Harnessing adaptive NK cell transfer to deplete viral reservoirs

项目摘要 自然杀伤（NK）细胞凭借其以下能力提供针对病毒和肿瘤的立即防御： 对感染的或恶性的细胞没有预先的抗原刺激作出反应。这是通过 来自活化和抑制性NK细胞受体（aNKRs和iNKRs）的信号的整合。在人类和其他 在灵长类物种中，这些包括C型凝集素受体，如CD 94/NKG 2A和CD 94/NKG 2C，以及C型凝集素受体。 高度多态性免疫球蛋白样受体（KIR），两者均与I类MHC相互作用 配体。这些受体-配体相互作用是NK细胞分化健康细胞的能力的基础。 从不健康的细胞，并提供了一个潜在的机制，特异性的发展“NK细胞 记忆”。NK细胞可以对HIV-1感染产生重大影响。KIR和HLA I类多态性 已经确定与较低的病毒载量和较慢的疾病进展过程相关， 某些NK细胞亚群可以杀死培养物中的HIV感染细胞。因此，基于NK细胞的疗法代表了一种新的治疗方法。 这是一种很有前途的方法，用于靶向HIV感染的细胞并减少病毒库的大小。我们假设 由aNKRs的MHC I类配体结合的病毒肽对NK细胞识别和杀伤 HIV/SIV感染的细胞和过继转移的离体活化NK细胞与潜伏期相结合， 逆转可以耗尽SIV感染的猕猴中的病毒储库，抑制性抗逆转录病毒疗法（ART）。 在目的1中，我们将确定由aNKRs的MHC I类配体结合的病毒肽对NK细胞的贡献。 细胞识别HIV和SIV感染的细胞。这些研究将利用高通量细胞分析， 快速筛选与aNKRs相互作用的MHC I类病毒肽，并鉴定破坏aNKRs相互作用的置换， 这些互动。将对HIV-1和SIV进行相应的修改，以评估它们对 NK细胞对病毒感染细胞的反应。在目标2中，我们将评估离体扩增的NK细胞的能力， 联合潜伏期逆转以消耗SIV感染、ART抑制的恒河猴中的病毒储库 猕猴这一目标将利用条形码SIV和一种有效的新型潜伏期逆转剂进行比较 以最大的敏感性，自体与同种异体NK细胞转移降低病毒感染率的能力， 在目标3中，我们将检验病毒库的耗竭 通过适应性NK细胞转移，可以通过具有抗体依赖性细胞免疫应答的Env特异性抗体来增强。 对SIV感染细胞的细胞毒性。这一目标将采用与目标2类似的方法来确定 其将NK细胞效应器功能与抗体的无与伦比的特异性结合， 耗尽这些前所未有的研究将提供一个更好的了解病毒肽在NK细胞中的作用， HIV和SIV感染细胞的细胞识别，以及NK细胞开发的重要概念验证 在慢性感染者中根除HIV-1宿主的治疗。

PROJECT SUMMARY Natural killer (NK) cells provide an immediate defense against viruses and tumors by virtue of their ability to respond to infected or malignant cells without prior antigenic stimulation. This is accomplished through the integration of signals from activating and inhibitory NK cell receptors (aNKRs & iNKRs). In humans and other primate species, these include C-type lectin receptors, such as CD94/NKG2A and CD94/NKG2C, and the highly polymorphic killer-cell immunoglobulin-like receptors (KIRs), both of which interact with MHC class I ligands. These receptor-ligand interactions are fundamental to the ability of NK cells to differentiate healthy cells from unhealthy cells and provide a potential mechanism of specificity for the development of “NK cell memory”. NK cells can have a significant impact on HIV-1 infection. KIR and HLA class I polymorphisms have been identified that are associated with lower viral loads and slower courses of disease progression and certain NK cell subsets can kill HIV-infected cells in culture. Thus, NK cell-based therapies represent a promising approach for targeting HIV-infected cells and reducing the size of viral reservoirs. We hypothesize that viral peptides bound by the MHC class I ligands of aNKRs are critical to NK cell recognition and killing of HIV/SIV-infected cells and that the adoptive transfer of ex vivo activated NK cells in combination with latency reversal can deplete viral reservoirs in SIV-infected macaques on suppressive antiretroviral therapy (ART). In Aim 1, we will determine the contribution of viral peptides bound by MHC class I ligands of aNKRs to NK cell recognition of HIV- and SIV-infected cells. These studies will utilize high-throughput cellular assays to rapidly screen viral peptides for MHC class I interactions with aNKRs and to identify substitutions that disrupt these interactions. The corresponding changes will be introduced into HIV-1 and SIV to assess their impact on NK cell responses to virus-infected cells. In Aim 2, we will assess the capacity of ex vivo expanded NK cells in combination with latency reversal to deplete viral reservoirs in SIV-infected, ART-suppressed rhesus macaques. This aim will take advantage of barcoded SIV and a potent new latency reversal agent to compare with maximal sensitivity the ability of autologous versus allogeneic NK cell transfer to reduce the rate of viral reactivation after discontinuing ART. In Aim 3, we will test the hypothesis that the depletion of viral reservoirs by adaptive NK cell transfer can be enhanced by an Env-specific antibody with antibody-dependent cellular cytotoxicity against SIV-infected cells. This aim will use a similar approach as Aim 2 to determine the extent to which coupling NK cell effector function to the unparalleled specificity of antibodies can maximize reservoir depletion. These unprecedented studies will provide a better understanding of the role of viral peptides in NK cell recognition of HIV- and SIV-infected cells and an important proof-of-concept for the development of NK cell therapies to eradicate HIV-1 reservoirs in chronically infected individuals.