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

项目总结： 大多数人类免疫缺陷病毒(HIV)感染者，如果不坚持高活性组合 抗逆转录病毒疗法(CART)，最终死于慢性感染。然而，有些人被称为“精英” 具有卓越病毒控制能力的控制器(ECs)，即使在 没有购物车。尽管ECs中的HIV特异性细胞毒性T淋巴细胞(CTL)对病毒控制至关重要 和进展状况，利用CTL来对抗艾滋病毒宿主仍然具有挑战性，因为几乎没有 病毒蛋白是在静止的CD4T细胞中产生的，这使得宿主CTL很难检测到这个储存库 免疫反应。目前可用于清除潜伏感染艾滋病毒的细胞的策略包括“休克并杀死”， 治疗性疫苗引起的广泛反应的TCR，广泛中和单抗， 程序性细胞死亡蛋白-1(PD-1)阻断，以及其他免疫检查点阻断。然而，一个也没有 这些战略中的一项有效地根除了艾滋病毒宿主。我们的初步数据显示， 将一个小的接头分子，鸡肿瘤病毒10号，蛋白激酶调节因子(Crk)，磷酸化， 由耗竭的HIV特异性CTL免疫突触中心的PD-1信号诱导。这一点至关重要 观察促使我们开发了一种新的策略，以共同的主信号分子为目标 恢复耗尽的CTL的功能以根除艾滋病毒宿主，这是一种优于 靶向个体抑制性免疫受体。我们假设慢性HIV会导致功能性 CTL通过上调抑制性受体以及由此导致的下游磷酸化而受损 而Crk的激活反过来又阻止了CTL的激活。我们认为外源性磷酸化Crk(PCrk)抑制剂可以 用于恢复用尽的CTL功能，以根除艾滋病毒宿主。我们提出以下两个目标： (目标1)通过以下方式恢复耗尽的HIV特异性CTL对生产性感染细胞的功能 抑制Crk的磷酸化。使用多管齐下的方法新发现的pCrk抑制剂(包括 将测试最先进的Alphascreen、热位移分析、体外激酶和细胞磷流分析) 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 被高效感染的细胞。(目标2)恢复耗尽的HIV特异性CTL的潜伏期功能 通过抑制Crk的磷酸化而感染细胞。为了进一步测试pCrk抑制剂的疗效，我们将 确定pCrk抑制剂是否能恢复耗尽的HIV特异性CTL的潜伏期功能 被感染的细胞。我们将从外周血和淋巴中产生潜伏感染HIV的初级CD4T细胞 比较它们对使用和不使用pCrk抑制剂的耗尽的HIV特异性CTL杀伤的敏感性。如果 成功地、治疗性地使用小分子pCrk抑制剂可以改善慢性艾滋病毒患者的护理， 恢复精疲力竭的患者CTL防御。这项研究的成功成果将引领该领域的发展 一种新的免疫疗法来根除艾滋病毒宿主和相关的临床试验。

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.