Induction of tumor specific cytotoxic T lymphocyte by using transfer of co-simulatory molecule gene

抗原提呈细胞上的共刺激分子B7-1(CD80)与T淋巴细胞上的CD28受体之间的相互作用在包括肿瘤在内的细胞免疫反应的诱导中起着关键作用。许多实体瘤缺乏B7-1的表达，这被认为是导致这些疾病免疫识别失败的原因之一。在此基础上，我们推测通过复制缺陷型腺病毒载体(Ad)在肺癌细胞上表达的B7-1或B7-2分子所传递的共刺激信号可诱导肿瘤特异性细胞毒性T淋巴细胞(CTL)。为了验证这一假说，我们构建了两个AdS：AdCMVhB7(含巨细胞病毒即刻早期启动子和增强子驱动的人B7-1基因的AdCMVhB7载体)和AdNull(与我们外源基因表达相同的载体)作为对照。理论上，Ad的优势在于能够高效地进行基因转导。使用这些ADS，产生肿瘤特异性CTL…在原代同基因混合淋巴细胞肿瘤培养中，我们用肺癌细胞和肺癌患者的外周血淋巴细胞进行了进一步的研究。用10倍体AdCMVhB7感染肺癌细胞，细胞表面快速高效表达B7-1分子(24小时达细胞的90%)。淋巴细胞杀伤活性测定(E/T=40)表明，经AdCMVhB7处理的HB7-1(+)肺癌细胞诱导的效应淋巴细胞对亲本肺癌细胞HB7-1(-)的杀伤率为45%，最大杀伤率为62%，且该效应淋巴细胞不能溶解无关的同基因成纤维细胞。而以AdNull为对照病毒或PBS为对照的肺癌细胞诱导的效应淋巴细胞对亲本肺癌细胞均无杀伤作用。此外，抗CD3抗体(10ug/ml)可抑制B7-1转导的肺癌细胞诱导的效应淋巴细胞的杀伤活性。这些结果提示，AdCMVhB7诱导的肺癌效应淋巴细胞具有肿瘤特异性CTL的特性，腺病毒介导的HB7-1基因转移有望成为过继免疫治疗肺癌基因治疗的一种有效手段。较少

Interaction between the costimulatory molecule B7-1 (CD80) on antigen-presenting cells and its counter-receptor CD28 on T lymphocyte plays a key role in the induction of cell-mediated immune responses including cancer. Many solid tumor lack expression of B7-1 and this has been suggested to contribute to the failure of immune recognition of these diseases. Based on this knowledge, we hypothesized that co-stimulatory signal delivered through B7-1 or B7-2 molecule expressed on lung cancer cells using replication deficient adenovirus vector (Ad) would induce tumor-specific cytotoxic T lymphocyte (CTL). To evaluate this hypothesis, we constructed two Ads ; AdCMVhB7 (am E1^- Ad5 based vector containing human B7-1 cDNA driven by cytomegalovirus immediate early promoter and enhancer) ; AdNull (above same vector withour expression of exogenous gene) as control. Theoretical advantages of Ad consist in capacity of high efficient gene transduction. Using these Ads, generation of tumor specific CTL … More was studied in a primary syngeneic mixed lymphocyte tumor culture by using both lung cancer cells and peripheral blood lymphocytes obtained from patients with lung cancer. Inoculation of lung cancer cells with 10 multiplicity of infection of AdCMVhB7 resulted in rapid and efficient cell surface expression of B7-1 molecule (>90% of cell at 24h). Cytolytic activity of lymphocytes by using ^<51>Cr-releasing assay (E/T=40) demonstrated that effector lymphocytes induced by hB7-1(+)lung cancer cells treated with AdCMVhB7 could lyse 45% parental lung cancer cells hB7-1(-) with a maximum lysis of 62%, in addition, the effector lymphocytes could not lyse the irrelevant syngeneic fibroblast. In countrast, effector lymphocytes induced by lung cancer cells treated with AdNull as control virus or PBS as control could not lyse parental lung cancer cells at all. Furthermore, cytolytic activity of the effector lymphocytes induced by B7-1 transduced lung cancer cells was inhibited by addition of anti-CD3 antibody (10ug/ml). These data suggested that effector lymphocytes induced by lung cancer treated with AdCMVhB7 have a character of tumor-specific CTL.Adenovirus mediated-hB7-1 gene transfer may be a useful means for gene therapy for lung cancer in application of adoptive immunotherapy. Less