B16 Melanoma in Mice Inoculated with Allogeneic Spleen Cells

Inbred C57BL/6 (B6) mice which had received an inoculation of allogeneic spleen cells showed remarkable antitumor activity against syngeneic tumor challenge with B16 melanoma cells 3 days after the allogeneic cell inoculation. This antitumor activity was not specific to the inoculated alloantigen, since the challenging B16 cells are syngeneic to B6 mice and since it was induced by BALB/c spleen cells as well as C3H/He spleen cells. The antitumor activity was sensitive to an in vivo treatment with anti-asialo GM1 (AGM1) antiserum or anti-Thy.1 monoclonal antibody (mAb) just before the tumor challenge and was resistant to an in vivo treatment with anti-CD8 (Ly. 2) mAb. These results suggest that AGM1+Thy.1+CD8– activated natural killer (NK) cells were generated by alloantigen inoculation and took an important part in the antitumor effect of the alloantigen inoculation.     

Complete tumour regressions induced by vaccination with IL-12 gene-transduced tumour cells in combination with IL-15 in a melanoma model in mice

In the present study, IL-12 gene-transduced B78-H1 melanoma cells (B78/IL-12) were used in combination with IL-15 to treat melanoma-bearing mice. Genetically modified B78/IL-12 cells, when injected subcutaneously, induced strong activation of antitumour mechanisms resulting in complete loss of tumourigenicity. In a therapeutic model, intratumoural injection of irradiated B78/IL-12 cells significantly delayed tumour growth and led to the regression of melanoma in one case. Similarly, consecutive daily injections of IL-15 markedly inhibited tumour progression with occasional curative effects. When used in combination, vaccination with B78/IL-12 cells and treatment with IL-15 caused eradication of established tumours in all treated mice. The combined treatment with B78/IL-12 cells and IL-15 activated not only a local response against tumour, but also induced systemic antitumour immunity that led to a delay or inhibition of tumour development at a distant site. In vitro studies demonstrated that when used together, B78/IL-12 cells and IL-15 induced a shift from a type Th2 to a type Th1 response. Activation of the antitumour immune response in double-treated mice resulted, in part, from stimulation of IFN- production and was accompanied by the development of cytotoxic effectors in the spleen. As shown in a macrophage tumouricidal assay, macrophages could also play a role in the antitumour effects. The results confirmed that vaccination with IL-12 gene-modified tumour cells is superior to the treatment with unmodified tumour cell vaccine and, additionally, showed that IL-15 is an excellent candidate for adjuvant therapy, inducing synergistically not only a delay of tumour growth but also its complete eradication.     

Immunity to melanoma in mice immunized with transfected allogeneic mouse fibroblasts expressing melanoma-associated antigens

Summary Transfection of genomic DNA from B16 mouse melanoma into LM(TK^–) fibroblasts led to the generation of several clones of transfected cells that strongly expressed B 16 melanoma-associated antigens (MAA). The transfected cells retained their H-2^k markers and served as allogeneic cells with expressive MAA in C57BL/6 mice, syngeneic with the melanoma. The cells were capable of eliciting primary anti-B16 immune responses in vitro in spleen cells from C57BL/6 mice. Immunization of C57BL/6 mice with the transfected cells led to the generation of anti-B16 cytotoxic activity in spleen cells, and C57BL/6 mice immunized with the MAA-positive transfected cells were partially resistant to a lethal challenge with B16 melanoma cells. Under similar conditions, B16 cells were nonimmunogenic. Therefore, transfected allogeneic LM(TK^–) fibroblast cells expressing MAA served as more potent anti-melanoma immunogens than the parental B16 tumor cells themselves.