T CELL RESPONSE TO GENETICALLY ENGINEERED AND MATURED DC

T 细胞对基因工程和成熟 DC 的反应

• 批准号: 6377490
• 负责人: BIJAY MUKHERJI
• 金额: $ 23.58万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377490
• 关键词: MHC class I antigen; MHC class II antigen; T lymphocyte; antigen presentation; apoptosis; biological signal transduction; cell population study; chimeric proteins; colony stimulating factor; cytokine receptors; cytotoxic T lymphocyte; dendritic cells; genetic manipulation; helper T lymphocyte; human tissue; immunogenetics; interleukin 2; intracellular transport; melanoma; neoplastic cell; receptor expression; tissue /cell culture; transfection /expression vector; tumor antigens

The major goal of this proposal is to test the hypothesis that "a more efficient anti-tumor T cell response can be generated through antigen presentation by specialized antigen presenting cells such as dendritic cells (DC) grown to immunogenic maturity in an environment of danger and/or tissue damage and made to present the relevant tumor associated antigen through the MHC class I and class II pathways". Using the human melanoma antigen MART-1 system as a prototype, the specific aims are: 1) to undertake a comprehensive analysis of both CD4+ and CD8+ T cell responses to MART-1 presented in vitro by engineered and immunogenically matured DC; 2) to define the role of and the mechanism by which CD4+ T cells facilitate and amplify CTL response; 3) to examine the molecular basis of "help" (IL-2 message/IL2R expression, signaling through cytokine common receptor gammac, and anti-apoptotic vs pro-apoptotic mechanism (Bcl/Bax); and 4) to examine the in vitro immunogenicity of compartmentalized epitope presentation by DC genetically engineered with VSV pseudotyped retrovector expressing EGFP-TAA fusion protein plus intracellular trafficking signal sequences and bacterial immuno-stimulatory sequences (ISS). Myeloid DC grown in GM-CSF and IL-4 will be transduced with an adenovector to express the MART-1 antigen and matured to "immunogenic competence" through CD40 signaling, with a variety of bacterial stimulants, or by making the DC capture MART-1 antigen from apoptotic cells. The conditioned DC will be used to generate CTL and helper T cell in vitro. T cell responses will be monitored in CTL assay, Fastimmune assay, and tetrameter binding assay. The role of the CD4+ T cells on the DC as well as on the CTL will be examined in appropriate co-cultures and the robustness of the CTL response will be determined in CTL assay, Fastimmune assay and in tetramer binding assay to obtain a quantitative assessment of CTL expansion. We shall also test a number of avenues to enhance antigen presentation by compartmentalized class I and/or class II loading of antigen via engineered DC. These are: a) endosomal localization with chimeric polypeptide expressing the TAA and intracellular trafficking signals: b) trafficking of TAA and heat shock -TAA fusions; c) TAA trafficking under a polarized Th1 condition engineered internally by expressing chimeric TAA and bacterial ISS sequences; and d) nuclear localization of EGFP:TAA chimeras. These studies will provide a much needed understanding of the rules of engagement of DC and CD4+ T cells with translational implications.

该提案的主要目标是检验以下假设：“通过专门的抗原呈递细胞（例如在危险和/或组织损伤的环境中生长至免疫原性成熟的树突状细胞（DC））的抗原呈递，可以产生更有效的抗肿瘤 T 细胞反应，并通过 MHC I 类和 II 类途径呈递相关的肿瘤相关抗原”。 以人类黑色素瘤抗原MART-1系统为原型，具体目标是：1）全面分析CD4+和CD8+T细胞对工程化和免疫原性成熟的DC在体外呈现的MART-1的反应； 2) 定义CD4+ T细胞促进和放大CTL反应的作用和机制； 3) 检查“帮助”的分子基础（IL-2 信息/IL2R 表达、通过细胞因子共同受体 gammac 发出的信号、以及抗凋亡与促凋亡机制 (Bcl/Bax)；4) 检查通过表达 VSV 假型逆转录病毒基因工程的 DC 进行区室化表位呈递的体外免疫原性 EGFP-TAA 融合蛋白加上细胞内运输信号序列和细菌免疫刺激序列 (ISS)。 在 GM-CSF 和 IL-4 中生长的髓样 DC 将用腺载体转导以表达 MART-1 抗原，并通过 CD40 信号传导、使用多种细菌刺激剂或通过使 DC 从凋亡细胞捕获 MART-1 抗原来成熟至“免疫原性能力”。条件化的 DC 将用于在体外生成 CTL 和辅助 T 细胞。 T 细胞反应将通过 CTL 测定、Fastimmune 测定和四联结合测定进行监测。将在适当的共培养物中检查 CD4+ T 细胞对 DC 和 CTL 的作用，并在 CTL 测定、快速免疫测定和四聚体结合测定中确定 CTL 反应的稳健性，以获得 CTL 扩增的定量评估。 我们还将测试多种通过工程化 DC 加载 I 类和/或 II 类抗原来增强抗原呈递的途径。 它们是：a)用表达TAA和细胞内运输信号的嵌合多肽进行内体定位；b)TAA运输和热休克-TAA融合体； c) 通过表达嵌合 TAA 和细菌 ISS 序列，在内部设计的极化 Th1 条件下进行 TAA 运输； d) EGFP:TAA嵌合体的核定位。 这些研究将为 DC 和 CD4+ T 细胞的参与规则及其翻译意义提供急需的理解。