T CELL RESPONSE TO GENETICALLY ENGINEERED AND MATURED DC

这项建议的主要目的是验证这样一种假设，即“通过特殊的抗原提呈细胞，如树突状细胞(DC)，在危险和/或组织损伤的环境中生长到免疫原性成熟，并通过MHC I类和II类途径递送相关的肿瘤相关抗原，可以产生更有效的抗肿瘤T细胞反应”。以人黑色素瘤抗原MART-1系统为原型，其具体目的是：1)全面分析经基因工程和免疫原性成熟的DC体外提呈的CD4+和CD8+T细胞对MART-1的反应；2)确定CD4+T细胞促进和放大CTL反应的作用和机制；3)研究HELP的分子基础(IL-2消息/IL2R的表达，通过细胞因子共同受体Gammac的信号传递，以及抗凋亡和促凋亡机制(BclBax)；4)检测表达EGFP-TAA融合蛋白的VSV伪型逆转录载体、细胞内转运信号序列和细菌免疫刺激序列(ISS)构建的DC的区隔表位呈递体外免疫原性。在GM-CSF和IL-4中生长的髓样DC将用腺病毒载体转导表达Mart-1抗原，并通过CD40信号、多种细菌刺激剂或使DC从凋亡细胞中捕获Mart-1抗原而成熟为“免疫原性”。经条件处理的DC将用于体外产生CTL和辅助T细胞。T细胞反应将通过CTL试验、Fastimmune试验和四聚体结合试验进行监测。CD4+T细胞在DC和CTL上的作用将在适当的共培养中进行检测，CTL反应的稳健性将在CTL试验、Fastimmune试验和四聚体结合试验中确定，以获得CTL扩增的定量评估。我们还将测试一些途径，通过通过工程树突状细胞进行I类和/或II类抗原负载来增强抗原提呈。它们是：a)表达TAA和细胞内转运信号的嵌合多肽的内体定位：b)TAA和热休克-TAA融合的转运；c)通过表达嵌合TAA和细菌ISS序列在内部工程的极化Th1条件下的TAA转运；以及d)EGFP：TAA嵌合体的核定位。这些研究将提供对DC和CD4+T细胞结合规则的迫切需要的理解，并具有翻译含义。

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.