Targeting of Master Signaling Molecule to Restore Functions of Exhausted HIV-specific CTLs

项目概要： 大多数人类免疫缺陷病毒（HIV）感染者，如果不坚持高活性的组合 抗逆转录病毒治疗（cART），最终死于慢性感染。然而，有些人被称为“精英 证明上级病毒控制的“控制者”（EC），即使在 没有CART。尽管内皮细胞中的HIV特异性细胞毒性T淋巴细胞（CTL）对于病毒控制至关重要， 和进展状态，利用CTL来对抗HIV储库仍然具有挑战性，因为很少或没有 病毒蛋白质在静止的CD 4 + T细胞中产生，使得宿主CTL难以检测到该储存库 免疫反应目前可用的清除潜伏感染艾滋病毒的细胞的策略包括“休克和杀死”， 由治疗性疫苗引发的广泛应答性TCR，广泛中和的单克隆抗体， 程序性细胞死亡蛋白-1（PD-1）阻断和其他免疫检查点阻断。但是没有一 这些策略有效地根除了艾滋病病毒库。我们的初步数据显示， 一个小的衔接分子，鸡肿瘤病毒10号激酶调节因子（Crk）的磷酸化， 在耗尽的HIV特异性CTL免疫突触的中心由PD-1信号传导诱导。这一关键 观察促使我们开发了一种新的策略，该策略针对一种常见的主信号分子， 恢复耗尽的CTL的功能以根除HIV储库，这是一种上级于 靶向单个抑制性免疫受体。我们假设慢性HIV导致功能性 通过抑制性受体的上调损害CTL，并且由此产生的下游磷酸化 Crk的表达反过来又阻止了CTL的激活。我们认为外源性磷酸化Crk（pCrk）抑制剂可以 用于恢复耗尽的CTL功能以根除HIV宿主。我们提出以下两个目标： (Aim 1）通过以下方式恢复针对生产性感染细胞的耗尽的HIV特异性CTL的功能： 抑制Crk磷酸化。使用多管齐下的方法新鉴定的pCrk抑制剂（包括 最先进的Alphascreen、热位移测定、体外激酶和细胞磷酸流测定）进行检测 为了确定这些pCrk抑制剂是否可以恢复耗尽的HIV特异性CTL的功能， 高效感染的细胞(Aim 2）恢复耗尽的HIV特异性CTL的功能， 通过抑制Crk磷酸化感染细胞。为了进一步测试pCrk抑制剂的功效，我们将 确定pCrk抑制剂是否可以恢复耗尽的HIV特异性CTL的功能， 被感染的细胞我们将从外周血和淋巴中产生潜伏性HIV感染的原代CD 4 + T细胞 节点，以比较它们对使用和不使用pCrk抑制剂的耗尽的HIV特异性CTL的杀伤的敏感性。如果 小分子pCrk抑制剂的成功治疗应用可以改善慢性HIV患者的护理， 恢复疲惫的患者CTL防御。这项研究的成功结果将导致开发 一种新的免疫疗法，以消除艾滋病毒水库和相关的临床试验。

Project Summary: Most human immunodeficiency virus (HIV)-infected people, if not adherent to highly active combination antiretroviral therapy (cART), ultimately succumb to chronic infection. However, some are known as “elite controllers” (ECs) who demonstrate superior virus control, maintaining virtually undetectable viral loads even in the absence of cART. Although HIV-specific cytotoxic T lymphocytes (CTLs) in ECs are critical for viral control and progression status, harnessing CTLs to combat HIV reservoirs remains challenging because little to no viral protein is produced in quiescent CD4+ T cells, rendering this reservoir difficult to detect by the host CTL immune response. Currently available strategies to purge latently HIV-infected cells include “Shock and Kill,” broadly responsive TCR elicited by therapeutic vaccines, broadly neutralizing monoclonal antibodies, programmed cell death protein-1 (PD-1) blockade, and other immune checkpoint blockades. However, none of these strategies efficiently eradicates HIV reservoirs. Our preliminary data showed that strong phosphorylation of a small adaptor molecule, chicken tumor virus number 10 regulator of kinase (Crk), was induced by PD-1 signaling at the center of exhausted HIV-specific CTL immunological synapses. This critical observation prompted us to develop a novel strategy that targets a common master-signaling molecule to restore the function of exhausted CTLs to eradicate HIV reservoirs, which is an approach that is superior to targeting individual inhibitory immunoreceptor. We hypothesize that chronic HIV leads to functional impairment of CTLs via up-regulation of inhibitory receptors and that the resulting downstream phosphorylation of Crk, in turn, prevents CTL activation. We propose that exogenous phosphorylated Crk (pCrk) inhibitor could be used to restore exhausted CTL functions to eradicate HIV reservoirs. We propose the following two aims: (Aim 1) Restore the function of exhausted HIV-specific CTLs against productively infected cells by inhibiting Crk phosphorylation. pCrk inhibitors newly identified using multipronged approaches (including the state-of-the-art Alphascreen, thermal shift assay, in vitro kinase and cellular phosphoflow assay) will be tested to determine whether these pCrk inhibitors can restore functions of exhausted HIV-specific CTLs against productively infected cells. (Aim 2) Restore the function of exhausted HIV-specific CTLs against latently infected cells by inhibiting Crk phosphorylation. To further test the efficacy of pCrk inhibitors, we will determine whether pCrk inhibitors can restore the function of exhausted HIV-specific CTLs against latently infected cells. We will generate latently HIV-infected primary CD4+ T cells from peripheral blood and lymph nodes to compare their sensitivity to killing by exhausted HIV-specific CTLs with and without pCrk inhibitors. If successful, therapeutic use of small-molecule pCrk inhibitors could improve care of chronic HIV patients, restoring exhausted patient CTL defenses. Successful outcomes of this research will lead to the development of a novel immunotherapy to eradicate HIV reservoirs and associated clinical trials.