Rescuing CTL from Activation Induced Death

我们最近发现，很大一部分Mart-1表位特异的初级CTL，在体外产生 多肽负载的DC为基础的CTL生成方案，在第一次二次相遇后经历AICD 同源抗原。这些CTL中的AICD不是由常见的外部死亡受体(Fas，TNFR等)触发的- 介导的信号转导，不依赖于caspase。我们的研究表明，这些CTL可以从 C-jun末端激酶(JNK)抑制剂SP600125诱导的AICD。在救援过程中，SP600125介入 具有合成干扰素的能力，但不阻断其细胞溶解功能。我们最近发现， 这些CTL中的AICD是由线粒体的激活介导的！以细胞凋亡机制为特征的 释放线粒体为基础的凋亡诱导因子(AIF)，而不释放任何细胞色素c。然后是AIF 移位到细胞核上，导致大规模(约50kBP)单链DNA断裂。JNK抑制剂 SP600125阻止AIF版本。我们已经检测到Bim的一小段磷酸化片段，可被 JNK抑制剂SP600125是在AICD期间在这些CTL中产生的。我们还发现JNK也在场 在线粒体上，并与几种基于线粒体的Bcl-2家族蛋白和线粒体相互作用 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 在自身黑色素瘤中，表位特异的CTL主要是由线粒体为基础的细胞凋亡的释放所致 效应蛋白，如AIF，由只有JNK-BH3的线粒体结合蛋白的激活而触发- VDAC轴和SP600125通过阻断JNK的激活将部分CTL从AICD中拯救出来。因此，一个漫长的- 活体CTL对肿瘤表位的反应可能是通过干扰JNK驱动的凋亡途径来协调的。 因此，本文的具体目的是：1)界定S在《自我而为》一书中所蕴含的反腐败和反腐败的规则 黑色素瘤表位特异性CTL和流感MP(一种非自身和危险抗原)特异性CTL；2)研究 黑色素瘤表位AICD中基于线粒体的凋亡机制的机制 重点阐明JNK-Bim/Bax-VDAC相互作用作为 触发AIF释放；3)扩大我们对AICD的发现，并在黑色素瘤特异性表位中拯救AICD RagW-小鼠体内异种移植模型中的CTL。这项工作将用各自的抗原进行 体外CTL生成方案中产生的特异性CTL，随后评估其对AICD的敏感性 在不同实验条件下建立Rag1-/-小鼠异种移植模型。这一机制 将通过药理学、遗传学(基于干扰RNA的沉默)探索AICD的发生和从AICD中拯救 JNK、BIM)和生化方法。这些研究将提供对如何CTL的迫切需要的理解 针对相关的肿瘤相关抗原产生的抗原可以保持更长的生命，因此将有助于 设计更有效的肿瘤免疫疗法。

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.