Increased immunogenicity of tumor vaccines complexed with anti-Gal: studies in knockout mice for alpha1,3galactosyltransferase.

A major prerequisite for the success of tumor vaccines is their effective uptake by antigen-presenting cells (APCs) and transport of these APCs to the draining lymph nodes, where the processed and presented tumor-associated antigens activate tumor-specific naive T cells. We previously suggested that the immunogenicity of autologus tumor vaccines in humans may be augmented by engineering vaccinating tumor cell membranes to express alpha-galactosyl (alpha-gal) epitopes (i.e., Galalpha1,3Galbeta1,4GlcNAc-R). Subsequent in situ binding of natural anti-Gal IgG molecules to these epitopes would result in the formation of immune complexes that target tumor vaccines for uptake by APCs, via the interaction of the Fc portion of anti-Gal with Fcgamma receptors on APCs. This hypothesis was tested in a unique experimental animal model of knockout mice for alpha1,3galactosyltransferase (alpha1,3GT) and the mouse melanoma B16-BL6 (referred to here as BL6). Like humans, these mice lack alpha-gal epitopes and produce anti-GaL BL6 melanoma cels are highly tumorigenic, and like human tumor cells, they lack alpha-gal epitopes. Expression of alpha-gal epitopes on these melanoma cells was achieved by stable transfection with alpha,3GT cDNA. The transfected melanoma cells (termed BL6alphaGT) express approximately 2 x 10(6) alpha-gal epitopes per cell and readily form immune complexes with anti-Gal. Vaccination of the mice with 2 x 10(6) irradiated melanoma cells that express alpha-gal epitopes, followed by challenge with 0.5 x 10(6) live parental melanoma cells, resulted in protection for at least 2 months (i.e, no tumor growth) in one-third of the mice, whereas all mice immunized with irradiated parental melanoma cells developed tumors 21-26 days post-challenge. The proportion of protected mice doubled when the mice were immunized twice with irradiated melanoma cells expressing alpha-gal epitopes and challenged with 0.2 x 10(6) live BL6 cells. Histological studies on the developing tumors in challenged mice that were immunized with melanoma cells expressing alpha-gal epitopes demonstrated extensive infiltration of T lymphocytes and macrophages, whereas no mononuclear cell infiltrates were observed in tumors of mice immunized with parental tumor cells. Overall, these studies imply that immunization of alpha1,3GT knockout mice with BL6 melanoma cells that express alpha-gal epitopes elicits, in a proportion of the population, protective immune response against the same tumor lacking such epitopes. These studies further suggest that similar immunization of cancer patients with autologous tumor vaccines that are engineered to express alpha-gal epitopes may increase the immune response to autologous tumor-associated antigens and, thus, may elicit immune-mediated destruction of metastatic cells expressing these antigens.     

THE HUMORAL ANTITUMOR RESPONSES INDUCED BY IL-4 GENE-MODIFIED TUMOR VACCINE

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Combining TGF-β1 knockdown and miR200c administration to optimize antitumor efficacy of B16F10/GPI-IL-21 vaccine

TGF-β1 secreted abundantly by tumors cells as well as present in the local microenvironment promotes neoplasm invasion and metastasis by triggering the epithelial to mesenchymal transition (EMT). MiR200c has been shown to suppress EMT and to regulate the cellular epithelial and interstitial state conversion, whereas the tumor vaccines are intended to specifically initiate or amplify a host response against evolving tumor cells. Our study aimed at optimizing the antitumor effects of the B16F10/glycosylphosphatidylinositol-interleukin 21 (B16F10/GPI-IL-21) tumor vaccine on melanoma bearing mice by combining the TGF-β1 knockdown and the administration of miR200c agomir. The mice were subcutaneously vaccinated with inactivated B16F10/GPI-IL-21 vaccine and challenged by B16F10 cells transfected with shTGF-β1 (B16F10/shTGF-β1 cells) or B16F10/shTGF-β1 cells with the administration of miR200c agomir. The later combination showed that, when compared with the mice in the control group that received no vaccination, vaccinated mice significantly increased NK and CTL activities, enhanced levels of IFN-γ, and reduced expression of TGF-β1, N-cadherin, Vimentin, Gli1/2, P-Smad2/3 and others involved in promoting expression of EMT-related molecules in tumor areas, and inhibited the melanoma metastasis in lungs and lymph nodes. Altogether, our findings demonstrate that this synergistic anti-cancer regimen effectively induces strong immune response and diminishes the melanoma progression.     

Antigen-specific cancer immunotherapy using a GM-CSF secreting allogeneic tumor cell-based vaccine

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cell-based vaccines are currently one of the major forms of cancer vaccines. However, the preparation of GM-CSF-transduced autologous tumor vaccines is time-consuming and technically challenging. In addition, the host antigen presenting cells, rather than the tumor vaccine cells themselves, present tumor-specific antigens and prime the host T cells. Therefore, we tested the efficacy of antigen-specific allogeneic tumor vaccines. We used human papillomavirus 16 (HPV-16) E7 protein as a model tumor antigen, which is associated with the development of most cervical carcinoma. B16, a C57BL/6 (H-2(b)) derived melanoma cell line, was genetically engineered to produce GM-CSF alone (B16GM), HPV-16 E7 alone (B16E7), or both (B16GME7). These vaccine cells were injected into BALB/c (H-2(d)) mice (10(6) cells/mouse). Two weeks later, mice were challenged with 10(5) live HPV-16 E7(+) BL-1 (H-2(d)) tumor cells and monitored for tumor progression twice weekly. To determine the effective cell population in the antitumor immunity elicited by B16GME7, we carried out in vivo antibody depletion experiments using CD4 and CD8 specific antibodies. In addition, as a measure of the immune responses produced by B16GME7, we performed an in vitro cytotoxic T lymphocyte assay using a standard chromium release method. We found that all of the mice vaccinated with B16GME7 remained tumor free 49 days post-BL-1 challenge. In contrast, mice vaccinated with B16GM and B16E7 did not show any tumor protection against a similar dose of BL-1 cells. Furthermore, the antitumor immunity produced by B16GME7 was dependent on both CD4 and CD8 T cells. In addition, E7-specific cytotoxic T lymphocyte activity could be readily demonstrated in mice immunized with B16GME7. These results suggest that allogeneic tumor cells transduced with GM-CSF and the tumor antigen, HPV-16 E7, cannot only generate an E7-specific cytotoxic T lymphocytes response in vitro, but can also elicit a potent antitumor immune response against an E7 expressing tumor in vivo.     

Effective anti-metastatic melanoma vaccination with tumor cells transfected with MHC genes and/or infected with newcastle disease virus (NDV)

The therapeutic efficacy of active immunization with B16-F10.9 melanoma cells transfected with syngeneic major histocompatibility complex (MHC) class-l genes, modified by infection with Newcastle Disease virus (NDV) or modified by both treatments, was compared. B16-F10.9 tumor-bearing mice were treated at various stages of tumor growth and metastasis with irradiated, modified tumor-cell vaccines. Irradiated tumor cells and H-2D^b transfectants did not stimulate anti-tumor immunity while H-2K^b transfectants and NDV-modified F10.9 cells showing low and high expression of MHC class-l genes efficiently prevented metastasis of small established tumors. NDV-modified parental-cell vaccines functioned optimally and improved overall survival by about 60%, also at early stages of metastasis establishment. A synergistic effect of H-2K^b expression and virus modification on rejection of micrometastases was observed in mice bearing advanced tumors. Postoperative vaccination of mice carrying multiple metastases with NDV-modified vaccines caused significant, but incomplete, reduction of metastatic tumor load. The therapeutic effect of NDV-modified tumor vaccines was dependent on multiple immune mechanisms. Depletion of CD8, CD4 or NK cells by in vivo treatment with monoclonal antibodies reversed the immunotherapeutic effects of the vaccine. Thus, tumor xenogenization and gene modification may act synergistically to vaccinate against advanced tumors, while single modalities can effectively vaccinate against metastasis at early stages of tumor growth.     

Relationship between alphaGal epitope expression and decrease of tumorigenicity in pancreatic adenocarcinoma model

The alphaGal epitope is a carbohydrate structure, Galalpha1,3Galbeta1,4GlcNAc-R, synthesized on glycoconjugates in many mammals by alpha1,3galactosyltransferase. Humans do not express this epitope and present in serum large amounts of naturally occuring antibodies, which recognize the alphaGal epitopes and participate in the hyperacute rejection of xenograft. Studies indicated that the fundamental mechanism of hyperacute rejection involving the alphaGal epitope expression can be used in cancer therapy. We have previously suggested that the alphaGal epitope expression by human pancreatic tumoral cells could decrease the tumorigenic behavior of these cells. To determine whether the expression of the alphaGal epitope can modify the tumorigenicity of pancreatic cancer cells, we used a Syrian golden hamster pancreatic adenocarcinoma experimental model. The expression of alphaGal epitopes in the Syrian golden hamster pancreatic cancer cell line HaP-T1 was obtained by selecting stable cell clones transfected with murine alpha1,3galactosyltransferase gene. The alphaGal epitope expression resulted in a delay in the tumoral development of HaP-T1 cells in vivo after allograft transplantation of Syrian golden hamsters (2.5-fold, P < 0.05) and of nude mice. This result is associated with an 100% increase in survival time of nude mice bearing tumors expressing the alphaGal epitope. Our results confirm that the cell surface expression of alphaGal epitope decreases the tumorigenic behavior of pancreatic cancer cells. This novel property may be useful for the development of cancer gene immunotherapy strategy.     

Effectiveness of a Simply Designed Tumor Vaccine in Prevention of Malignant Melanoma Development

We investigated the efficacy of a simple syngeneic tumor vaccine to induce specific antitumor immunity in female C57B1/6 mice. Tumor vaccine was prepared by mixing irradiated B-16 melanoma tumor cells with the pleiotropic biological response modifier—maleic anhydride divinyl ether (MVE-2). Experimental animals were pretreated with the vaccine in order to prevent the development of intraperitoneal (i.p.) B-16 melanoma tumors after inoculation of viable tumor cells. More than 40% of prevaccinated animals challenged i.p. with 5×10⁵ viable tumor cells were completely protected from tumor development and remained tumor-free 100 days after tumor cell inoculation. The percentage of tumor-free animals (survivors) rose to as much as 90% when the application of tumor vaccine was repeated two weeks after the first vaccination (i.e. one week after the inoculation of viable tumor cells). The induced antitumor response depended predominantly upon macro-phage function, since vaccinated animals which were depleted of peritoneal macrophages died within the same time range as animals in the control group. Also, tumor-type specificity of the vaccine was confirmed by the fact that the animals vaccinated with B-16 melanoma vaccine were not protected from the development of another type of tumor. In conclusion, comparison of the experimental data with the data from the literature suggests that our simple tumor vaccine may be as effective as genetically engineered tumor vaccines. At the same time, this kind of vaccine is easier to control and thus safer to apply in humans when compared to genetically engineered vaccines.     

Recombinant E.coli LLO/OVA Vaccination Effectively Inhibits Murine Melanoma Metastasis to Lung by CD8^＋T Cells Immunity

Objective: To construct recombinant E.coli LLO/OVA and investigate its tumor metastatic inhibition effect in B16 OVA melanoma challenged mice.Methods: Recombinant E.coli LLO/OVA was constructed and the expression of listeriolysin O (LLO) and ovalbumin (OVA) of the vaccine was determined by coomassie brilliant blue staining and western blotting. After 3 subcutaneous injections of E.coli LLO/OVA, the percentages of CD3+CD4+T, CD4+CD25+T, CD3+CD8+T and OVA257(264 SIINFEKL specific CD8+T cells were determined by flow cytomytry, and the tumor metastatic inhibition effect in B16 OVA melanoma challenged mice was observed.Results: Recombinant E.coli LLO/OVA was successfully constructed, and the expression of LLO and OVA of the vaccine was confirmed. After 3 subcutaneous injections of E.coli LLO/OVA and E.coli OVA in mice, the percentages of CD3+CD4+T, CD4+CD25+T and CD3+CD8+T cells were equivalent in the two groups of mice. However, there were significantly more OVA257(264 SIINFEKL specific CD8+T cells in E.coli LLO/OVA vaccinated mice than that in E.coli OVA vaccinated mice. The prophylactic E.coli LLO/OVA vaccination effectively prevented the tumor metastasis to lungs in B16 OVA melanoma challenged mice. Depletion of CD8+T cells significantly impaired the tumor inhibition effect of the vaccine in B16 OVA challenged mice. The therapeutic vaccination of E.coli LLO/OVA significantly prevented melanoma metastasis to lungs in B16 OVA challenged mice too.Conclusion: Recombinant E.coli LLO/OVA vaccination is highly effective in inhibiting murine malignant melanoma metastasis by promoting CD8+T cell immunity.     

Decrease of human pancreatic cancer cell tumorigenicity by alpha1,3galactosyltransferase gene transfer

The enzyme alpha1,3galactosyltransferase synthesizes the alphaGal epitope, a carbohydrate structure (Galalpha1,3Galbeta1,4GlcNAc-R), on glycoconjugates in lower mammals. The enzyme is absent in humans but large amounts of natural antibodies that recognize alphaGal epitopes are present in human serum. It is likely that these antibodies contribute to the host defense and participate in the hyperacute rejection of xenograft. Previous studies indicated that the glycosyltransferase gene transfer into tumoral cells can modify the structure of glycoconjugates at the cell surface and, as a consequence, modulates the metastatic and tumorigenic behaviors of these cells. The aim of our study was to determine whether the expression of alphaGal epitope can modify the tumorigenicity of human pancreatic cancer cells. The expression of alphaGal epitopes in the human pancreatic cancer cell lines BxPC-3 and Panc-1 was obtained by selecting stable cell clones transfected with murine alpha1,3galactosyltransferase gene. The expression of the enzyme activity in BxPC-3 and Panc-1 cells resulted in the formation at the cell surface of alphaGal epitopes that are recognized by human anti-alphaGal antibodies. alphaGal epitope expression at the surface of pancreatic cancer cells was associated with the fixation of complement 1q to human anti-alphaGal antibodies. The alphaGal epitope expression also resulted in a delay in the tumoral development of BxPC-3 and Panc-1 cells in vivo after xenograft transplantation of nude mice. In addition to the impairment of the metastatic potential of murine tumor cell lines and the activation of immune response, our study provides evidence that the cell surface expression of alphaGal epitopes also modulates the tumorigenic behavior of human pancreatic cancer cells.     

Deoxyribonucleic acid (DNA) encoding a pan-major histocompatibility complex class II peptide analogue augmented antigen-specific cellular immunity and suppressive effects on tumor growth elicited by DNA vaccine immunotherapy

Vaccine immunotherapy must induce helper and cytotoxic cell-mediated immunity to generate the powerful antitumor immune responses needed to suppress cancer progression. We reported previously that a 16-amino acid peptide analogue derived from pigeon cytochrome c can bind broad ranges of MHC class II types and activate helper T cells in mice. To determine whether DNA encoding the Pan-MHC class II IA peptide (Pan-IA) can increase the efficacy of tumor suppression by DNA vaccine immunotherapy targeting tumor antigens, Pan-IA DNA was administered with ovalbumin (OVA) DNA to C57BL/6 mice bearing the OVA-expressing tumor cell line E.G7. Specific proliferative responses to and cytotoxic activities against OVA-expressing targets were induced in mice vaccinated with both OVA and Pan-IA DNA but not in those vaccinated with OVA DNA alone or control DNA plus Pan-IA DNA. Growth of E.G7 cells was suppressed only by combined vaccination with OVA and Pan-IA DNA, and tumors in five of the nine mice that received this combined vaccination were eradicated completely. In those mice, the frequency of CD8-positive T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased in the tumor site. Furthermore, immunofluorescent study of the inoculated tumors revealed increased accumulation of both CD4- and CD8-positive T cells producing IFN-gamma in the tumor only by this vaccine protocol. The data suggest that Pan-IA DNA can augment suppressive effects of DNA vaccines on tumor growth by increasing numbers of antigen-specific CTLs and helper T cells. This is the first study in which established tumors have been eradicated successfully by vaccination with DNA corresponding to CTL epitopes and helper T cell epitopes. Our animal model may contribute to the development of therapeutic DNA vaccines against cancer.     

Intrasplenic vaccination against experimental melanoma

The immunodominant component of a formalinized extracellular antigen (fECA) vaccine prepared from B16 F10 melanoma cells is the melanoma-associated antigen B700. We now demonstrate that a single prophylactic intrasplenic inoculation of B700 antigen (1-10 mu g) stimulates the production of antibodies which have antiproliferative effects on B16 F10 melanoma cells in vitro. In addition, potential cytotoxic effects of splenocytes from B700 antigen inoculated mice were evaluated for two cellular immune effector functions, natural killer (NK) cell activity and lymphokine activated killer (LAK) cell activity; both activities were increased following B700 antigen inoculation. Intrasplenic injection of B700 antigen elicited an increase in the expression of the CD25 surface antigen (IL-2 R alpha) by T lymphocytes and up-regulated the expression of IL-2 R alpha mRNA. Thus both humoral and cellular cytotoxic immune responses might play roles in the decreased growth of primary tumors in B700 antigen inoculated mice and in the higher survival rate in this group of animals.     

BAP31, a promising target for the immunotherapy of malignant melanomas

Purpose

Malignant melanoma’s (MM) incidence is rising faster than that of any other cancer in the US and the overall survival at 5 years is less than 10%. B cell associated protein 31 (BAP31) is overexpressed in most MMs and might be a promising target for immunotherapy of this disease.

Experimental design

Firstly, we investigated the expression profiles of human BAP31 (hBAP31) and mouse BAP31 (mBAP31) in human and mouse normal tissues, respectively. The expression level of hBAP31 in human MMs and mBAP31 in B16 melanoma cells was also analyzed. Then we constructed novel mBAP31 DNA vaccines and tested there ability to stimulate mBAP31-specific immune responses and antitumor immunity in B16 melanoma-bearing mice.

Results

For the first time, we found that protein expression of hBAP31 were dramatically upregulated in human MMs when compared with human normal tissues. Predominant protein expression of mBAP31 was found in mouse B16 melanoma cells but not in mouse important organs. When mice were immunized with mBAP31 DNA vaccines, strong cellular response to mBAP31 was observed in the vaccinated mice. CTLs isolated from immunized mice could effectively kill mBAP31-positive target mouse B16 melanoma tumor cells in vitro and vaccination with mBAP31 DNA vaccines had potent anti-tumor activity in therapeutic model using B16 melanoma cells.

Conclusions

These are the first data supporting a vaccine targeting BAP31 that is capable of inducing effective immunity against BAP31-expressing MMs and will be applicable to human MMs and hBAP31 DNA vaccine warrants investigation in human clinical trials.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0153-6) contains supplementary material, which is available to authorized users.     

Effectiveness of a simply designed tumor vaccine in prevention of malignant melanoma development.

We investigated the efficacy of a simple syngeneic tumor vaccine to induce specific antitumor immunity in female C57Bl/6 mice. Tumor vaccine was prepared by mixing irradiated B-16 melanoma tumor cells with the pleiotropic biological response modifier-maleic anhydride divinyl ether (MVE-2). Experimental animals were pretreated with the vaccine in order to prevent the development of intraperitoneal (i.p.) B-16 melanoma tumors after inoculation of viable tumor cells. More than 40% of prevaccinated animals challenged i.p. with 5 x 10(5) viable tumor cells were completely protected from tumor development and remained tumor-free 100 days after tumor cell inoculation. The percentage of tumor-free animals (survivors) rose to as much as 90% when the application of tumor vaccine was repeated two weeks after the first vaccination (i.e. one week after the inoculation of viable tumor cells). The induced antitumor response depended predominantly upon macrophage function, since vaccinated animals which were depleted of peritoneal macrophages died within the same time range as animals in the control group. Also, tumor-type specificity of the vaccine was confirmed by the fact that the animals vaccinated with B-16 melanoma vaccine were not protected from the development of another type of tumor. In conclusion, comparison of the experimental data with the data from the literature suggests that our simple tumor vaccine may be as effective as genetically engineered tumor vaccines. At the same time, this kind of vaccine is easier to control and thus safer to apply in humans when compared to genetically engineered vaccines.     

Antitumor effects of polyvalent and monovalent vaccines coupled with interleukin-2 in a metastatic melanoma model

We have previously reported that preimmunization of mice with formalinized extracellular antigens (fECA) derived from melanoma cells, in combination with interleukin 2 (IL-2) treatment and surgical resection, decreased subsequent tumor growth and increased survival of mice in a new model for spontaneous metastasis of melanoma. In this study, we have modified the sequence of tumor growth and therapy to more closely mimic the clinical situation. Mice were challenged subcutaneously in the tail with 5 x 10(5) B16 F10 melanoma cells and, by day 21, all of them had developed localized melanoma tumors. The primary tumor-bearing tails of control and experimental animals were then resected distal to the base of the tail, and therapy of the mice was initiated the following day. Groups of mice received different polyvalent and monovalent murine melanoma vaccines (including native or formalin treated extracellular antigens, intact melanoma cells, or purified B700 antigen), with or without concomitant low doses of IL-2. The results demonstrate that the vaccine therapies elicited significant increases in survival of the mice, accompanied by reductions in the size of lymph nodes and in the number of pulmonary metastases. These effects, particularly with the intact melanoma cell vaccine, could be improved even further with concomitant IL-2 treatment.     

Potent antitumor effect elicited by superantigen-linked tumor cells transduced with heat shock protein 70 gene

Heat shock proteins (HSP) induce antitumor-specific immunity via a unique mechanism, but HSP alone fails to produce a satisfactory antitumor efficacy. We considered that the potent immune-activation of superantigen (SAg) might assist HSP to elicit a strong tumor-antigen-specific immunity. We initially prepared B16 melanoma cells linked to SAg SEA via a fusion protein with a trans-membrane sequence (TM), and demonstrated that SEA thus anchored on the tumor cell surface could elicit strong antitumor immunity. We then prepared cells transduced with an inducible heat shock protein 70 (HSP70 ) gene, and bearing SEA-TM fusion protein on the cell surface, and used these cells as a dual-modified vaccine. In this study, either in a therapeutic setting or in a pre-immune model, the SEA-anchored vaccine or the HSP70 gene-modified vaccine induced marked tumor suppression, prolonged survival, augmented lymphocyte proliferation and higher NK and CTL activity in C57BL/6 mice compared with their controls ( P <0.01), though they were less effective than the dual-modified vaccine. Among these vaccines, the dual-modified vaccine showed the best therapeutic efficacy in B16 melanoma-bearing mice and gave the greatest protection against wild-type B16 melanoma challenge. The results indicated that the dual-modified vaccine could induce a potent tumor-antigen-specific immune response in addition to an increase of non-specific immunity. This study offers a novel approach to bridging specific and non-specific immunity for cancer therapy.     

Antigen-specific immunotherapy for murine lung metastatic tumors expressing human papillomavirus type 16 E7 oncoprotein

An important goal of cancer immunotherapy is to prevent and treat tumor metastasis. We have previously reported a recombinant vaccinia-based vaccine (Sig/E7/LAMP-1) that demonstrated significant anti-tumor effect in a subcutaneous tumor challenge model. In this study, we investigated the potency of the Sig/E7/LAMP-1 vaccine in preventing and treating metastatic tumors. A tumor metastasis model was generated by injecting human papillomavirus type 16 (HPV-16) E6/E7 expressing tumor cells, designated TC-1, into the tail vein of syngeneic C57BL/6 mice. All the naive mice injected with 1 × 10⁶ TC-1 cells developed tumors confined exclusively to the lungs within 1 month. For in vivo tumor prevention experiments, mice were vaccinated with Sig/E7/LAMP-1 followed by tumor challenge. While tumor growth was observed in all of the mice (10/10) in the control groups, 8 of 10 vaccinated mice (80%) remained tumor-free 2 months post-tumor challenge. For in vivo treatment experiments, mice were first inoculated with TC-1 cells and then vaccinated with Sig/E7/LAMP-1. Treatment with Sig/E7/LAMP-1 was effective in eliminating preexisting tumor cells in 4 of 5 vaccinated mice. Most importantly, treatment with Sig/E7/LAMP-1 resulted in regression of fully established lung tumors in 50% (5/10) of vaccinated mice. Our data suggest that the Sig/E7/LAMP-1 vaccine is effective in controlling the hematogenous spread of TC-1 tumor cells. In addition, the TC-1 lung metastasis model can be used to test the efficacy of various E6/E7-specific vaccines and immunotherapeutic strategies. Int. J. Cancer 78:41–45, 1998.© 1998 Wiley-Liss, Inc.     

4-1BBL联合Hsp70-肿瘤抗原肽抑制小鼠黑色素瘤肺转移

背景与目的正调节性共刺激分子表达偏低是肿瘤逃避机体免疫系统攻击的重要原因之一,增加这些分子的表达是促进抗肿瘤免疫的研究热点。小鼠黑色素瘤B16-F1细胞株是弱免疫原性癌细胞株。本研究旨在观察4-1BBL联合Hsp70-抗原肽对小鼠黑色素瘤肺转移的抑制作用,并探讨其机制。方法建立小鼠黑色素瘤肺转移模型,Hsp70-B16抗原肽小鼠皮下免疫,同时从尾静脉注射4-1BBL表达质粒p4-1BBL。于接种后第17天,解剖小鼠取肺组织,解剖显微镜下计数黑色素瘤肺转移结节的数目,并检测小鼠部分免疫学指标及肝、肾功能。结果4-1BBL联合Hsp70-B16抗原肽治疗组小鼠黑色素瘤肺转移结节数降至50±8,明显少于二者单独治疗组的500±80和450±40;联合治疗组小鼠血清IL-2和IFN-γ的分泌水平分别增加了4倍和3倍,与对照组相比均存在显著性差异(P<0.01);单独应用4-1BBL基因或与Hsp70-B16抗原肽联合应用对小鼠肝、肾的功能均无明显影响。结论表达4-1BBL联合应用Hsp70-B16抗原肽主要通过增强外周T淋巴细胞功能活性而有效抑制小鼠黑色素瘤肺转移。     

CpG-ODN-based immunotherapy is effective in controlling the growth of metastasized tumor cells

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN) act as potent immune stimulators by activating innate immunity through toll-like receptor 9. These immunomodulatory effects of CpG-ODN have been reported to be associated with anti-tumor immunity. In this study, we used a murine B16F10 melanoma model and a CT26 colon cancer model to assess whether CpG-ODN-based immunotherapy was effective in inhibiting tumor cells that have already metastasized to distant organs. Systemic administration of CpG-ODN after melanoma cell injection resulted in a significant inhibition of pulmonary colonization. When CpG-ODN was administered after tumor cell injection, it also inhibited pulmonary metastasis of the tumor cells, albeit to a lesser degree in the latter case. Systemic administration of CpG-ODN after subcutaneous inoculation of CT26 colon cancer cells diminished pulmonary metastasis from the primary tumor sites. Additionally, CpG-ODN also inhibited the growth of pulmonary colonization of the colon tumor cells when CpG-ODN was administered after the primary tumors had been surgically removed. These data indicate that CpG-ODN was effective in inhibiting pulmonary metastasis of the B16F10 melanoma and CT26 colon cancer cells, as well as the growth of metastasized tumor cells. Our results suggest that CpG-ODN-based immunotherapy may be beneficial in controlling micrometastasis after surgery in clinical settings.     

Depletion of CD25(+) CD4(+) T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon alpha-induced, CD8(+) T-cell-dependent immune defense of B16 melanoma.

Transduction of B16 melanoma cells with IFN alpha (B16-IFN alpha) enhances CD8(+) T-cell-dependent tumor immunity in mice, resulting in delayed outgrowth in vivo. Here we provide evidence that CD4(+) T cells down-regulate the IFN alpha-induced tumor immune defense. Importantly, depletion of regulatory CD25(+) CD4(+) T cells prevented growth of B16-IFN alpha in most mice and promoted long-lasting protective tumor immunity. Rejection of B16-IFN alpha could also be achieved with therapeutic injections of dendritic cells genetically engineered to express the melanoma antigen tyrosinase-related protein 2. These results support the development of novel strategies for the immunotherapy of melanoma using IFN alpha in combination with elimination of regulatory T cells or antigen-specific immunization.