Rescuing CTL from Activation Induced Death

描述(申请人提供)：我们最近发现，在体外基于DC的多肽负载CTL生成方案中产生的MART-1表位特异性初级CTL中，有很大一部分在同源抗原的第一次二次相遇后经历AICD。这些CTL中的AICD不是由通常的外部死亡受体(Fas，TNFR等)介导的信号触发的，也不依赖于caspase。我们的研究表明，这些CTL可以被c-Jun N末端激酶(JNK)抑制剂SP600125从AICD中拯救出来。在解救过程中，SP600125干扰了它们合成IFNG的能力，但没有阻断它们的细胞溶解功能。我们最近发现，这些CTL中的AICD是由线粒体凋亡机制的激活所介导的，其特征是释放基于线粒体的凋亡诱导因子(AIF)，而不释放任何细胞色素c。然后，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可阻断AIF的释放。我们已经检测到，在AICD期间，这些CTL中产生了一段短的Bim磷酸化片段，该片段可被JNK抑制剂SP600125抑制。我们还发现JNK存在于线粒体上，并与几个基于线粒体的Bcl-2家族蛋白以及线粒体孔蛋白电压依赖的阴离子通道(VDAC)相互作用。这些观察结果促使我们假设，自身但黑色素瘤表位特异性CTL中的AICD主要是由线粒体JNK-BH3-Only促凋亡蛋白-VDAC轴激活触发的基于线粒体的凋亡效应蛋白的释放所致，SP600125通过阻断JNK激活来挽救AICD中的一些CTL。因此，长寿的CTL对肿瘤表位的反应可能是通过干扰JNK驱动的凋亡途径来进行的。因此，本研究的具体目的是：1)在“自身”但黑色素瘤表位特异性CTL和流感MP(一种非自身且危险的抗原)特异性CTL中定义AICD和AICD救治的规则(S)；2)研究黑色素瘤表位特异性原代CTL中AICD所涉及的基于线粒体的凋亡机制，重点阐明JNK-BIM/BAX-VDAC相互作用在AIF释放中的潜在作用；3)扩展我们在Rag1-/-小鼠体内移植模型中黑色素瘤表位特异性CTL中AICD和AICD救治的发现。这项工作将在体外CTL生成程序中产生各自的抗原特异性CTL，然后在Rag1-/-小鼠的异种移植模型中评估它们在不同实验条件下对AICD的敏感性。将通过药理学、遗传学(干扰基于RNA的JNK、Bim沉默)和生化方法来探索AICD的机制和从AICD中拯救出来。这些研究将提供对针对相关肿瘤相关抗原产生的CTL如何保持更长时间生存的迫切需要的理解，因此将有助于设计更有效的肿瘤免疫治疗。

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.