MiR-22 regulated T cell differentiation and hepatocellular carcinoma growth by directly targeting Jarid2

MiR-22 has been demonstrated to inhibits tumor growth in several cancers. However, its function in the tumor microenvironment is still unclear, especially for T cell differentiation. Here, miR-22 expression in the circulating T cells from hepatocellular carcinoma (HCC) patients and healthy controls was analyzed with quantitative polymerase chain reaction (qPCR). Diethylnitrosamine (DEN)/phenobarbital (PB)-mediated primary HCC and Hepa1-6 subcutaneous tumor mouse models were established and subjected to lenti-miR-22 injection. Mice immunoreconstituted with miR-22-overexpressing T cells were employed to investigate the antitumor effect of miR-22 in mice. Luciferase assay, immunofluorescent staining, in vitro Th17 cell differentiation assay, and rescue experiments were employed to investigate the mechanism underlying the miR-22-mediated regulation of Th17 cell differentiation and liver tumor growth. Results confirmed the dramatic downregulation of miR-22 expression in malignant tissues and circulating T cells from patients with HCC. MiR-22 expression correlated with good prognosis of patients. Overexpression of miR-22 impaired the DEN/PB-induced primary HCC formation and the growth of Hepa1-6 subcutaneous tumors by promoting Th17 differentiation. Injection of miR-22-overexpressing T cells in irradiated mice resulted in the inhibition of Hepa1-6 subcutaneous tumor growth via Th17 differentiation promotion. MiR-22 could directly bind to Jarid2, which played an important role during the miR-22-mediated regulation of Th17 differentiation. Taken together, our study expands the understanding of miR-22 function and provides a therapy target for HCC.     

应用细胞因子缓释微球的肿瘤疫苗治疗肝癌的研究

目的 探讨采用细胞因子缓释微球的肿瘤疫苗预防和治疗肝癌的疗效及抗癌机制。方法 我们研制开发了一种肿瘤疫苗,其组成是固定的肿瘤细胞或组织碎片、细胞因子缓释微球和免疫辅助药。采用多聚甲醛固定的小鼠Hepal-6细胞或肿瘤碎片、微球包装的GM—CSF和／IL—2和合成TiterMax Gold等不同成份的瘤苗皮内接种C57BL／6J小鼠,随后肝内接种活体Hepal-6细胞。结果 对照组15只小鼠全部发展成肝肿瘤;含有固定Hepal-6细胞和IL-2及GM—CSF微球的肿瘤疫苗,80％小鼠获得保护。再加入免疫辅助剂TiterMax Gold的肿瘤疫苗,则87％小鼠获得保护。将Hepal-6细胞接种于左躯干皮下。肿瘤长至直径5mm时,皮内接种肿瘤疫苗2次。结果显示,对照组肿瘤继续生长。疫苗组在第2次接种后7—10天,10只小鼠中9只肿瘤生长受到抑制,随后明显缩小。60％小鼠的肿瘤完全消散。细胞毒性实验结果显示,未接种疫苗的小鼠脾细胞不能杀灭Hepal-6细胞和其他肿瘤细胞;而接种疫苗的小鼠脾细胞对Hepal-6细胞杀瘤活性达41％,但对B16—Fl,Lewis肺癌细胞(LLC),肾癌细胞(Renca),膀胱癌细胞(MBT-2)则无效。疫苗的Ⅰ期临床实验结果显示肝癌疫苗能有效地预防肝癌术后复发,诱导DTH反应。结论 肝癌疫苗能有效预防和治疗原发性肝癌,其抗瘤机制是诱导内源性抗原特异性CTL反应,其杀瘤特性是由典型的：MHC—Ⅰ限制的CD8^ T细胞所介导的。     

Vaccination with allogeneic GM-CSF gene-modified lung cancer cells: antitumor activity comparing with that induced by autologous vaccine

OBJECTIVE: The aim of this study was to investigate the whole allogeneic (differing tissue-type) tumor cells as vaccine in the mouse lung cancer model. The immunogenic and antitumor activity of allogeneic vaccine was compared with that of autologous cancer cell vaccine. METHODS: C57/BL mice inoculated with Lewis lung cancer (LLC) cells were used as the animal model to test the effects of allogeneic vaccination. LA795 and LLC lung cancer cell lines, which were transfected with the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, were administered as allogeneic and autologous tumor vaccine, respectively. The irradiated tumor cells were administered as subcutaneous vaccines before the tumor challenge. The immunity of cancer vaccine was tested by mouse interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) lactate dehydrogenase (LDH) assays. The serum level of IFN-gamma and interleukin (IL)-4 was tested using the enzyme-linked immunosorbent assay method. RESULTS: Prophylactic vaccination with allogeneic LA795 cells protected against the LLC tumor challenge in C57/BL. The tumor growth was inhibited and the survival was accordingly prolonged. The cytotoxicity of the spleen cells or the purified CD(8)(+) T-cells against LLC cells in the mice immunized with either the autologous or allogeneic cancer cell vaccine was significantly increased, relative to that of the control, untreated group (p<0.05). ELISPOT IFN-gamma assays showed that spleen cells from mice immunized with LA795 cells could be activated after coculture with irradiated LLC cells. In addition, the serum level of Th1-king cytokine IFN-gamma significantly increased after vaccination; however, no statistically difference was found in Th2-kind cytokine IL-4. CONCLUSIONS: The allogeneic cancer vaccine could induce immune responses and protection against lung cancer, which had no significant difference with that of autologous vaccine.     

An attenuated Salmonella oral DNA vaccine prevents the growth of hepatocellular carcinoma and colon cancer that express alpha-fetoprotein

Antitumor vaccination therapies using attenuated Salmonella typhimurium carrying plasmid DNA encoding tumor-associated antigens are currently under preclinical development. In the present study, we first established a useful method to facilitate in vivo monitoring of attenuated S. typhimurium uptake using a bioluminescent lux gene operon plasmid. Following transformation with the lux gene operon construct, mice were fed with various amounts of attenuated S. typhimurium-lux to monitor in vivo clearance over a period of 24 h. We found that the ingested attenuated S. typhimurium-lux cells were almost cleared out 9 h postfeeding, as judged by a significant decrease in bioluminescence. We further examined the therapeutic efficacy of vaccination using attenuated S. typhimurium carrying the mouse alpha-fetoprotein (AFP) gene against a cancer line CT26-murine alpha-feto protein (mAFP) that stably expresses AFP and mouse hepatocellular carcinoma (HCC) Hepa1-6. Attenuated S. typhimurium oral DNA vaccine was found to promote protective immunity against both CT26-mAFP and Hepa1-6 tumor cells growth. The oral DNA vaccine significantly increased the life span of tumor-challenged mice in both tumor models. Together, these results suggest that vaccination with the attenuated S. typhimurium oral DNA vaccine that carries the AFP gene could be a promising strategy to prevent HCC development.     

Growth suppression of the hepatocellular carcinoma cell line Hepa1-6 by an activatable interferon regulatory factor-1 in mice

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis and few therapeutic options. The aim of the study was to evaluate the potential of IFN regulatory factor-1 (IRF-1) for cytokine gene therapy of HCC using an IRF-1/human estrogen receptor fusion protein (IRF-1hER), which is reversibly activatable by beta-estradiol (E2). IRF-1hER stably expressing murine Hepa1-6 HCC cells (HepaIRF-1hER) were characterized by lowMHC 1, highCD54, and lack of MHC II, CD80, and CD86 expression. Activation of HepaIRF-1hER cells induced a highMHC I, lowMHC II, and highCD54 phenotype. Furthermore, they were characterized by IFN-beta secretion, decreased anchorage-independent growth in a soft agar assay, and diminished cell growth. Tumor growth in E2-treated syngeneic C57L/J mice, but not in E2-untreated mice, was suppressed. These E2-treated mice were protected against rechallenge with HepaIRF-1hER and wild-type Hepa1-6 tumors even in the absence of E2, suggesting induction of tumor specific immunity. In fact, significant CTL activity against Hepa1-6 tumors and the endogenously expressed HCC-specific self antigen alpha-fetoprotein was observed. Antitumoral effects, however, were only partially dependent on both CD4+ and CD8+ T cells. IRF-1 treatment of mice bearing HepaIRF-1hER tumors resulted in growth arrest of tumors, and a significant survival benefit was observed in comparison to E2-untreated mice. In conclusion, our data demonstrate that IRF-1 suppresses HCC growth through both a direct antitumor growth effect and enhanced immune cell recognition of the tumor and is a promising candidate for gene therapy of HCC.     

自体肿瘤瘤苗对荷瘤鼠Th1、Th2型细胞因子及细胞毒作用的影响

目的研究经处理的H22肝癌细胞肿瘤瘤苗作为全细胞瘤苗对H22荷瘤小鼠体内Th1/Th2细胞比例和细胞因子的影响以及CTL的杀伤活性.方法用加重组白细胞介素2、重组粒细胞单核细胞集落刺激因子及福氏不完全佐剂制成疫苗,建立荷瘤小鼠模型,用51Cr释放法测定瘤苗免疫组、荷瘤组、正常组小鼠脾细胞对亲本H22肝癌细胞的杀伤活性;流式细胞仪检测单个核细胞中的Th1和Th2细胞,并取血检测血清中IL-10、IFN-γ水平.结果效靶比为200:1时,免疫小鼠脾细胞体外杀伤亲本H22肝癌细胞的杀伤率为38.3%,显著高于荷瘤组的13.6%,正常组的7.5%,以及对S180细胞的9.1%(P均＜0.05).瘤苗免疫组Th1细胞及Th1/Th2细胞的比值显著升高(P＜0.01),血清IFN-γ较对照组明显升高(P＜0.01);血清IL-10较对照组明显降低(P＜0.01).结论肿瘤细胞加小剂量IL-2和GM-CSF及佐剂组成的肿瘤细胞瘤苗可激发特异性细胞介导的免疫反应,改善抗肿瘤免疫反应.     

GM-CSF基因修饰肺癌细胞疫苗的抗肿瘤活性及其与化疗的协同作用

目的评价粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因修饰肿瘤疫苗的抗肿瘤活性,探讨其与化疗联合应用的抗肿瘤效果。方法以小鼠肺癌Lewis细胞系接种C57BL/6小鼠建立动物模型。利用含GM-CSF基因的重组质粒GM-CSF-pIRES2-EGFP,转染小鼠肺癌细胞系Lewis和LA795细胞,分别制备自体(Lewis细胞)和同种异体(LA795细胞)肿瘤疫苗。小鼠皮下接种Lewis细胞(1×107个)后5 d,采用GM-CSF分泌性肿瘤疫苗接种3次,同时设PBS组和单纯疫苗组作为对照,检测疫苗治疗前后脾细胞对Lewis细胞杀伤活性的变化,以及血清白介素4(IL-4)和γ干扰素(IFN-γ)的水平。观察GM-CSF分泌性肿瘤疫苗接种及联合化疗对小鼠生存期的影响。结果GM- CSF分泌性自体或同种异体肿瘤疫苗,均可诱导小鼠脾细胞对Lewis细胞杀伤活性增高,第3次接种后分别为(42.0±2.5)%和(39.6±7.3)%;同时血清中Th1类细胞因子IFN-γ的水平升高,而Th2类因子IL-4的水平无显著变化。GM-CSF分泌性肿瘤疫苗治疗组小鼠生存期与对照组比较,差异无统计学意义(P>0.05);而化疗联合疫苗组小鼠生存期较单独治疗组及对照组明显延长(P<0.05)。结论GM-CSF基因修饰的肿瘤疫苗可刺激机体产生特异性免疫反应;化疗的联合应用有助于提高肿瘤疫苗的治疗效果。     

Irreversible electroporation enhances immunotherapeutic effect in the off-target tumor in a murine model of orthotopic HCC

Irreversible electroporation (IRE) has been postulated to have an off-target effect on lesions not in the tumor-ablative field, possibly through heightened immunologic response. In this study, we evaluated whether combination IRE and immunotherapy would lead to increased tumor necrosis and T cell recruitment to both the treated tumors and tumors outside the local ablative field. An in vitro cell-IRE model was established to evaluate the ability of T lymphocytes (EL4 cell and HH cells) migration in response to Hepatocellular carcinoma (HCC) cells (Hepa1-6 and HepG2) with IRE treatment. An orthotopic HCC mouse model was established by implantation of 1mm^3 sections of Hepa1-6 tumor tissues into the right and left lobes of the liver. The Hepa1-6 cells and HepG2 cells with IRE treatment increased the migration ability of EL4 cell and HH cells, specifically when they were pretreated with immunotherapeutic agents in vitro. In the orthotopic HCC mouse model, IRE+immunotherapy treatment enhanced the necrosis and subpopulation of infiltrated CD8 positive cells, but attenuated the tumor associated inflammatory cells in both IRE target tumor tissues and IRE off-target tumor tissues from the mice with 4 weeks of immunotherapy following IRE. This study provided the evidence that combination of IRE and immunotherapy enhances tumor necrosis and immune responses, not only in the IRE-treated tumor but also in the off-target tumor.     

Experiments evaluating antitumor immunity induced by cholesterol hemisuccinate-treated syngeneic cell vaccines

Evaluation of the efficiency of immunizations with syngeneic tumor vaccines prepared from cells treated with cholesterol hemisuccinate (CHS) was performed in five animal models: P815 mastocytoma in DBA/2 mice, MCA-103 fibrosarcoma in C57BL/6N mice, L1210 leukemia in DBA/2 mice, Ehrlich ascites carcinoma in C57BL/6N mice, and CBP pancreatic cancer in CB/SsLak hamsters. Animals received two to four weekly intraperitoneal immunizations with 10(6) or 10(7) tumor cells, followed by challenges with syngeneic viable tumor cells. Survival and tumor growth rates were observed. No significant differences were observed among animals immunized with CHS-treated irradiated tumor vaccines, nontreated irradiated tumor vaccines, and nonimmunized controls in the P815, MCA-103, L1210, and CBP models. Mice immunized with nontreated irradiated Ehrlich ascites cell vaccines showed longer survival than those immunized with CHS-treated irradiated cell vaccines and nonimmunized controls. Results indicated that CHS-treated tumor cell vaccines were not effective in protecting against tumor challenges in five different syngeneic tumor models.     

Development of a Listeria monocytogenes-based vaccine against hepatocellular carcinoma

Live attenuated Listeria monocytogenes (LM) is a promising bacterial vector able to induce a T-cell response to tumor-associated antigens and demonstrates great potential for use in vaccine development. A novel recombinant LM-based vaccine (Lmdd (LM ΔdalΔdat)-MPFG (multiple peptide fusing genes)) was developed with the ability to express and secrete hepatocellular carcinoma (HCC)-related tumor-associated antigens fragments due to the insertion of hepatitis B virus (HBV)-X protein (HBx)-derived epitopes HBx(52-60) and HBx(140-148), the universal T-helper epitope, alpha-fetoprotein (AFP) epitope AFP(158-166), and melanoma antigen gene (MAGE)-3(271-279) into the HBV core protein. Following immunization with the Lmdd-MPFG vaccine, macrophages exhibited uptake of the bacteria; the vaccine was then nearly cleared 3 days after the first administration. It disappeared even more quickly following subsequent vaccinations. However, recombinant Lmdd-MPFG allowed for the full development of an antitumor response towards the human leukocyte antigen (HLA)-A0201 epitopes of MPFG. Each epitope stimulated an augmented T-cell proliferation and enhanced the supernatant level of interferon (IFN)-γ in vitro. In addition, IFN-γ-producing CD8(+) T cells as well as in vivo cytolytic activity were significantly increased in HLA-A2 transgenic mice. Additionally, the Lmdd-MPFG developed a strong antitumor response, as indicated by the significant resistance of immunized mice to MPFG-positive Hepa1-6 cell challenge in both a prophylactic and therapeutic setting. Tumor regression was accompanied by an enhanced cytotoxic T lymphocyte response and a decrease of regulatory T cells in the tumor. Collectively, these results suggest that utilizing attenuated LM as a vaccine vector, able to carry the MPFG gene, presents a potentially feasible strategy for prevention of HCC.     

Induction of antitumor immunity with combination of HER2/neu DNA vaccine and interleukin 2 gene-modified tumor vaccine.

The therapeutic effects of both cytokine-secreting tumor vaccine and DNA vaccine were studied using mouse MBT-2 bladder cancer cells as a model. Cytokine-secreting MBT-2 cells were obtained by infecting cells with retroviral particles containing interleukin (IL) 2-, IL-4-, or granulocyte-macrophage colony-stimulating factor (GM-CSF)-expression vector. The MBT-2-IL-2 cells were not tumorigenic in syngenic C3H mice at all. Tumor formation decreased significantly for the MBT-2-GM-CSF cells. MBT-2-IL-2, -IL-4, and -GM-CSF cells were killed by irradiation and tested as tumor vaccines. The irradiated MBT2-IL-2 cells could complete protect mice from the growth of the preexisting tumor cells, and the immune memory lasted for 8 months. On the other hand, irradiated MBT-2-IL-4 and MBT-2-GM-CSF cells were less effective. When the loading tumor mass increased, all tumor vaccines lost protective effects. DNA vaccine encoding the tumor antigen neu was additionally tested to improve the therapeutic efficacy. Coinjection of 60 microg pSV-neu DNA was effective in enhancing the antitumor effects of MBT2-IL-2; however, DNA vaccine alone cannot prevent the progression of the preexisting tumor. Immunohistochemical analysis of tumor infiltrate revealed massive increase of CD4+ lymphoid cells in the group of mice treated with both DNA vaccine and IL-2-secreted tumor vaccine. Western blotting demonstrated the presence of anti-neu antibody in the serum from immunized mice. In contrast, combination of DNA vaccine and MBT-2-GM-CSF has no additive effect. The results indicate the combination of DNA vaccine and IL-2-secreting tumor vaccine can additionally improve therapeutic efficacy, and the efficacy is correlated with the increase of CD4+ T lymphocytes and anti-neu antibody.     

树突状细胞体外诱导抗肿瘤免疫通过诱导肿瘤细胞凋亡抑制裸鼠移植瘤生长

研究树突状细胞（ＤＣ）体外诱导的细胞免疫抑制裸鼠移植瘤生长作用及其机理。方法：联合应用粒／巨噬细胞集落刺激因子ＫＭ－ＣＳＦ）及白介素－４（ＩＬ－４）直接从肝癌患者外周血中培养出ＤＣ,以人肝癌细胞系ＨｅｐＧ２肿瘤细胞的肿瘤抗原粗提物刺激ＤＣ,ＤＣ激活同源的Ｔ淋巴细胞,建立裸鼠人肝癌细胞系ＨｅｐＧ２移植瘤模型,以被激活的Ｔ淋巴细胞治疗裸鼠ＨｅｐＧ２移植瘤并观察治疗效果,检测移植瘤标本肿瘤细胞凋亡情况。结果：ＤＣ诱导的Ｔ细胞免疫能够诱导裸鼠人肝癌细胞系ＨｅｐＧ２移植瘤肿瘤细胞大量凋亡从而抑制裸鼠ＨｅｐＧ２移植瘤生长。结论：经肿瘤抗原激活的ＤＣ作为一新概念上的抗肿瘤疫苗有可能在肿瘤的治疗中发挥重要作用。     

人AFP腺病毒载体感染的树突状细胞诱导小鼠抗肝癌免疫

目的探讨复制缺损型腺病毒载体(Ad)介导异种AFP修饰的树突状细胞(DCs)诱发抗肝癌免疫、打破肿瘤免疫耐受的效果。方法从HepG2和Hepa16细胞中克隆人和小鼠AFP,插入Ad中构建AdhAFP和AdmAFP。用AdhAFP或AdmAFP感染小鼠骨髓来源的DC后,在无或有删除CD4或CD8情况下免疫C57BL/6小鼠,7d后取脾细胞行51Cr释放实验,检测特异性CTL杀伤活性;或给免疫小鼠接种Hepa16肝癌细胞,观察荷瘤小鼠成活情况。结果AdhAFP/DCs免疫小鼠1周后其细胞毒性T淋巴细胞杀伤活性明显强于AdmAFP/DCs。AdhAFP/DCs免疫小鼠后1周接种5×106Hepa16肝瘤细胞,2个月后仍然有80%的小鼠无瘤生长;而接种1×106Hepa16细胞至AdmAFP/DCs免疫小鼠,2个月后小鼠成活率为20%。删除小鼠CD4或CD8T细胞均使AdhAFP/DCs诱发的抗肿瘤免疫反应消失。结论Ad介导异种AFP修饰的DCs能有效地打破肿瘤的免疫耐受,诱发强烈的抗原特异性细胞免疫反应,这种特异性细胞免疫反应是CD4和CD8依赖性的。     

In vivo and in vitro growth stimulation of murine hepatoma cells by glucocorticoid

An increased level of glucocorticoid receptors has been found in hepatoma. The aim of this study was to determine the in vivo effect of glucocorticoid on the growth of murine hepatoma cells. In vitro cell growth was determined by MTT assay, while cell cycle progression was assayed with flow cytometry. For in vivo experiments, ML-3 and Hepa 1-6 cells were implanted in BALB/c and SCID mice, respectively. Each mouse received 4 subcutaneous injections of hydrocortisone and/or RU486 after tumor implantation. We found that glucocorticoid treatment of ML-3 and Hepa 1-6 cells in vitro resulted in an increase in cell growth which was partially inhibited by RU486, a glucocorticoid antagonist. Glucocorticoid treatment enhanced the cell cycle progression of ML-3 cells. The incidence of ML-3 tumor in the hydrocortisone group was significantly higher than that of the saline, RU486, or hydrocortisone plus RU486 groups. RU486 partially blocked the hydrocortisone-stimulated growth of hepatoma. Further study showed that glucocorticoid treatment of SCID mice also stimulated Hepa 1-6 tumor growth. In conclusion, our results indicated that glucocorticoid directly stimulated hepatoma growth and this stimulation was not the result of immune suppression. Glucocorticoids may play an important role in regulating the growth of hepatoma.     

Inhibition of heme oxygenase 1 expression by small interfering RNA decreases orthotopic tumor growth in livers of mice

Endogenous overexpression of the antiapoptotic protein heme oxygenase 1 (HO-1) has been shown to occur in various cancer diseases and might contribute to cancer progression. We compared the expression levels of HO-1 in human liver to expression levels in hepatocellular carcinoma (HCC), as well as the effect of HO-1 inhibition by small interfering RNA (siRNA) on cellular survival and apoptosis in the mouse hepatoma cell lines Hepa129 and Hepa1-6 and on orthotopic tumor growth in immune-competent C3H/HeN mice. Our results show that HO-1 is frequently overexpressed in human HCC. Downmodulation of HO-1 by siRNA resulted in increased cellular damage and apoptosis, reduced proliferation, reduced growth of orthotopic HCC and reduced angiogenesis. Livers and kidneys of treated animals did not reveal signs of damage by this treatment. In conclusion, a specific knockdown of HO-1 might represent a novel therapeutic approach in HCC therapy.     

表达VEGFR-2的减毒鼠伤寒沙门氏菌疫苗诱导特异性抗胶质瘤血管免疫应答

背景与目的:血管内皮生长因子(vascular endothelialgrowth factor,VEGF)及其主要受体血管内皮生长因子受体-2(vascular endothelialgrowth factorreceptor-2,VEGFR-2)在肿瘤新生血管和肿瘤基质形成过程中起着重要作用。本研究的目的是观察表达鼠VEGFR-2的重组减毒沙门氏疫苗菌诱导的抗血管特异性免疫应答及抗胶质瘤作用。方法:构建真核表达载体pcDNA3.1-VEGFR2,通过电转化法将pcDNA3.1-VEGFR2导入减毒鼠伤寒沙门氏菌SL7207中,经由胃管饲予C57BL/6J小鼠,对小鼠进行基因免疫。采用ELISA法检测免疫小鼠血清中特异性抗VEGFR2-IgG抗体,分离免疫小鼠的脾细胞,分析重组疫苗菌免疫后小鼠体内的特异性细胞毒性T细胞(cytotoxic T lym phocyte,CTL)应答。用携带pcDNA3.1-VEGFR2的重组沙门氏菌免疫治疗胶质瘤荷瘤小鼠,通过测量荷瘤小鼠肿瘤大小,检测肿瘤微血管密度及肿瘤细胞凋亡,评价重组疫苗菌的抗血管及肿瘤生长抑制作用。结果:重组疫苗菌免疫后小鼠产生了高水平的抗VEGFR2-IgG抗体,诱导小鼠脾淋巴细胞产生针对VEGFR2的特异性CTL活性。重组疫苗菌的免疫能够明显抑制胶质瘤的生长。NaH CO3对照组、载体对照组、重组疫苗菌组的平均微血管密度分别为26.5±5.8、27.2±4.5、8.8±1.9,平均凋亡细胞数分别为4.41.2、3.     

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.     

Induction of immune tolerance toward tumor-associated-antigens enables growth of human hepatoma in mice.

Adoptive transfer of immunity against hepatitis B surface antigen (HBsAg) was previously shown to facilitate suppression of experimental human hepatocellular carcinoma (HCC) expressing HBsAg in athymic mice. We have shown that oral tolerance induces antigen‐specific immune suppression of HBsAg by feeding hepatitis B virus (HBV) antigens. In the present study we evaluated the effect of oral tolerance induction toward HBV or HCC antigens on the growth of experimental HCC‐expressing HBsAg in mice. Tolerance induction was induced in mice by 5 oral feedings of 1 μg HBV antigens or HCC‐extracted proteins (50 μg protein) before vaccination with recombinant HBsAg. Splenocytes (2 × 10⁶) from these mice were transferred to sublethally irradiated athymic BALB/c mice previously transplanted subcutaneously with 10⁷ human hepatoma Hep3B cells. Adoptive transfer of splenocytes immunized toward HBsAg prevented tumor growth. At 4 weeks after splenocyte transplantation, tumor volume and serum alpha‐fetoprotein (AFP) levels in athymic mice transplanted with splenocytes immunized to HBsAg were undetectable as compared with 1,048 ± 738 mm³ and 2,500 ± 1,431 ng/ml in recipients of naïve splenocytes (p < 0.0001). Mice receiving splenocytes tolerized toward Hep3B cells, as manifested by reduced serum HBs antibody levels, reduced HBV‐specific stimulation index and reduced HBV‐specific‐IFNγ spot‐forming cells, had early tumor growth evident by elevated AFP serum levels, weight loss and mortality, which were suppressed at 6 weeks. Mice transplanted with splenocytes tolerized toward HBV antigens did not have direct evidence of tumor growth. Induction of oral tolerance toward HCC‐extracted proteins enabled transient tumor growth in this model. This effect was mediated through downregulation of the anti‐HBV immune response. © 2002 Wiley‐Liss, Inc.