Rescuing CTL from Activation Induced Death

我们最近发现，在体外培养中产生的大部分Mart-1表位特异性初级CTL， 负载肽的基于DC的CTL产生方案，在第一次二次遇到CTL后经历AICD。 同源抗原这些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，并且不是半胱天冬酶依赖性的。我们的研究表明，这些CTL可以从 AICD的c-jun N末端激酶（JNK）抑制剂，SP 600125。在抢救过程中，SP 600125干扰 具有合成IFNg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！细胞凋亡机制的特征是 基于细胞凋亡诱导因子（AIF）的释放而没有任何细胞色素c释放。AIF然后 易位到细胞核上并引起大规模（约50 kbp）单链DNA断裂。的JNK抑制剂 SP 600125阻止AIF发布。我们已经检测到Bim的一个短的磷酸化片段， JNK抑制剂SP 600125在AICD期间在这些CTL中产生。我们还发现JNK存在于 并与几种基于线粒体的Bcl-2家族蛋白和线粒体的线粒体蛋白相互作用。 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，表位特异性CTL主要是由基于细胞凋亡的细胞因子的释放引起的。 效应蛋白，如AIF触发的线粒体halJNK-BH 3-only促凋亡蛋白的激活， 因此，SP 600125通过阻断JNK激活从AICD中拯救了这些CTL中的一些。因此，一个长期的- 对肿瘤表位的活的CTL应答可能通过干扰JNK驱动的凋亡途径来协调”。 因此，具体的目标是：1）定义AICD的基本规则，并从AICD中拯救“自我，但 黑色素瘤表位特异性CTL和流感病毒MP（非自身和危险抗原）特异性CTL; 2）研究 黑色素瘤表位中AICD所涉及的基于细胞凋亡机制的潜在机制 特异性初级CTL，重点是阐明JNK-Bim/JNK-VDAC相互作用的潜在作用， 3）为了扩展我们在黑色素瘤表位特异性中AICD和AICD拯救的发现， RagW-小鼠体内异种移植模型中的CTL。将使用相应的抗原进行工作 在体外CTL产生方案中产生特异性CTL，随后评估其对AICD的敏感性 在Rag 1-/-小鼠的异种移植模型中，在体外和体内的不同实验条件下。机制 将通过药理学、遗传学（基于干扰RNA的沉默） 的JNK，Bim）和生物化学方法。这些研究将提供急需的理解如何CTL 产生的抗相关肿瘤相关抗原的抗体可以保持更长的存活时间，因此将促进肿瘤细胞的增殖。 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.