T CELL RESPONSE TO GENETICALLY ENGINEERED AND MATURED DC

该提案的主要目标是验证这样一种假设，即“通过抗原呈递，可以产生更有效的抗肿瘤T细胞反应，这种抗原呈递是由在危险和/或组织损伤的环境中生长到免疫原性成熟的树突状细胞（DC）等特化抗原呈递，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，具体目的是：1)通过工程和免疫原性成熟的DC，对体外CD4+和CD8+ T细胞对MART-1的反应进行全面分析；2)明确CD4+ T细胞促进和增强CTL反应的作用和机制；3)研究“帮助”(IL-2信息/IL2R表达，通过细胞因子共同受体γ - mac信号传导，抗凋亡/促凋亡机制（Bcl/Bax）的分子基础；4)用表达EGFP-TAA融合蛋白、细胞内运输信号序列和细菌免疫刺激序列（ISS）的VSV伪型逆转录载体进行DC基因工程，检测区隔表位呈递的体外免疫原性。在GM-CSF和IL-4中生长的髓系DC将用腺载体转导以表达MART-1抗原，并通过CD40信号、各种细菌刺激物或使DC从凋亡细胞中捕获MART-1抗原而成熟为“免疫原性能力”。体外培养的DC将用于生成CTL和辅助T细胞。T细胞反应将在CTL试验、fasttime试验和四参数结合试验中进行监测。CD4+ T细胞在DC和CTL上的作用将在适当的共培养中进行检查，CTL反应的稳健性将通过CTL测定、fasttimmune测定和四聚体结合测定来确定，以获得CTL扩增的定量评估。我们还将测试一些途径，通过工程DC将抗原分成I类和/或II类负载来增强抗原呈递。这些是：a)表达TAA和细胞内运输信号的嵌合多肽的内体定位；b) TAA的运输和热休克-TAA融合；c)通过表达嵌合的TAA和细菌ISS序列，在Th1极化条件下进行TAA转运；d) EGFP - TAA嵌合体的核定位。这些研究将提供对DC和CD4+ T细胞结合的规则的理解，并具有翻译意义。

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.