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：
7216216
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/7216216
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 Class Condition DNA DNA Single Strand Break Data Dendritic Cells Effectiveness Epitopes Face Generations Genetic Goals Homeostasis Immunotherapy In Vitro Influenza Life Lymphocyte Activation MAPK14 gene Mediating Mitochondria Mus Nuclear Numbers Pathway interactions Peptides 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 manganese(III)-tetrakis(4-benzoic acid)porphyrin melanoma mimetics mitochondrial membrane neoplastic cell porin prevent receptor response stress-activated protein kinase 1 tumor

项目摘要

DESCRIPTION (provided by applicant): 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 caspase dependent. Our studies indicated that these CTLs could be rescued from AICD by the c-jun N terminal 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 found that the AICD in these CTLs is mediated by the activation of a mitochondrial 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 DNA breaks. The JNK inhibitor SP600125 blocks the AIF release. We have detected that a short phosphorylated fragment 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 epitope-specific CTLs mostly results from the release of the mitochondria-based apoptotic effector proteins such as AIF triggered by the activation of the mitochondrial JNK-BH3-only proapoptotic protein- VDAC axis and that SP600125 rescues some of these CTLs from AICD by blocking JNK activation. Thus, a long-lived CTL response to tumor epitopes might be orchestrated by 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 Rag1-/- 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 how CTLs generated against a relevant tumor associated antigen can be kept alive longer and will therefore facilitate the design of more effective tumor immunotherapy.

描述（由申请人提供）：我们最近发现，在基于体外肽的基于DC的CTL生成方案中产生的很大一部分MART-1表位特定的主要CTL，在Cognate抗原的第一次二次接触之后，经历了AICD。这些CTL中的AICD不是由通常的外部死亡受体（FAS，TNFR等）触发的 - 介导的信号传导，并且不依赖于caspase。我们的研究表明，这些CTL可以由C-Jun N末端激酶（JNK）抑制剂SP600125从AICD中救出。在救援过程中，SP600125干扰了它们合成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是通过线粒体凋亡机制的激活来介导的，其特征是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，AIF易位到核上，并引起大规模（约50 kbp）的单链DNA断裂。 JNK抑制剂SP600125阻止了AIF释放。我们已经检测到，在AICD期间，在这些CTL中生成了JNK抑制剂SP600125的BIM的短磷酸化片段。我们还发现，JNK存在于线粒体上，并与几种基于线粒体的Bcl-2家族蛋白以及Mitichondrial Porin依赖性阴离子通道（VDAC）相互作用。这些观察结果促使我们假设AICD在自我但甲状腺瘤的表位特异性CTL中主要是由于基于线粒体的凋亡效应蛋白的释放而引起的，例如AIF，例如AIF，例如激活线粒体的激活，即线粒体jnk-bh3- kh3-on in cropoptoptopic propoptopic oppoptopic propoptotic proptipotial of sop proptip protins of spt62 propty of sp600 of sp600 of sp600 of sp600 of sp600 of sp600 sp600 sp600 sap600 sap600 sap600。通过阻止JNK激活通过AICD。因此，可以通过干扰这种JNK驱动的凋亡途径来策划对肿瘤表位的长期CTL反应。 2）研究黑色素瘤表位特异性主要CTL中基于线粒体的凋亡机制的基础，重点是阐明JNK-BIM/BAX-VDAC相互作用的潜在作用rag1 - / - 小鼠在体内将使用在体外CTL生成方案中产生的相应的抗原特异性CTL，然后在rag1-/ - / - / - / - / - / - / - / - / - / - / - / - / - / - / - / - / - / - / - / - / - /cesportion conderiogical conderication conterical conterical conterical conterical contimist conterigant conterical contimist conterical conterical conterical conterical conterigange conts ctl生成方案。基于RNA的JNK，BIM）和生化方法的沉默。这些研究将为相关肿瘤抗原产生的CTL如何保持更长的时间，从而提供急需的了解，从而促进更有效的肿瘤免疫疗法的设计。