DC Th Crosstalk in CTL Response to Mart 1

描述：（申请人摘要）本提案的主要目标是测试 “针对人肿瘤相关抗原（TAA）的CTL应答”这一假说， 由基于树突状细胞（DC）的刺激诱导的免疫应答受以下调节： DC-T辅助（Th）细胞串扰。更好地理解规则 参与的DC-Th串扰，支配的产生和控制， 抗TAA CTL应答将对基于DC的疫苗设计产生重大影响。“瞄准 1将对人类进行批判性和全面的重新审视 体外髓样DC成熟过程。目标2将研究 DC/Th为基础的crosstalk在CTL产生过程中，在体外， Mart-1是人类TAA的原型。目标3将审查 基于DC/Th的CTL启动调节与启动抑制（耐受性） 诱导），在体外，在Mart-1系统中。在GM-CSF中生长的髓样DC和 IL-4将进一步极化为免疫原性DC（DC 1）或致耐受性DC (DC2)CD 4 + T细胞将极化为Th 1和Th 2表型， 不依赖于抗原（即，多克隆激活的）和抗原依赖性（即， 产生抗原特异性CD 4 + Th 1细胞系）的方式。Th细胞和DC， 通过用Adeno/Mart-1载体转导而工程化以表达Mart-1，或 用Mart-1编码的肽脉冲，将与CD 8 + T细胞组装，或 用Mart-1四聚体分选的CTL前体在体外CTL产生系统中 检查DC 1/Th 1串扰是否可以产生鲁棒且持续的 Mart-1特异性CTL应答，并且如果DC 2/Th 2二联体能够抑制和/或 终止CTL应答。DC/Th结合规律及分子机制 调节机制将通过将CTLp或CTL暴露于 调节串扰（即，通过DC 2/Th 2）和通过确定 激活/失活状态（IL-2/IL-2 R）或死亡与生存机制 (Fas/FasL、TNFR、Bcl/Bax基因和蛋白质）的CTL的表达， 在单细胞水平上。DC/Th细胞对CTL前体的作用将是 在适当的共培养物中检查，CTL应答的稳健性将 在CTL测定、Fastimmune测定和四聚体结合测定中测定， 获得CTL扩增的定量评估。这些研究将提供一个 急需了解DC和CD 4 + T细胞的参与规则， 将有助于设计更有效的DC和基于抗原的免疫疗法， 癌

DESCRIPTION: (Applicant's Abstract) The major goal of this proposal is to test the hypothesis that "CTL response against human tumor associated antigen (TAA) induced by dendritic cell (DC)-based stimulation is subject to regulation by DC-T helper (Th) cell crosstalks. A better understanding of the rules of the engagement of DC- Th crosstalks that govern the generation and the control of anti-TAA CTL response will have a major impact in DC-based vaccine design." Aim 1 will undertake a critical and comprehensive re-examination of the human myeloid DC maturation process, in vitro. Aim 2 will study the role of DC/Th-based crosstalks in the process of CTL generation, in vitro, against Mart-1 as a prototype human TAA. Aim 3 will examine the mechanism(s) underlying DC/Th-based regulation of CTL priming versus inhibition of priming (tolerance induction), in vitro, in the Mart-1 system. Myeloid DCs grown in GM-CSF and IL-4 will be further polarized to immunogenic DCs (DC1) or to tolerogenic DCs (DC2) and CD4+ T cells will be polarized to Th1 and Th2 phenotypes in an antigen independent (i.e., polyclonally activated) and antigen dependent (i.e., generating antigen specific CD4+ Th1 lines) manner. The Th cells and the DCs, engineered by transduction with an Adeno/Mart-1 vector to express Mart-1 or pulsed with Mart-1 encoded peptides, will be assembled with CD8+ T cells or with Mart-1 tetramer sorted CTL precursors in an in vitro CTL generation system to examine if the DC1/Th1 crosstalk could generate a robust and sustained Mart-1 specific CTL response and if the DC2/Th2 duo could inhibit and/or terminate CTL responses. The rules of the DC/Th engagements and the molecular mechanisms of regulation will be defined by exposing the CTLp or CTLs to the regulatory crosstalk (i.e., by DC2/Th2) and by determining the activation/inactivation status (IL-2/IL-2R) or the death vs survival machinery (Fas/FasL, TNFR, Bcl/Bax genes and proteins) of the CTL at population level and at single cell level. The role of the DC/Th cells on the CTL precursors 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. These studies will provide a much needed understanding of the rules of engagement of DC and CD4+ T cells and will help design a more effective DC and antigen-based immunotherapy for cancer.