Rescuing CTL from Activation Induced Death

拯救 CTL 免遭激活诱导的死亡

基本信息

批准号：
7578930
负责人：
BIJAY MUKHERJI
金额：
$ 25.51万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7578930
关键词：
Adjuvant Adverse effects Antigen-Presenting Cells Antigens Apoptosis Apoptotic Automobile Driving Biochemical Cancer Patient Cancer Vaccines Caspase Cell Death Cell Nucleus Cells Cessation of life DNA Data Dendritic Cells Effectiveness Epitopes Face Generations Genetic Goals Homeostasis Immunotherapy In Vitro Influenza JUN gene Life Lymphocyte Activation MAPK14 gene Mediating Mitochondria Mus Nuclear Pathway interactions Peptides Phosphotransferases Process Proliferating Protein Family Proteins Protocols documentation Rag1 Mouse Role SP600125 Signal Transduction Stimulus Superoxide Dismutase T-Lymphocyte Tumor Antigens Tumor Necrosis Factor Receptor Vaccination Voltage-Dependent Anion Channel Work Xenograft Model apoptosis inducing factor base cytochrome c design improved in vivo in vivo Model inhibitor/antagonist kinase inhibitor melanoma mimetics mitochondrial membrane neoplastic cell porin prevent receptor response stress-activated protein kinase 1 tumor

项目摘要

We have recently found that a large fraction of Mart-1 epitope specific primary CTLs, generated in an in vitro peptide-loaded DC-based CTL generation protocol, undergoes AICD after the very first secondary encounter of the cognate antigen. The AICD in these CTLs is not triggered by the usual external death receptor (FAS, TNFR, etc.)- mediated signaling and is not caspasedependent. Our studies indicated that these CTLs could be rescued from AICD by the c-jun Nterminal kinase (JNK) inhibitor, SP600125. In the process of rescuing, SP600125 interfered with their capacity to synthesize IFNg but did not block their cytolytic function. We have recently foundthat the AICD in these CTLs is mediated by the activation of a mitochondria! apoptotic machinery characterized by the release of the mitochondria-based apoptosis inducing factor (AIF) without any cytochrome c release. AIF then translocates onto the nuclei and causes large scale (around 50 kbp) single stranded DMAbreaks. The JNK inhibitor SP600125 blocks the AIF release. We have detected that a short phosphorylatedfragment of Bim, inhibitable by the JNK inhibitor SP600125, is generated in these CTLs during AICD. We have also found that JNK to be present on mitochondria and to interact with several mitochondria-based Bcl-2 family proteins and with the mitichondrial porin voltage dependent anion channel (VDAC). These observations have prompted us to hypothesize that AICD in self-but-melanoma epftope-specific CTLs mostly results from the release of the mitochondria-based apoptotic effector proteins such asAIF triggeredby the activation of the mitochondhalJNK-BH3-only prpapoptotic protein- VDAC axis and that SP600125 rescues some of these CTLs from AICD by blocking JNK activation. Thus, a long- lived CTLresponse to tumor epitopes might be orchestratedby interfering with this JNK-driven apoptotic pathway". Hence, the specific aims are :1) To define the rule(s) underlying AICD and rescue from AICD in "self but melanoma epitope specific CTLs and influenza MP (a non-self and dangerous antigen)-specific CTLs; 2) To study the mechanism underlying the mitochondria-based apoptotic machinery involved in AICD in the melanoma epitope specific primary CTLs with emphasis on elucidating a potential role of JNK-Bim/Bax-VDAC interaction as the trigger for AIF release; 3) To extend our findings of AICD and rescue from AICD in the melanoma epitope specific CTLs in a xenograft model in RagW- mice, in vivo. The work will be carried out with the respective antigen specific CTLs generated in an in vitro CTL generation protocol followed by assessing their sensitivity to AICD under different experimental conditions in vitro and in vivo in a xenograft model in Rag1-/- mice. The mechanism of AICD and rescue from AICD will be explored through pharmacologic, genetic (interfering RNA-based silencing of JNK, Bim), and biochemical approaches. These studies will provide much needed understanding of howCTLs generated against a relevant tumor associated antigen can be kept alive longer and will therefore facilitate the design of more effective tumor immunotherapy.

我们最近发现，在体外产生的很大一部分MART-1表位特异性主要CTL 基于肽的DC基于DC的CTL生成协议，在第一次二次相遇之后进行AICD 同源抗原。这些CTL中的AICD并未由通常的外部死亡受体（FAS，TNFR等）触发 - 介导的信号传导，不是依赖性的。我们的研究表明，这些CTL可以从 C-Jun N端激酶（JNK）抑制剂SP600125的AICD。在营救过程中，SP600125干扰了它们可以合成IFNG但没有阻止其细胞溶解功能的能力。我们最近发现这些CTL中的AICD是通过线粒体的激活介导的！凋亡机械为特征释放基于线粒体的凋亡诱导因子（AIF），而没有任何细胞色素C释放。然后易位到细胞核上，并引起大规模（约50 kbp）的单链Dmabreaks。 JNK抑制剂 SP600125阻止了AIF版本。我们已经检测到BIM的短磷酸化剥离，可抑制 JNK抑制剂SP600125在AICD期间在这些CTL中产生。我们还发现JNK在场在线粒体上并与几种基于线粒体的Bcl-2家族蛋白和Mitichondrial相互作用孔蛋白电压依赖性阴离子通道（VDAC）。这些观察结果促使我们假设AICD 在自我 - 甲状腺瘤特异性CTL中，主要是由于基于线粒体的凋亡的释放而引起的效应子蛋白（例如aSAIF）触发线粒体蛋白质的激活，仅促凋亡蛋白 VDAC轴和SP600125通过阻止JNK激活从AICD中拯救了其中一些CTL。因此，长期对肿瘤表位的生命可能是通过干扰这种JNK驱动的凋亡途径的精心策划的。因此，具体目的是：1）定义AICD的规则并从AICD中救出的规则黑色素瘤表位特异性CTL和流感MP（一种非自身和危险的抗原）特异性CTL； 2）学习黑色素瘤表位中AICD涉及的基于线粒体的凋亡机制的基础机制特定的主要CTL强调阐明JNK-BIM/BAX-VDAC相互作用的潜在作用触发AIF释放； 3）扩展了我们对AICD的发现，并从黑色素瘤特异性的AICD中救出 ragw-鼠的异种移植模型中的CTL，体内。该工作将使用相应的抗原进行在体外CTL生成方案中产生的特定CTL，然后评估其对AICD的敏感性在不同的实验条件下，在Rag1 - / - 小鼠中的异种移植模型中的体外和体内。机制 AICD和AICD的营救将通过药物，遗传（干扰基于RNA的沉默）进行探索 JNK，BIM）和生化方法。这些研究将提供对HOWCTL的急需理解针对相关肿瘤相关抗原产生的可以保持更长的时间，因此将促进设计更有效的肿瘤免疫疗法。