Optimizing the Depletion of innate immune Effector Cells in Nonhuman Primates

项目摘要 天然免疫在宿主抗HIV感染的应答中起着重要作用。这一对策的关键是 先天性免疫效应细胞，包括NK细胞、单核细胞、巨噬细胞和粒细胞，其具有 开展多项抗艾滋病活动。值得注意的是，单核细胞和巨噬细胞也可能有助于艾滋病毒的持久性 在抑制治疗期间通过携带有复制能力的HIV前病毒DNA的整合拷贝。细胞毒性 NK细胞和其他表达Fcγ-RIIIA（CD 16）的细胞可以结合特异性抗体，以使抗体-RIIIA（CD 16）结合成为可能。 依赖性细胞毒性（ADCC），可介导HIV感染细胞的杀伤。但 先天免疫效应子活性的非冗余作用通常难以在体内确定。一个关键的障碍是， 用于功能丧失研究的耗尽这些先天免疫细胞的方式（例如，Janus激酶3抑制剂，抗- IL-15单克隆抗体[mAb]，抗-MHC-II mAb）也可以影响适应性免疫系统的细胞， 即T细胞。例如，我们先前使用抗IL-15单克隆抗体在恒河猴体内中和IL-15信号传导， 猕猴（RM），以评估NK细胞在急性和慢性感染过程中的作用，使用CCR 5嗜性 SIVmac239.我们观察到血液和组织中NK细胞的大量耗竭（DeGotarrdi等人，J. Immunol. 2016年），令人惊讶的是对SIV复制的影响最小，以及致癌γ疱疹的显著再激活 病毒（Okoye等人，2019）。特别是，我们注意到恒河猴RM Rhadinovirus（RRV）的重新激活， 一种与人疱疹病毒8型/卡波西肉瘤相关疱疹病毒密切相关的猿猴γ-疱疹病毒， 这可能与NK细胞的耗竭直接相关，或者由于IL-15的影响， 对其它淋巴细胞群如CD 4+和CD 8+效应记忆T细胞的信号传导阻断。同样地， 使用抗CD 16耗竭mAb评估CD 16/Fcγ-RIIIA在SIV感染的RM中的作用， 并且经常是不完全的功能丧失（Choi等人，J. Virol. 2008年）。这里提出的实验将克服 通过使用嵌合抗原受体（CAR）T细胞来消除NKG 2A+和CD 16+细胞， 在RM。我们的初步数据清楚地显示了我们在这种方法中的专业知识，使用CD 20导向的CAR T细胞， 耗竭RM血液和淋巴组织中的B细胞。我们将使用类似的技术来优化两个CAR 分子，我们已经设计并初步验证。每一种都是针对 在RM中的绝大多数NK细胞（NKG 2A）或可介导ADCC活性的细胞（CD 16）上表达， 这又将在体内诱导它们的特异性功能缺陷。CD 16 CAR T细胞可以提供附加价值 作为一种耗尽骨髓储备的方法。如果成功，该项目有可能大幅推进 利用非人灵长类动物模型研究先天免疫效应子活性在HIV中的特异性作用 发病机理、疫苗开发和治愈策略。

PROJECT SUMMARY Innate immunity plays an important role in the host response against HIV infection. Crucial to this response are innate immune effector cells including NK cells, monocytes, macrophages and granulocytes, which possess numerous anti-HIV activities. Notably, monocytes and macrophages may also contribute to HIV persistence during suppressive therapy by harboring integrated copies of replication-competent HIV proviral DNA. Cytotoxic NK cells and other Fcγ-RIIIA (CD16) expressing cells can bind to specific antibodies to enable antibody- dependent cellular cytotoxicity (ADCC), which can mediate killing of HIV-infected cells. However, the nonredundant role of innate immune effector activity is often difficult to define in vivo. A key barrier is that modalities to deplete these innate immune cells for loss-of-function studies (e.g., Janus kinase 3 inhibitors, anti- IL-15 monoclonal antibodies [mAb], anti-MHC-II mAb) can also impact cells of the adaptive immune system, namely T cells. For example, we previously used anti-IL-15 mAbs to neutralize IL-15 signaling in vivo in rhesus macaques (RM) to assess the role of NK cells during acute and chronic infection, using the CCR5-tropic SIVmac239. We observed a massive depletion of NK cells in blood and tissues (DeGotarrdi et al., J. Immunol. 2016), surprisingly minimal impact on SIV replication, and a significant reactivation of oncogenic gammaherpes viruses (Okoye et al., J. Immunol. 2019). In particular, we noted a reactivation of rhesus RM rhadinovirus (RRV), a simian γ-herpesvirus closely related to human herpesvirus type 8/Kaposi's sarcoma–associated herpesvirus, which may have been directly related to the depletion of NK cells, or alternatively due to the impact of IL-15 signaling blockade on other lymphocyte populations such as CD4+ and CD8+ effector memory T cells. Likewise, use of an anti-CD16 depleting mAb to assess the role of CD16/Fcγ-RIIIA in SIV-infected RM resulted in transient and often incomplete loss-of-function (Choi et al., J. Virol. 2008). The experiments proposed here will overcome these limitations by using chimeric antigen receptor (CAR) T cells designed to deplete NKG2A+ and CD16+ cells in RM. Our preliminary data clearly shows our expertise in this approach using CD20-directed CAR T cells to deplete B cells in blood and lymphoid tissues of RM. We will use similar techniques to optimize two CAR molecules, which we have already designed and preliminarily validated. Each is designed to target markers that are expressed on the vast majority of NK cells in RM (NKG2A) or cells that can mediate ADCC activity (CD16), which will in turn induce their specific functional deficiency in vivo. CD16 CAR T cells may provide added value as an approach to deplete myeloid reservoirs. If successful, this project has the potential to substantially advance the utility of nonhuman primate models to study the specific role of innate immune effector activity in HIV pathogenesis, vaccine development and cure strategies.