DC Th Crosstalk in CTL Response to Mart 1

描述：(申请人摘要)这项提案的主要目标是测试 假设“针对人类肿瘤相关抗原(TAA)的CTL反应 以树突状细胞(DC)为基础的刺激诱导受 DC-T辅助(Th)小区串扰。更好地理解世界银行的规则 DC-Th串扰的接合控制产生和控制 抗TAA CTL反应将对DC疫苗设计产生重大影响。 1将对人类进行关键和全面的重新检查 髓系树突状细胞体外成熟过程。目标2将研究 DC/Th为基础的串扰在CTL产生过程中，在体外，对抗 MART-1作为人类TAA的原型。目标3将研究(S)背后的机制 基于DC/Th的CTL启动调控与抑制启动(耐受性 诱导)，在体外，在Mart-1系统中。粒细胞集落刺激因子诱导培养的髓系DC IL-4将进一步极化为免疫原性DC(Dc1)或耐受性DC (DC2)和CD4+T细胞将极化为Th1和Th2表型 不依赖于抗原(即，多克隆激活)和依赖于抗原(即， 产生抗原特异性的CD4+Th1系)方式。Th细胞和DC， 通过转导Adeno/Mart-1载体表达Mart-1或 用MART-1编码的多肽冲击，将与CD8+T细胞或 MART-1四聚体分选CTL前体在体外CTL生成系统中的应用 检查DC1/Th1串扰是否能产生强健且持续的 MART-1特异性CTL反应以及DC2/Th2二重奏是否能抑制和/或 终止CTL响应。Dc/Th相互作用规则和分子 将通过将CTLp或CTL暴露于 监管串扰(即，通过DC2/Th2)和通过确定 激活/失活状态(IL-2/IL-2R)或死亡与生存机制 (Fas/FasL，TNFR，BclBax基因和蛋白)在群体水平和 在单细胞水平上。DC/Th细胞对CTL前体的作用将是 在适当的共培养中检查和CTL反应的稳健性将 在CTL试验、Fastimmune试验和四聚体结合试验中被检测到 获得CTL扩展的定量评估。这些研究将提供一个 亟需了解DC和CD4+T细胞的结合规则和 将有助于设计一种更有效的DC和基于抗原的免疫疗法 癌症。

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.