Optimizing the Depletion of innate immune Effector Cells in Nonhuman Primates

项目总结 先天免疫在宿主对HIV感染的反应中起着重要作用。对这一反应至关重要的是 天然免疫效应细胞包括NK细胞、单核细胞、巨噬细胞和粒细胞，它们具有 大量的抗艾滋病毒活动。值得注意的是，单核细胞和巨噬细胞也可能与艾滋病毒的持久性有关。 在抑制治疗期间，通过窝藏复制能力强的艾滋病毒前病毒DNA的完整拷贝。细胞毒性 NK细胞和其他表达Fcγ-RIIIA(CD16)的细胞可以结合特定的抗体，使抗体- 依赖性细胞毒性(ADCC)，可介导对艾滋病毒感染细胞的杀伤。然而， 先天免疫效应器活性的非多余作用在体内往往很难定义。一个关键的障碍是 耗尽这些天然免疫细胞以进行功能丧失研究的方式(例如，Janus Kinase 3抑制剂、抗肿瘤药物 IL-15单抗(单抗，抗MHC-II单抗)也可以影响适应性免疫系统的细胞， 即T细胞。例如，我们之前在恒河猴体内使用了抗IL-15单抗来中和IL-15信号 猕猴(RM)利用趋化CCR5来评估NK细胞在急性和慢性感染中的作用 SIVmac239。我们观察到血液和组织中的NK细胞大量耗尽(DeGotarrdi等人，J. 2016)，令人惊讶的是，对SIV复制的影响微乎其微，致癌伽马疱疹病毒显著重新激活 病毒(Okoye等人，J.2019年)。特别是，我们注意到恒河猴狂犬病病毒(RRV)的重新激活， 一种与人类疱疹病毒8型/卡波西肉瘤相关疱疹病毒关系密切的猴γ疱疹病毒， 这可能与NK细胞的耗尽直接相关，也可能是由于IL-15的影响 信号阻断其他淋巴细胞群，如CD4+和CD8+效应记忆T细胞。同样， 使用抗CD16mAb评估CD16/Fcγ-RIIIA在SIV感染RM中的作用导致一过性 而且常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细胞来耗尽NKG2A+和CD16+细胞的这些限制 在Rm。我们的初步数据清楚地显示了我们在使用CD20导向的CAR T细胞来实现这一目标方面的专长 去除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辆车 分子，我们已经设计并初步验证了它。每个标记都被设计成以 在RM(NKG2A)中的绝大多数NK细胞或能够介导ADCC活性的细胞(CD16)上表达， 这反过来又会导致它们在体内的特异性功能缺陷。CD16汽车T细胞可能提供附加值 作为一种耗尽髓系储存库的方法。如果成功，这个项目有可能取得实质性的进展 非人灵长类动物模型在研究先天免疫效应器活性在HIV中的特殊作用中的作用 发病机制、疫苗开发和治疗策略。

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.