B7.1、GM-CSF基因修饰的EL- 4细胞激发小鼠抗淋巴瘤免疫反应

目的 研究B7.1，GM-CSF基因修饰的淋巴瘤瘤苗进行恶性淋巴瘤免疫基因治疗的有效性。方法 用逆转录病毒载体分别将B7.1和GM-CSF基因导入小鼠淋巴瘤细胞EL-4中，得到EL-4/B7和EL-4/GM转基因瘤苗细胞，观察EL-4/B7和EL-4/GM瘤苗细胞对荷淋巴瘤小鼠的免疫治疗作用，以及预先接种过EL-4/B7或/和EL-4/GM瘤苗的小鼠，对野生型EL-4细胞致瘤性作用的影响，并检测接种EL-4/B7或/和EL-4/GM瘤苗的小鼠血浆IL-2及TNF-α的水平，及其脾淋巴细胞对野生型EL-4的细胞毒效应，另外，检测经EL-4/B7，EL-4/GM刺激后的小鼠脾淋巴细胞的增殖程度，结果 （1）经EL-4/B7、EL-4/GM细胞注射后，荷瘤小鼠的淋巴瘤生长速度变慢，生存时间延长，淋巴瘤组织中出现大量的炎症细胞；（2）接种一定数量的EL-4/B7，EL-4/GM细胞的小鼠，能抵抗野生型EL-4细胞的攻击；（3）接种瘤苗的小鼠血浆IL-2的水平明显升高，而TNF-α的水平无明显变化；（4）接种瘤苗的小鼠脾淋巴细胞，对野生型EL-4细胞有明显的细胞毒效应；EL-4/B7、EL-4/GM细胞能刺激小鼠脾淋巴细胞明显增殖。结论 B7.1,GM-CSF基因修饰的EL-4细胞，能有效地激发C57BL/6小鼠的抗淋巴瘤免疫反应，且B7.1基因与GM-CSF基因具有协同作用。     

IL—12和B7—1协同抗肿瘤作用和疫苗效应的研究

研究了IL-12和B7-1基因导入小鼠EL-4胸腺瘤细胞后在小鼠体内诱导的协同抗肿瘤免疫作用。方法将逆转录病毒载体重组的小鼠IL-12,B7-1基因表达质粒分别导入小鼠EL-4胸腺瘤细胞,利用转基因细胞治疗小鼠观察其抗肿瘤免疫效果。结果转基因细胞的肿瘤原性较EL-4/Wt和EL-4/Neo组明显降低(P<0.001)。同时免疫原性明显增强,以     

小鼠白介素—12基因转染对EL4细胞在小鼠体内成瘤性的抑制作用

目的探讨逆转录病毒(RV)载体介导的小鼠白介素12(mIL-12)融合基因转染,对低免疫原性的小鼠T细胞淋巴瘤细胞EL4体内生长的影响。方法用共培养法导入基因,以PCR法检测基因的导入。以脾细胞增殖法测定mIL-12的生物学活性。观察导入mIL-12基因的肿瘤细胞EL4/12于小鼠皮下接种后的成瘤性。对预先接种野生型肿瘤细胞EL4的小鼠,用60Co照射的肿瘤疫苗EL4/12接种于瘤周,观察瘤苗的抗肿瘤作用等。结果在EL4/12细胞中,用PCR可特异地检测到mIL-12p35和p40亚基的cDNA片段。5×105EL4/12细胞48h表达的mIL-12达(10.2±3.2)ng。EL4/12细胞在小鼠体内的成瘤性明显下降。EL4/12瘤苗治疗组约50%的小鼠可长期无瘤生存,而对照组小鼠则在短期内全部成瘤死亡。部分长期生存的小鼠产生了有效的抗肿瘤免疫,能耐受野生型肿瘤细胞的二次攻击。与对照组相比较,疫苗治疗组中其余小鼠的成瘤时间延迟(P∨0.01),小鼠存活时间延长(P∨0.01);死亡时肿瘤体积小(P∨0.05),形成的肿瘤有完整包膜,瘤周和肿瘤内部有较多的淋巴细胞和浆细胞浸润等。结论转染mIL-12基因能抑制EL4肿瘤细胞的成瘤性。mIL-12基因修饰的肿瘤疫苗对野生型肿瘤细胞具有治疗作用。     

小鼠白介素-12基因转染对EL细胞在小鼠体内成瘤性的抑制作用

目的探讨逆转录病毒(RV)载体介导的小鼠白介素12(mIL-12)融合基因转染,对低免疫原性的小鼠T细胞淋巴瘤细胞EL4体内生长的影响.方法用共培养法导入基因,以PCR法检测基因的导入.以脾细胞增殖法测定mIL-12的生物学活性.观察导入mIL-12基因的肿瘤细胞EL4/12于小鼠皮下接种后的成瘤性.对预先接种野生型肿瘤细胞EL4的小鼠,用60Co照射的肿瘤疫苗EL4/12接种于瘤周,观察瘤苗的抗肿瘤作用等.结果在EL4/12细胞中,用PCR可特异地检测到mIL-12p35和P40亚基的cDNA片段.5×105EL4/12细胞48h表达的mIL-12达(10.2±3.2)ng.EL4/12细胞在小鼠体内的成瘤性明显下降.EL4/12瘤苗治疗组约50%的小鼠可长期无瘤生存,而对照组小鼠则在短期内全部成瘤死亡.部分长期生存的小鼠产生了有效的抗肿瘤免疫,能耐受野生型肿瘤细胞的二次攻击.与对照组相比较,疫苗治疗组中其余小鼠的成瘤时间延迟(P＜0.01),小鼠存活时间延长(P＜0.01);死亡时肿瘤体积小(P＜0.05),形成的肿瘤有完整包膜,瘤周和肿瘤内部有较多的淋巴细胞和浆细胞浸润等.结论转染mIL-12基因能抑制EL4肿瘤细胞的成瘤性.mIL-12基因修饰的肿瘤疫苗对野生型肿瘤细胞具有治疗作用.     

小鼠IL-18基因修饰瘤苗的抗白血病作用研究

我们在前期工作中成功制备了IL-18基因修饰的白血病疫苗,为了探讨IL-18基因修饰瘤苗的体内抗白血病作用,实验采用L1210小鼠淋巴细胞白血病模型,在小鼠体内接种IL-18基因修饰瘤苗,观察瘤苗对L1210细胞致瘤性的影响及免疫保护作用,并进一步对其抗白血病作用机制进行了探索。结果显示,IL-18基因修饰瘤苗能够明显延长荷瘤小鼠存活时间,大部分小鼠达到长期生存,且长生存小鼠用野生型L1210细胞二次攻击后大多数仍能长期生存,表明IL-18基因修饰瘤苗有显著的抗白血病作用,并可诱导小鼠产生免疫记忆和免疫保护。机制探讨发现,接种IL-18基因修饰瘤苗后,小鼠脾脏淋巴细胞对L1210肿瘤细胞的CTL及NK细胞杀伤活性明显高于对照组(P<0.05),提示IL-18基因修饰瘤苗能够显著增强抗肿瘤CTL和NK细胞反应。接种瘤苗可使小鼠IFN-γ水平升高,但与对照相比无统计学意义,提示IFN-γ可能在IL-18基因修饰瘤苗诱导的抗肿瘤免疫应答中作用不大。     

ZnPcS2P2介导的光动力疗法联合mGM-CSF、mB7.1基因修饰的瘤苗治疗小鼠淋巴瘤

目的：研究两亲性酞菁锌光敏剂ZnPcS2P2介导的光动力疗法（ZnPc-PDT）联合GM—CSF、B7．1基因修饰的EL-4细胞，对小鼠淋巴瘤的抑制作用。方法：应用反复多次转染的方法，分别将逆转录病毒pLXSN、pLXSNmGM—CSF、pLXS—NmB7．1导入EL-4细胞，分别命名为EL-4／pLXSN、EL-4／GM和EL-4／B7。进行ZnPc—PDT时，经荷瘤小鼠的尾静脉注射ZnPcS2P2，4h后局部用波长670nm的激光照射瘤体；光源为670nm的半导体激光仪。将40只荷瘤小鼠分为5组，每组8只，即对照组（不做任何处理）、EL-4／B7＋EL-4／GM对照组、ZnPc—PDT对照组、ZnPc—PDT＋EL-4／pLXSN对照组、ZnPc—PDT＋EL-4／B7＋EL-4／GM实验组。隔天测量瘤块的大小，观察荷瘤小鼠淋巴瘤的生长情况，并计算瘤块的相对体积（RTV）。观察荷瘤小鼠的生存期及瘤组织的病理学改变。结果：实验组的生存率高于各个对照组（P〈0．01）。ZnPc—PDT组小鼠的生存期略高于EL-4／B7＋EL-4／GM组（P=0．01）。结论：mB7．1、mGM—CSF、基因修饰的瘤苗可显著增强ZnP c-PDT的抗肿瘤效应。     

B7-1基因转染小鼠肝癌细胞诱导的抗瘤效应研究

目的：研究共刺激分子B7-1在抗肿瘤免疫中的作用.方法：体外观察转染B7-1基因及空载体的小鼠肝癌细胞株H22与同源小鼠脾细胞混合培养后,测定淋巴细胞增殖指数、CTL,于小鼠皮下接种不同H22细胞后观察肿瘤生长情况.结果：转染B7-1基因的H22细胞体外可刺激淋巴细胞增殖,增强CTL的杀伤活性,接种小鼠后成瘤潜伏期和荷瘤鼠存活期明显延长（P＜0.05）.结论：转入B7-1基因的小鼠肝癌细胞H22能增强免疫原性,能有效诱导CTLs介导的抗肿瘤免疫反应.     

B7共刺激分子肿瘤免疫基因治疗研究进展

转染B7基因使肿瘤细胞表达B7共刺激分子后,瘤细胞提供双信号活化T细胞,使具有免疫原性的瘤细胞诱导有效的抗肿瘤免疫。以此瘤细胞作为疫苗治疗已形成的肿瘤取得了部分疗效。共转染B7基因和细胞因子基因等使抗肿瘤免疫得到进一步加强,对肿瘤的疗效也有进一步的提高。转染B7基因诱导免疫反应的作用在人肿瘤细胞系中也得到了证实。对肿瘤免疫基因治疗的深入研究中,发现了一些制约免疫反应的因素,这些问题的解决将最终为肿瘤免疫基因治疗的临床应用扫除障碍。     

B7-H4基因对SKOV3细胞生长功能的影响

目的： 初步探讨B7-H4基因对人卵巢癌细胞株SKOV3细胞生长和致瘤性的影响。方法：RT-PCR法扩增编码基因, 构建真核表达载体PEGFP-N1/B7-H4,以脂质体为载体将重组质粒PEGFP-N1和PEGFP-N1/B7-H4分别导入SKOV3细胞中,筛选建立高表达的细胞株。MTT法绘制体外培养细胞生长曲线,将转染前后的肿瘤细胞分别接种于SCID小鼠的腹部皮下观察其致瘤性。结果：成功构建了人B7-H4真核表达载体,SKOV3/B7-H4细胞高表达B7-H4,转染后肿瘤细胞体外生长速度明显增快。SKOV3/B7-H4细胞在小鼠体内的致瘤性较SKOV3/neo细胞和野生型SKOV3细胞明显升高。结论： B7-H4基因导入细胞后在体外和体内具有明显加快肿瘤生长的作用,可作为肿瘤基因治疗的靶向基因。     

表达前列腺特异性膜抗原的DNA疫苗对肿瘤细胞的抑制作用

目的构建表达前列腺特异性膜抗原（PSMA）的DNA疫苗，观察其在体外对肿瘤细胞的免疫攻击和在体内对肿瘤细胞攻击的免疫保护作用。方法通过稳定转染构建表达PSMA的小鼠黑色素瘤细胞系B16-PSMA，将DNA疫苗pCDNA3．1-PSMA通过肌肉注射导入C57BL／6小鼠体内，分离小鼠脾细胞，检测细胞毒性T淋巴细胞（cytotoxic T lymphocytes，CTL）反应。以B16-PSMA细胞攻击免骺小鼠，观察免疫动物的无瘤生存期和肿瘤体积增长情况，评价DNA疫苗的抗肿瘤作用。结果DNA疫苗可诱导小鼠脾淋巴细胞CTL活性，经过免疫后的小鼠成瘤率降低，无瘤生存期延长，肿瘤生长缓慢，肿瘤组织内有较多淋巴细胞浸润，表明产生较强的抗肿瘤反应。结论表达PSMA的DNA疫苗能够诱导小鼠产生特异性免疫反应，对表达PSMA的肿瘤细胞的攻击产生免疫保护作用，为前列腺癌的预防和免疫治疗提供了新的思路。     

Co-expression of B7-1 and ICAM-1 on tumors is required for rejection and the establishment of a memory response

Although the transfection of B7-1 cDNA into a few mouse tumor cell lines can induce anti-tumor T cell immunity, its expression alone is ineffective in many other tumor cell lines tested. We were interested to study what factors limit B7-1 co-stimulatory activity, and decided to investigate whether B7-1 requires the cooperation of ICAM-1 to provide the minimal co-stimulatory signal for establishing an efficient anti-tumor immunity. We show that the transfection of B7-1 cDNA into three ICAM-1⁺ (plasmocytoma J558L, T lymphomas EL-4 and RMA), but not into two ICAM-1^? tumor cell lines (adenocarcinoma TS/A and melanoma B16.F1), is sufficient to induce their complete rejection in syngeneic mice. The expression of ICAM-1 is necessary for the rejection of the B7 expressing tumors, since the primary response elicited by B7-1⁺ EL-4 and RMA clones expressing reduced levels of ICAM-1 is severely reduced. Furthermore, super-transfection of ICAM-1 cDNA into B7-1⁺ adenocarcinoma and melanoma clones optimizes their primary rejection. Histologic examination of transfected tumors reveals that B7-1 and ICAM-1 exert a potent pro-inflammatory activity. The intra-tumor infiltration is composed of both eosinophils and lymphomono-cytes, and is already massive 5 days after the tumor challenge. The primary rejection of the B7-1⁺ ICAM-1⁺ tumors depends critically on CD8⁺ T cells, natural killer cells and granulocytes, but is independent of CD4⁺ T cells. Remarkably, in addition to its effects on the early phases of the immune response, the co-expression of ICAM-1 and B7-1 on tumors is also necessary for the efficient induction of a memory response. In fact, only the primary challenge with B7-1⁺, ICAM-1⁺ tumor cells protects the majority of the mice from a second injection of parental tumor cells. Collectively, our findings indicate that B7-1 and ICAM-1 are fundamental components for triggering the primary rejection of tumors and establishing a protective memory response. These findings may help to define new strategies for the rational application of co-stimulation in tumor immunotherapy.     

The diversity of T-cell co-stimulation in the induction of antitumor immunity

Summary: T-cell activation has now been shown to require at least two signals. The first signal is antigen-specific, is delivered through the T-cell receptor (TCR) via the peptide/major histocompatibility complex (MHC). and causes the T cell to enter the ceil cycle. The second, or co-stimulatory, signal is required for cytokine production and proliferation, and is mediated through ligand interaction on the surface of the T cell. This chapter deals with: 1) comparative studies on the use of a dual-gene construct of a recombinant vaccinia (rV) vector containing a tumor-associated antigen (TAA) gene and a co-stimulatory molecule gene vs che use of admixtures of rV-TAA and rV containing the co-stimulatory molecule to induce and-tumor immunity; 2) the use of an admixture of vaccinia viruses containing a lAA gene and the B7-1 co-stimulatory molecule gene to induce a therapeutic response in a lung metastasis tumor model: 3) the antitumor efficacy of whole-tutnor-cell vaccines in which the B7-1 co-stimulatory molecule is expressed in a tumor-cell vaccine via a vaccinia vs a retroviral vector; 4) the use of recombinant poxviruses containing the genes for the co-stimulatory molecules tCAM-l or LFA-3 to induce antitumor immunity: and 5) the use of poxvirus vectors containing a triad of co-stimulatory molecules (B7-1. ICAM-1 and LFA-3) that synergize to enhance both CD4⁺ and CD8+ T-cell responses to a new threshold.     

细胞因子处理联合B7—l基因转染制备有效的肝癌疫苗

在体外用IFN-Y和TNF-。等细胞因子处理B7-1表达阳性的Hepal-6,观察其对小鼠肝癌表面MHC Class Ⅰ和ICAM-1表达的调节作用和对体内外抗肿瘤免疫反应的影响。结果发现:单独B7-1基因转染的Hepal-6的致瘤性消失,但仅能诱导部分细胞毒性和部分保护性免疫反应,而B7-1基因转染与细胞因子处理联合应用则能诱导强烈的细胞毒杀伤活性和完全的抗Hepal-6保护性免疫反应。结论B7-1共刺激分子在小鼠肝癌的表达是激活抗肿瘤免疫所必需的但不充分,而B7基因转染联合IFN-7等细胞因子处理能诱导彻底的抗肿瘤免疫反应。     

Effective induction of therapeutic antitumor immunity by dendritic cells coexpressing interleukin-18 and tumor antigen

Dendritic cell (DC) based cancer vaccine can induce potent antitumor immunity in murine models; however, objective clinical responses have been observed only in a minority of cancer patients. To improve the antitumor effect of DC vaccine, Th1-biasing cytokine interleukin (IL) 18 and melanoma-associated antigen gp100 were cotransfected into bone marrow-derived DC (IL-18/gp100-DC), which were used as vaccine to induce the protective and therapeutic immunity in a B16 melanoma model. Immunization with IL-18/gp100-DC resulted in tumor resistance in 87.5% of the mice challenged with B16 cells; however, 12.5% and 25% of mice immunized with gp100 gene-modified DC (gp100-DC) or IL-18 gene-modified DC (IL-18-DC) were tumor free, respectively. Most importantly, IL-18/gp100-DC immunization led to the generation of potent therapeutic immunity that significantly inhibited the tumor growth and improved the survival period of mice bearing established melanoma. Immune cell depletion experiments identified that CD4(+) T cells also played an important role in the priming phase of antitumor immunity and CD8(+) T lymphocytes were the primary effectors. gp100-specific CTL response were induced most markedly in the tumor-bearing mice immunized with IL-18/gp100-DC. Administration with such vaccine also significantly increased the production of Th1 cytokine (IL-2 and interferon-gamma) and induced infiltration of inflammatory cells inside and around the tumors. In addition, natural killer cell activity was also augmented. These results indicate that immunization with DC vaccine coexpressing Th1 cytokine IL-18 and tumor antigen gene may be an effective strategy for a successful therapeutic vaccination.     

Expression of MHC Class II and B7–1 and B7–2 costimulatory molecules accompanies tumor rejection and reduces the metastatic potential of tumor cells

Mouse tumor cells transfected with syngeneic MHC class II genes are highly immunogenic in the autologous host, and induce a potent tumor-specific immunity against wild type tumor. Previous studies with sarcoma tumor cells expressing transfected class II gene products with truncated cytoplasmic domains suggested that during the process of tumor rejection costimulatory molecules are induced on the tumor cells, contributing to the cells' ability to stimulate immunity. In the present study we directly demonstrate that tumor cells containing full-length class II heterodimers are induced to express B7–1 and B7–2 costimulatory molecules during the rejection process. In contrast, tumor cells expressing class II heterodimers truncated for their cytoplasmic tails are not induced to express B7–1 and/or B7–2. Blocking the interaction of the induced costimulatory molecules with their corresponding receptors on T cells prevents tumor rejection. These results support the hypothesis that the cytoplasmic domain of the MHC class II molecule is involved in induction of costimulatory molecule expression, perhaps via intracellular signalling pathways. Because class II, B7 transfected tumor cells are such effective immunogens against ascites and solid tumors, they have also been tested in metastatic disease. K1735 and B16BL6 mouse melanomas, when transfected with syngeneic MHC class II and B7–1 genes, are significantly less metastatic than parental cells, and immunization with the transfectants protects against subsequent challenge with wild type tumor.     

Apoptotic and necrotic cells induced by different agents vary in their expression of MHC and costimulatory genes

We have recently reported, in a murine tumor model, that apoptotic cells induced by different agents may vary in their ability to elicit host immunity. The basis for this observation is unclear but may involve varying efficiencies of cross-presentation and/or direct activation of immunity by different apoptotic preparations. As a first step in addressing this issue, we compared expression patterns of selected immune genes (MHC class I, class II, CD40, B7-1, B7-2) on viable and apoptotic populations induced by four different agents. The histone deacetylase inhibitor trichostatin A (TSA) induced MHC class II expression on viable and apoptotic cell populations, while LPAM, H2O2 and gamma-irradiation did not activate class II. Each agent employed elicited a different expression pattern of costimulatory molecules (CD40, B7-1, B7-2) on both apoptotic and 7-AAD+ 'necrotic' populations. In striking contrast to the TSA induction of MHC class II, class I cell surface protein was diminished on the apoptotic populations. These effects were not a result of changes in the cell cycle produced by the various treatments. The data demonstrate that distinctive gene expression patterns on viable and apoptotic cells are elicited by different apoptosis inducing agents. We discuss how expression patterns on dead or dying tumor cells could potentially affect the tumor's ability to elicit immunity.     

Therapy of established tumour with a hybrid cellular vaccine generated by using granulocyte–macrophage colony-stimulating factor genetically modified dendritic cells

Dendritic cells (DCs) are the most powerful of all antigen-presenting cells and play a critical role in the induction of primary immune responses. DC-based vaccination represents a potentially powerful strategy for cancer immunotherapy. In this study, a new approach for a DC-based melanoma vaccine was described. Splenic DCs from C57BL/6 mice were fused with B16 melanoma cells, and the resultant B16/DC hybrid cells expressed major histocompatibility complex (MHC) molecules - B7 as well as the B16 tumour marker M562 - which were enriched by Ia-mediated positive selection with a MiniMACS column. The fusion rates were 12.7-26.8%. To generate hybrid tumour vaccines with potentially greater potent therapeutic efficacy, we genetically engineered DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) prior to cell fusion. Recombinant adenovirus vector was used to mediate gene transfer into DCs with high efficiency and DCs expressed GM-CSF at 96-138 ng/105 cells/ml 24 hr after GM-CSF gene transfer. GM-CSF gene-modified DCs (DC.GM) exhibited higher expression of B7 and co-stimulatory capacity in mixed lymphocyte reaction (MLR). Fusion of DC.GM with B16 cells generated B16/DC.GM hybrid cells secreting GM-CSF at 59-63 ng/105 cells/ml. Immunization of C57BL/6 mice with the B16/DC hybrid vaccine elicited a specific cytotoxic T-lymphocyte (CTL) response and protected the immunized mice from B16 tumour challenge, reduced pulmonary metastases and extended the survival of B16 tumour-bearing mice. The B16/DC.GM hybrid vaccine was able to induce a CTL response and protective immunity more potently and tended to be therapeutically more efficacious than the B16/DC vaccine. In vivo depletion of T-cell subsets demonstrated that both CD8+ and CD4+ T cells were essential for the therapeutic effects of B16/DC and B16/DC.GM hybrid vaccines. Additionally, other non-specific effector cells may also contribute to tumour rejection induced by the B16/DC.GM hybrid vaccine. These data indicate that a DC-based hybrid tumour vaccine may be an attractive strategy for cancer immunotherapy, and that GM-CSF gene-modified DCs may lead to the generation of hybrid vaccines with potentially increased therapeutic efficacy.