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

项目总结 感染艾滋病毒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，它表达在活化的T细胞或NK细胞上。我们已经研究了小路 参与HIV感染约15年，发现(I)尽管表达TRAIL受体，HIV 被感染的细胞对TRAIL的致死效应具有矛盾的抵抗力，(Ii)我们发现了一种剪接 TRAIL的变体，由HIV+细胞产生，我们称之为TRAILShort。TRAILShort绑定到TRAIL 并阻止正常的(全长)TRAIL杀死这些细胞。 因此，我们创造了完全人源化的抗TRAILShort特异性抗体，隔离了TRAILShort，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) 导致更多的艾滋病毒感染细胞在急性感染期间死亡，从而减少艾滋病毒的复制。 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 仅限于患有活动性传染病和/或人类恶性肿瘤的捐赠者的样本。从那以后我们就 已公布约40%的人类癌症表达TRAIL Short(通过免疫组织化学和原位 杂交)，原发的B细胞恶性肿瘤被自体T细胞低效杀伤，但在 TRAIL短小抗体的存在，杀伤力显著增强。 我们还观察到，暴露于同源抗原的T细胞在存在抗TRAIL短小的情况下增殖更多 提示TRAILShort也可能直接影响T细胞功能和 扩散。这里提供的蛋白质组学数据表明，TRAIL短期处理原始T细胞会导致 细胞内T信号，细胞磷酸组的变化，T细胞激活和调节因子的变化 功能(如p38、ERK、JNK和Akt)。在TRAILShort蛋白处理的原代T细胞中，我们观察到p38 蛋白印迹法检测180/182位残基的磷酸化，并抑制T细胞受体诱导的T细胞活化 (TCR)结扎(CD25和69减少，CFSE稀释减少，ZAP70 Lat磷酸化减少 印迹)，表明TRAILShort对T细胞具有免疫抑制作用。 我们将通过(I)检测TRAILShort对HIV特异性T细胞功能的影响来加深我们对TRAILShort影响的理解 TRAIL短拮抗在恢复HIV特异性T细胞杀伤增殖性HIV感染细胞和 潜伏感染HIV的CD4T细胞被诱导从潜伏期重新激活，(Ii)使用先进的磷酸蛋白质组 以及了解TRAIL与TRAIL受体2的短结合如何改变T细胞的生化技术 动态平衡和(Iii)研究TCR在存在或不存在TRAIL Short的情况下诱导细胞激活，以确定 TCR信号的缺陷，使用遗传和小分子方法逆转缺陷，以及 产生较短的抗体。

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.