Activated NK CAR Cells to Cure HIV

病毒特异性CD 8 T细胞在体内外均具有抗HIV-1/SIV的活性。但尽管 在HIV-1感染的人类和SIV感染的猕猴中， 复制的这可能是由于大多数病毒复制发生在B细胞滤泡内的CD 4 + T细胞中， 次级淋巴组织;其中病毒特异性CD 8 T细胞数量相对较少。事实上，我们发现 体内效应病毒特异性CD 8 T细胞与靶SIV RNA+细胞比率（E：T）在体内低40倍以上， 与淋巴组织中的B细胞滤泡外相比。这些发现表明，B细胞滤泡是一种 免疫特权位点，其中低水平的病毒特异性CD 8 T细胞允许进行中的病毒复制。 此外，我们发现很少有病毒特异性CD 8 T细胞表达滤泡归巢分子CXCR 5，这可能是由于病毒特异性CD 8 T细胞表达CXCR 5。 解释了它们在B细胞卵泡中的低水平。这些数据表明，病毒特异性CD 8 T细胞不能表达病毒特异性T细胞。 完全抑制病毒复制可能是由于B细胞滤泡中这些T细胞的缺乏。这些发现 使我们得出了我们的中心假设，即针对B细胞滤泡的HIV特异性免疫疗法将导致持久的 艾滋病毒感染的缓解。为了支持这一假设，我们已经表明，增加病毒特异性 B细胞滤泡中的CD 8 T细胞与较低的病毒载量相关。尽管许多免疫疗法利用患者 T细胞产生CAR-T疗法，在治疗艾滋病毒方面有特殊考虑。一个主要的空头 CAR-T方法的缺点是，由于移植物抗宿主的风险，T细胞需要是自体的。 GvHD是一种免疫缺陷病毒性疾病（GvHD），需要复杂/昂贵的患者细胞制造过程。这也是 在HIV环境中具有挑战性，因为患者T细胞已经受损，并且处理细胞可能 含有活性病毒是有风险的。或者，自然杀伤（NK）细胞非常适合同种异体使用，因为它们 不会引起GvHD，因此作为现成的细胞产品具有显著的临床潜力。当然，我们建议 评估靶向病毒特异性CAR NK细胞（表达CD 4-MBL-CAR和CXCR 5）的NK免疫疗法 到B细胞滤泡。此外，我们将使用CRISPR/Cas9敲除NK细胞功能的负调控因子，如 PD 1，我们之前已经证明它可以增强NK细胞的功能。我们的长期目标是发展一个 这将导致使用CAR NK细胞持久缓解HIV感染的干预。为了验证我们的假设，我们 提出以下目标。1)开发试剂和方法，以产生人类和恒河猴 CAR/CXCR 5/PD 1 KO NK细胞。2)确定CAR/CXCR 5/PD 1 KO NK细胞迁移至B细胞的能力 卵泡的SIV感染恒河猴，并诱导和维持病毒抑制。我们建议的研究 将CAR NK细胞靶向卵泡将通过提供对细胞运输的见解而对该领域产生广泛影响， 持久性和NK免疫疗法的预处理方案。此外，我们的工程方法 恒河猴NK细胞将使得能够进行评估NK细胞在临床前NHP模型中的治疗用途的研究。 此外，这些研究可能会产生一种有效的策略，以诱导长期持续缓解艾滋病毒。

ABSTRACT Virus-specific CD8 T cells exert antiviral activity against HIV-1/SIV in vitro and in vivo. Yet, despite these responses in HIV-1-infected humans and SIV- infected macaques, they are unable to fully suppress virus replication. This is likely due to the majority viral replication occurring in CD4+ T cells within B-cell follicles in secondary lymphoid tissues; where virus-specific CD8 T cells are relatively few in number. In fact, we found that in vivo effector virus-specific CD8 T cell to target SIV RNA+ cell ratios (E:T) were over 40-fold lower inside compared to outside of B cell follicles in lymphoid tissues. These findings indicate that B cell follicles are an immune privileged site in which low levels of virus-specific CD8 T cells permit ongoing viral replication. Furthermore, we found that few virus-specific CD8 T cells express the follicular homing molecule CXCR5, likely explaining their low levels in B cell follicles. These data suggest that the inability of virus specific CD8 T cells to fully suppress virus replication may be due to a deficiency of these T cells in B-cell follicles. These findings have led us to our central hypothesis that targeting HIV-specific immunotherapy to B cell follicles will lead to durable remission of HIV infection. In support of this hypothesis we have shown that increased levels of virus-specific CD8 T cells in B cell follicles is associated with lower viral loads. Although many immunotherapies utilize patient T cells to generate CAR-T therapies, there are special consideration in the treatment of HIV. One major short comings of CAR-T approaches is the fact that T cells need to be autologous due to the risk of graft versus host disease (GvHD), requiring complicated/expensive manufacturing processes of patient cells. This is also challenging in the HIV setting as the patient T cells are already compromised and processing cells which may contain active virus is risky. Alternatively, Natural Killer (NK) cells are highly suited for allogeneic use as they do not cause GvHD and thus hold significant clinical potential as an off-the-shelf cellular product. Thuts, we propose to evaluate NK immunotherapy that targets virus-specific CAR NK cells (expressing CD4-MBL-CAR and CXCR5) to B cell follicles. Moreover, we will use CRISPR/Cas9 to knockout negative regulators of NK cell function, such as PD1, which we have previously shown to enhance NK cell function. Our long-term goal is to develop an intervention that will lead to durable remission of HIV infection using CAR NK cells. To test our hypotheses, we propose the following aims. 1) Develop reagents and methods to generate human and rhesus macaque CAR/CXCR5/PD1KO NK cells. 2) Determine the ability of CAR/CXCR5/PD1KO NK cells to migrate into B cell follicles of SIV-infected rhesus macaques and to induce and maintain viral suppression. Our proposed studies targeting CAR NK cells to follicles will have a broad impact on the field by providing insights into cell trafficking, persistence, and pre-conditioning regimens for NK immunotherapy. Moreover, our methods for engineering rhesus macaque NK cells will enable studies assessing the therapeutic use of NK cells in preclinical NHP models. Moreover, these studies could result in an effective strategy to induce long-term sustained remission of HIV.