Evaluating the role of the novel apoptosis inhibitor TRAILshort, in maintaining HIV persistence

项目摘要 HIV感染的细胞逃避正常的宿主免疫反应。免疫系统用来杀死 病毒感染的细胞是TRAIL，其在活化的T细胞或NK细胞上表达。我们研究了TRAIL 参与HIV感染约15年，并发现（i）尽管表达TRAIL受体，HIV 感染的细胞对TRAIL的促死亡作用具有矛盾的抵抗力，并且（ii）我们发现了一种剪接， 由HIV+细胞产生的TRAIL变体，我们称之为TRAILshort。TRAIL短结合至TRAIL 受体并阻止正常（全长）TRAIL杀死这些细胞。 因此，我们创建了螯合TRAIL短的完全人源化的抗TRAIL短特异性抗体，并且 在急性体外艾滋病毒感染中进行了测试。抗TRAIL短抗体（或TRAIL短产生的遗传抑制） 导致更多的HIV感染细胞在急性感染期间死亡，从而减少HIV病毒的复制。 接下来，我们分析了来自253，200个人类样本的公开可用的RNAseq数据集，并确定了TRAILshort 仅在来自患有活动性传染病和/或人类恶性肿瘤的供体的样品中。我们已经 发表了约40%的人类癌症表达TRAIL短（通过免疫组织化学和原位 杂交），且原发性B细胞恶性肿瘤不能被自体T细胞有效地杀死，然而， 在TRAIL短抗体的存在下，该杀伤显著增强。 我们还观察到，暴露于同源抗原的T细胞在抗TRAIL短肽存在下增殖更多。 这表明TRAIL短也可能直接影响T细胞功能， 增殖本文提供的蛋白质组学数据显示，原代T细胞的TRAIL短处理导致细胞凋亡。 细胞内T信号传导，细胞磷质组的变化，T细胞活化调节因子的改变， 功能（例如p38、ERK、JNK和Akt）。在用TRAIL短蛋白处理的原代T细胞中，我们观察到p38 通过蛋白质印迹，在残基180/182处磷酸化，并且由T细胞受体诱导的T细胞活化受损 (TCR)连接（通过Western印迹分析，减少的CD 25和69，较少的CFSE稀释和减少的Zap 70 Lat磷酸化） 印迹），共同表明TRAIL短对T细胞具有免疫抑制性。 我们将通过（i）测试TRAILshort对HIV特异性T细胞功能的影响， TRAIL短拮抗作用对恢复HIV特异性T细胞杀伤生产性HIV感染细胞的作用， 潜伏性HIV感染的CD 4 T细胞诱导从潜伏期重新激活，（ii）使用先进的磷酸化蛋白质组学 和生物化学技术来了解TRAIL短结合TRAIL受体2如何改变T细胞 （iii）在存在或不存在TRAIL短的情况下研究TCR诱导的细胞活化，以确定 TCR信号传导中的缺陷，使用遗传和小分子方法逆转缺陷，以及 TRAIL短抗体。

PROJECT SUMMARY HIV infected cells escape normal host immune responses. One pathway that the immune system uses to kill virally infected cells is TRAIL, which is expressed on activated T cells or NK cells. We have studied TRAIL involvement in HIV infection for ~15 years and discovered that (i) despite expressing TRAIL receptors, HIV infected cells are paradoxically resistant to the pro-death effects of TRAIL, and (ii) we discovered a splice variant of TRAIL that is produced by HIV+ cells which we have called TRAILshort. TRAILshort binds to TRAIL receptors and prevents normal (full length) TRAIL from killing these cells. We therefore created fully-humanized anti-TRAILshort-specific antibodies that sequester TRAILshort, and tested it in acute in vitro HIV infection. Anti TRAILshort antibody (or genetic inhibition of TRAILshort production) causes more HIV infected cells to die during acute infection, resulting in reduced HIV viral replication. We next analyzed publicly available RNAseq datasets from 253,200 human samples and identified TRAILshort exclusively in samples from donors with active infectious diseases, and/or human malignancy. We have since published that ~40% of human cancers express TRAILshort (by immunohistochemistry and in situ hybridization), and that primary B cell malignancies are inefficiently killed by autologous T cells, yet in the presence of TRAILshort antibody, that killing is significantly enhanced. We also observed that T cells exposed to cognate antigen proliferated more in the presence of anti-TRAILshort antibody, than in the absence, suggesting that TRAILshort might also directly impact T cell function and proliferation. Proteomic data presented herein show that TRAILshort treatment of primary T cells results in intracellular T signaling, changes in the cellular phosphorome, alterations in regulators of T cell activation and function (e.g. p38, ERK, JNK and Akt). In primary T cells treated with TRAILshort protein, we observe p38 phosphorylation at residues 180/182 by western blot, and impaired T cell activation induced by T-cell receptor (TCR) ligation (reduced CD25 and 69, less CFSE dilution and reduced Zap70 Lat phosphorylation by western blot), altogether indicating that TRAILshort is immunosuppressive to T cells. We will advance our understanding of the effect of TRAILshort on HIV specific T cell function by (i) testing the effect of TRAILshort antagonism on restoring HIV-specific T cell killing of productively HIV infected cells and latently HIV infected CD4 T cells induced to reactivate from latency, (ii) using advanced phospho-proteomic and biochemical techniques to understand how TRAILshort binding to TRAIL receptor 2 alters T cell homeostasis and (iii) study TCR-induced cell activation in the presence or absence of TRAILshort, to define defects in TCR signaling, reversing the defect using genetic and small molecule approaches, as well as TRAILshort antibodies.