Dendritic cells fused with mastocytoma cells elicit therapeutic antitumor immunity

Characterization of the spontaneous immune response that frequently occurs in tumor-bearing animals, as well as immunization using dendritic cells pulsed with tumor antigens, suggests that a limiting factor of the tumor-specific immune response may be a defect in the co-stimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach to improve the antigen-presenting capacity of tumor cells, which does not require a source of purified tumor-associated antigen. We fused P815 mastocytoma cells with bone marrow-derived dendritic cells. We obtained one hybrid that displayed the phenotypic and functional properties of dendritic cells and expressed mRNA coding for the tumor-associated antigen P815 A/B. Injections of irradiated hybrid cells prevented the growth of pre-implanted mastocytoma and induced long-lasting tumor resistance. Int. J. Cancer76:250–258, 1998.© 1998 Wiley-Liss, Inc.     

Dendritic cells as adjuvants in antitumor immune therapy

While there has been considerable progress in the development of techniques to identify tumor-associated antigens, the traditional methods for delivering these antigens in the context of a tumor vaccine are, in many cases, crude and inadequate. Most adjuvants that are in principle available for such a vaccine have been discovered empirically and their mechanism of immune-stimulatory action is poorly understood. In addition, preclinical studies suggest that most of the conventional adjuvants often fail to elicit activation of both the humoral and the cellular arm of the immune system. Among other reasons, these findings have led to the application of dendritic cells (DCs) as adjuvants. In such experiments DCs were pulsed in vitro with tumor antigens which, upon in vivo application, caused tumor rejection in experimental mouse tumor systems, and such preparations indeed increased antitumor immunity in cancer patients. Recent advances in the understanding of the function of DCs and their first clinical applications in antitumor immune therapy are described.     

Hep-2细胞总RNA转染树突状细胞诱发抗喉癌特异性免疫效应的研究

目的:研究转染喉鳞状细胞癌总RNA对树突状细胞(DC)免疫功能的影响,观察肿瘤总RNA修饰后的DC在体外诱导高效而特异的抗喉癌免疫效应.方法:采用分离脐血单核细胞体外诱导DC,Trizol法提取喉鳞状细胞癌细胞株Hep-2总RNA后转染入DC,流式细胞仪分析DC表面分子CD40、CD80、CD83、CD86的表达,LDH法评估转染肿瘤总RNA的DC瘤苗的细胞毒性T淋巴细胞反应.结果:转染后的DC表面分子表达CD40(60.6±0.15)%,CD80(60.2±0.21)%,CD83(85.1±0.06)%,和CD86(91.9%±0.09)%,而在未转染的未成熟DC中低表达;转染RNA的DC瘤苗对于Hep-2细胞株有特异性杀伤效应,在E/T为40:1时,杀伤效率达45.6%,而对对照组靶细胞杀伤效率较低.结论: 喉鳞状细胞癌总RNA转染修饰的DC能特异性诱导细胞毒性T淋巴细胞,显著提高DC的抗原提呈功能,在体外能诱导高效而特异的抗喉癌免疫效应.     

Dendritic cells transduced with tumor-associated antigen gene elicit potent therapeutic antitumor immunity: comparison with immunodominant peptide-pulsed DCs

Several studies have shown that vaccine therapy using dendritic cells (DCs) pulsed with specific tumor antigen peptides can effectively induce antitumor immunity. Peptide-pulsed DC therapy is reported to be effective against melanoma, while it is still not sufficient to show the antitumor therapeutic effect against epithelial solid tumors such as gastrointestinal malignancies. Recently, it has been reported that vaccine therapy using DCs transduced with a surrogate tumor antigen gene can elicit a potent therapeutic antitumor immunity. In this study, we investigated the efficacy of vaccine therapy using DCs transduced with the natural tumor antigen in comparison with peptide-pulsed DCs. DCs derived from murine bone marrow were adenovirally transduced with murine endogenous tumor antigen gp70 gene, which is expressed in CT26 cells, or DCs were pulsed with the immunodominant peptide AH-1 derived from gp70. We compared these two cancer vaccines in terms of induction of antigen-specific cytotoxic T lymphocyte (CTL) responses, CD4+ T cell response against tumor cells, migratory capacity of DCs and therapeutic immunity in vivo. The cytotoxic activity of splenocytes against CT26 and Meth-A pulsed with AH-1 in mice immunized with gp70 gene-transduced DCs was higher than that with AH-1-pulsed DCs. CD4+ T cells induced from mice immunized with gp70 gene-transduced DCs produced higher levels of IFN-gamma by stimulation with CT26 than those from mice immunized with AH-1-pulsed DCs (p < 0.0001), and it was suggested that DCs transduced with tumor-associated antigen (TAA) gene induced tumor-specific CD4+ T cells, and those CD4+ T cells played a critical role in the priming phase of the CD8+ T cell response for the induction of CD8+ CTL. Furthermore, DCs adenovirally transduced with TAA gene showed an enhancement of expression of CC chemokine receptor 7 and improved the migratory capacity to draining lymph nodes. In subcutaneous models, the vaccination using gp70 gene-transduced DCs provided a remarkably higher therapeutic efficacy than that using AH-1-pulsed DCs. These results suggested that vaccine therapy using DCs adenovirally transduced with TAA gene can elicit potent antitumor immunity, and may be useful for clinical application.     

Dendritic cells genetically engineered to simultaneously express endogenous tumor antigen and granulocyte macrophage colony-stimulating factor elicit potent therapeutic antitumor immunity.

Recently, several studies have shown that vaccine therapy using dendritic cells (DCs) genetically engineered to express a surrogate tumor antigen can effectively induce antitumor immunity. In this study, murine bone marrow DCs were adenovirally transduced with murine endogenous tumor antigen gp70 expressed in CT26 cells and granulocyte macrophage colony-stimulating factor (GM-CSF), and we examined whether antigen-specific CTL responses and therapeutic immunity could be induced in mice immunized with those genetically modified DCs. The cytotoxic activity against CT26 in mice immunized with gp70-transduced DCs was significantly higher than that in control (P < 0.01) and was enhanced by GM-CSF-cotransduction (P < 0.001). GM-CSF gene transfer into DCs expressing tumor-associated antigen enhances CC chemokine receptor 7 expression on DCs, leading to improved migratory capacity of DCs to draining lymph nodes. Consequently, an effective antitumor immune response would be induced. Vaccination using gp70-transduced DCs provided remarkable therapeutic efficacy in s.c. models. Moreover, it could be sufficiently augmented by GM-CSF-cotransduction of DCs. These results support that vaccination therapy using DCs simultaneously transduced with tumor-associated antigen can elicit potent CTL response, and GM-CSF-cotransduction of DCs could optimize therapeutic response. Further investigation is needed to optimize this vaccine therapy to achieve the obvious benefit in clinical application.     

Dendritic cells transfected with tumor RNA for the induction of antitumor CTL in colorectal cancer

Dendritic cells (DC) are the most potent antigen-presenting cells known, currently tested for vaccination studies in cancer patients. The use of tumor-derived RNA to load DC overcomes the requirement of defined HLA types and the identification of tumor antigens expressed by the tumors. Here, we show that human monocyte-derived DC generated under serum-free conditions by GM-CSF, IL-4 and TNF-alpha acquire a mature phenotype and expression of the chemokine receptor CCR-7, which plays a pivotal role in DC migration to the afferent lymph nodes. We demonstrate the feasibility of total RNA transfection into such DC using the renal cell carcinoma (RCC) cell line N43-EGFP, which was stably transfected with an EGFP-encoding vector. Moreover, we show that DC transfected with RNA from colorectal cancer cells present HLA class I-restricted antigenic epitopes to induce a primary antitumor CTL response in vitro. Interestingly, the CTL induced by SW480 RNA also recognized another colon cancer line, HCT116, and the RCC line A498. Our results confirm the feasibility of total RNA transfection of serum-free generated DC for the induction of CTL against colon cancer and RCC cells, and support the relevance of shared tumor rejection epitopes between colorectal cancer and RCC.     

Addition of an induction regimen of antiangiogenesis and antitumor immunity to standard chemotherapy improves survival in advanced malignancies

Studies have shown that cancer requires two conditions for tumor progression: cancer cell proliferation and an environment permissive to and conditioned by malignancy. Chemotherapy aims to control the number and proliferation of cancer cells, but it does not effectively control the two best-known conditions of the tumor-permissive environment: neoangiogenesis and tolerogenic immunity. Many malignant diseases exhibit poor outcomes after treatment with chemotherapy. Therefore, we investigated the potential benefits of adding an induction regimen of antiangiogenesis and antitumor immunity to chemotherapy in poor outcome disease. In a prospective, randomized trial, we included patients with advanced, unresectable pancreatic adenocarcinomas, non-small cell lung cancer, or prostate cancer. Two groups of each primary condition were compared: group 1 (G1), n?=?30, was treated with the standard chemotherapy and used as a control, and group 2 (G2), n?=?30, was treated with chemotherapy plus an induction regimen of antiangiogenesis and antitumor immunity. This induction regimen included a low dose of metronomic cyclophosphamide, a high dose of Cox-2 inhibitor, granulocyte colony-stimulating factor, a sulfhydryl (SH) donor, and a hemoderivative that contained autologous tumor antigens released from patient tumors into the blood. After treatment, the G2 group demonstrated significantly longer survival, lower blood level of neoangiogenesis and immune-tolerance mediators, and higher blood levels of antiangiogenesis and antitumor immunity mediators compared with the G1 group. Toxicity and quality of life were not significantly different between the groups. In conclusion, in several advanced malignancies of different primary localizations, an increase in survival was observed by adding an induction regimen of antiangiogenesis and antitumor immunity to standard chemotherapy.     

Engineered fusion hybrid vaccine of IL-18 gene-modified tumor cells and dendritic cells induces enhanced antitumor immunity

Dendritic cell (DC)-tumor fusion hybrid vaccines that facilitate antigen presentation represent a novel powerful strategy in cancer immunotherapy. In our study, we investigated the antitumor immunity derived from the vaccination of fusion hybrids between engineered J558/IL-18 myeloma cells secreting Th1 cytokine IL-18 and DCs. DC/J558/IL-18 could secret a higher level of IL-18 than DCs, efficiently expressed J558 tumor antigen P1A, and enhanced ability of allogeneic T cell stimulation when compared to J558/IL-18. Our data showed that the immunization of BALB/c mice with DC/J558/IL-18 hybrids induced the most potent protective immunity against 1 x 10(6) cells with a J558 tumor challenge, compared to those immunized with the mixture of DCs and J558/IL-18, J558/IL-18, or J558. Furthermore, the immunization of mice with engineered DC/J558/IL-18 hybrids elicited stronger NK activity and J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro. In addition, DC/J558/IL-18 tumor cells into syngeneic mice induced a Th1 dominant immune response to J558 and resulted in tumor regression, which indicated that the antitumor effect mediated by DC/J558/IL-18 appeared to be dependent on TH1 cytokine production. These results demonstrate that the engineered fusion hybrid vaccines that combine Th1 gene-modified tumor with DCs may be an attractive strategy for cancer immunotherapy.     

A tumor lysate is an effective vaccine antigen for the stimulation of CD4+ T-cell function and subsequent induction of antitumor immunity mediated by CD8+ T cells

To develop a potent cancer vaccine, it is important to study how to prepare highly immunogenic antigens and to identify the most appropriate adjuvants for the antigens. Here we show that a tumor lysate works as an effective antigen to prime CD4⁺ T-cell help when baculovirus is employed as an adjuvant. When immunized intradermally with the combination (BLP) of baculovirus, a CT26 tumor lysate, and a cytotoxic T-cell epitope peptide before a tumor challenge, 60% of mice rejected tumors. In contrast, all mice vaccinated with baculovirus plus a tumor lysate (BL) developed tumors. In addition, flow cytometry showed that tumor-specific, interferon γ-producing CD8⁺ cytotoxic T lymphocytes (CTLs) were robustly activated by intradermal immunization with BLP. When BLP was administered therapeutically to tumor-bearing mice, antitumor efficacy was better compared to BL. The established tumor was completely eradicated in 50–60% of BLP-treated mice, and induction of tumor-specific CTLs was observed, suggesting that the antitumor efficacy of BLP is mediated by CD8⁺ T cells. Numerous CD4⁺ T cells infiltrated the tumors of BLP-treated mice, whereas the antitumor effect of BLP almost disappeared after removal of the tumor lysate from BLP or after depletion of BLP-immunized mice of CD4⁺ T cells. Thus, the combination of a peptide, lysate, and baculovirus provides stronger antitumor immunity than does a peptide plus baculovirus or a lysate plus baculovirus; effectiveness of BLP is determined by functioning of CD4⁺ T cells stimulated with a tumor lysate.     

Dendritic cells pulsed with an anti-idiotype antibody mimicking carcinoembryonic antigen (CEA) can reverse immunological tolerance to CEA and induce antitumor immunity in CEA transgenic mice

In this report, we have studied the immunogenicity of the nominal antigen, carcinoembryonic antigen (CEA), and that of an anti-idiotype antibody, 3H1, which mimics CEA and can be used as a surrogate for CEA. We have demonstrated that immunization of CEA transgenic mice with bone marrow-derived mature dendritic cells (DC) loaded with anti-idiotype 3H1 or CEA could reverse CEA unresponsiveness and result in the induction of CEA-specific immune responses and the rejection of CEA-transfected MC-38 colon carcinoma cells, C15. Immunized mice splenocytes proliferated in an antigen-specific manner by a mechanism dependent on the functions of CD4, MHC II, B7-2, CD40, CD28, and CD25. However, immune splenic lymphocytes isolated from 3H1-DC-vaccinated mice when stimulated in vitro with 3H1 or CEA secreted significantly higher levels of Th1 cytokines than did CEA-DC vaccinated mice. DC vaccination also induced antigen-specific effector CD8+ T cells capable of expressing interleukin-2, IFN-gamma, and tumor necrosis factor (TNF)-alpha and displayed cytotoxic activity against C15 cells in an MHC class I-restricted manner. 3H1-DC vaccination resulted in augmented CTL responses and the elevated expression of CD69, CD25, and CD28 on CD8(+) CTLs. The immune responses developed in 3H1-DC-immunized mice resulted in rejection of C15 tumor cells in nearly 100% of experimental mice, whereas only 40% of experimental mice immunized with CEA-DC were protected from C15 tumor growth. These findings suggest that under the experimental conditions used, 3H1-DC vaccination was better than CEA-DC vaccination in breaking immune tolerance to CEA and inducing protective antitumor immune responses in this murine model transgenic for human CEA.     

Dendritic cells strongly boost the antitumor activity of adoptively transferred T cells in vivo

Dendritic cells (DCs) have been well characterized for their ability to initiate cell-mediated immune responses by stimulating naive T cells. However, the use of DCs to stimulate antigen-activated T cells in vivo has not been investigated. In this study, we determined whether DC vaccination could improve the efficacy of activated, adoptively transferred T cells to induce an enhanced antitumor immune response. Mice bearing B16 melanoma tumors expressing the gp100 tumor antigen were treated with cultured, activated T cells transgenic for a T-cell receptor specifically recognizing gp100, with or without concurrent peptide-pulsed DC vaccination. In this model, antigen-specific DC vaccination induced cytokine production, enhanced proliferation, and increased tumor infiltration of adoptively transferred T cells. Furthermore, the combination of DC vaccination and adoptive T-cell transfer led to a more robust antitumor response than the use of each treatment individually. Collectively, these findings illuminate a new potential application for DCs in the in vivo stimulation of adoptively transferred T cells and may be a useful approach for the immunotherapy of cancer.     

Identification of arctigenin as an antitumor agent having the ability to eliminate the tolerance of cancer cells to nutrient starvation

Tumor cells generally proliferate rapidly and the demand for essential nutrients as well as oxygen always exceeds the supply due to the unregulated growth and the insufficient and inappropriate vascular supply. However, cancer cells show an inherent ability to tolerate extreme conditions, such as that characterized by low nutrient and oxygen supply, by modulating their energy metabolism. Thus, targeting nutrient-deprived cancer cells may be a novel strategy in anticancer drug development. Based on that, we established a novel screening method to discover anticancer agents that preferentially inhibit cancer cell viability under the nutrient-deprived condition. After screening 500 medicinal plant extracts used in Japanese Kampo medicine, we found that a CH(2)Cl(2)-soluble extract of Arctium lappa exhibited 100% preferential cytotoxicity under the nutrient-deprived condition at a concentration of 50 microg/mL with virtually no cytotoxicity under nutrient-rich condition. Further bioassay-guided fractionation and isolation led to the isolation of arctigenin as the primary compound responsible for such preferential cytotoxicity; the compound exhibited 100% preferential cytotoxicity against nutrient-deprived cells at a concentration of 0.01 microg/mL. Furthermore, arctigenin was also found to strongly suppress the PANC-1 tumor growth in nude mice, as well as the growth of several of the tested pancreatic cancer cell lines, suggesting the feasibility of this novel antiausterity approach in cancer therapy. Further investigation of the mechanism of action of arctigenin revealed that the compound blocked the activation of Akt induced by glucose starvation, which is a key process in the tolerance exhibited by cancer cells to glucose starvation.     

Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity

Dendritic cell (DC)-tumor fusion hybrid vaccine which facilitates antigen presentation represents a new powerful strategy in cancer therapy. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between wild-type J558 or engineered J558-IL-4 myeloma cells secreting cytokine interleukin-4 (IL-4) and immature DCs (DC(IMAT)) or relative mature DCs (DC(RMAT)). DC(RMAT) displayed an up-regulated expression of immune molecules (Ia(d), CD40, CD54, CD80 and CD86) and certain cytokines/chemokines, and enhanced ability of allogeneic T cell stimulation when compared to DC(IMAT). These DCs were fused with myeloma cells by polyethylene glycol (PEG). The fusion efficiency was approximately 20%. Our data showed that immunization of C57BL/6 mice with DC(RMAT)/J558 hybrids induced protective immunity against a high dose of J558 tumor challenge (1x10(6) cells) in 3 out of 10 immunized mice, compared with no protection seen in mice immunized with DC(IMAT)/J558 hybrids. Furthermore, immunization of mice with engineered DC(RMAT)/J558-IL-4 hybrids elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro and induced more efficient protective immunity (10/10 mice; tumor free) against J558 tumor challenge in vivo than DC(RMAT)/J558 hybrid vaccines. The results demonstrate the importance of DC maturation in DC-tumor hybrid vaccines and indicate that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.     

Intradermal vaccination of dendritic cell-derived exosomes is superior to a subcutaneous one in the induction of antitumor immunity

Because dendritic cell (DC)-derived exosomes (EXO) harbor many important DC molecules involved in inducing immune responses, EXO-based vaccines have been extensively used to induce antitumor immunity in different animal tumor models. However, it is not clear which route of EXO administration can induce more efficient antitumor immune responses. In this study, we compared the antitumor immunity derived from EXO vaccine by way of the two common administration routes, the subcutaneous (s.c.) and the intradermal (i.d.) administrations. Our data showed that the i.d. EXO administration resulted in more EXO-absorbed DC migrating into the T-cell areas of draining lymph nodes than the s.c. administration. Interestingly, the i.d. EXO administration also resulted in an enhanced ovalbumin (OVA)-specific CD8(+) T-cell proliferation and CD8(+) CTL effector responses in vivo, compared to the s.c. administration. Similarly, compared to the s.c. vaccination, the i.d. vaccination induced stronger antitumor immunity in the animal tumor model. Therefore, the i.d. EXO vaccination is superior to the s.c. one and should be considered when EXO-based vaccine is designed.     

Dendritic epidermal T-cell involvement in induction of CD8+ T cell-mediated immunity against an ultraviolet radiation-induced skin tumor

Murine epidermis contains 2 distinct cell populations which contribute to the skin immune system, Langerhans cells (LC), and dendritic epidermal T cells (DETC). LCs are important in the induction of immunity against a wide range of antigens; however, the function of DETC is unclear. To investigate the roles of these epidermal cells (EC) in protective antitumor immunity, an in vivo model of an ultraviolet radiation-induced fibrosarcoma, UV-13-1, was used. Mice were immunized with tumor antigen-pulsed EC followed 10 days later by an injection into the ear of 10⁵ tumor cells, which did not lead to formation of a detectable tumor, but was intended to simulate the influence of a developing tumor on the ensuing immune response. The mice were then challenged with 2 × 10⁶ viable tumor cells in each flank, sufficient to result in growth of a measurable tumor. Protective immunity was induced by DETC, and shown to be long-lasting, with tumors inoculated 160 days after immunization being effectively rejected. The effector cells responsible for protective immunity were CD8⁺ T cells. Delayed-type hypersensitivity generated by tumor antigen-pulsed EC was dependent on Lcs, with no involvement of DETCs. This response, in contrast to that of DETC, required prior culture of EC with GM-CSF, but failed to inhibit tumor growth or incidence. Thus DETC and LC can both activate antitumor immune responses, although only the DETC-dependent response results in protective immunity in the presence of a developing tumor. © 1997 Wiley-Liss, Inc.     

CD1d-restricted T cells license B cells to generate long-lasting cytotoxic antitumor immunity in vivo

Although resting B cells are known for being poorly immunogenic and for inducing T-cell tolerance, we have here attempted to test whether their immunogenicity could be enhanced by CD1d-restricted invariant T cells (iNKT) to a point where they could be used in cellular vaccines. We found that the addition of the iNKT ligand alpha-galactosylceramide (alphaGalCer) to peptide-loaded B cells overcame peptide-specific T-cell unresponsiveness and allowed for the generation of peptide-specific memory CTL immunity. This CTL was induced independently of CD4 T and natural killer cells but required iNKT and CD8 T cells. B cells directly primed CTL, and the alphaGalCer and the peptide must be presented on the same cell. Importantly, our B-cell-based vaccine is comparable in efficiency with dendritic cell-based vaccines, inducing similar CTL responses as well as providing an effective regimen for preventing and suppressing s.c. and metastatic tumors. Therefore, with the help of iNKT, peptide-pulsed B cells can establish long-lasting antitumor immunity and so show promise as the basis for an alternative cell-based vaccine.