Harnessing Stem-Like CD8 T Cells for Immunotherapies to Eradicate HIV Reservoirs

项目摘要 虽然联合抗逆转录病毒疗法（cART）有效地抑制人类免疫缺陷病毒， (HIV)但是，它不能治愈艾滋病毒感染，需要昂贵的终身治疗。面临的一大挑战 治愈艾滋病毒感染是长期潜伏的艾滋病毒库，它逃避免疫识别， cART中断后不久病毒反弹。大量的研究工作集中在消除 潜伏感染的细胞通过所谓的"休克和杀死"的战略，重新激活潜伏的艾滋病毒使用潜伏- 逆转剂（LRA），以允许通过细胞溶解或免疫介导的清除进行"杀伤"。特别是根除 作为一种"活的"药物，抗HIV T细胞在患者体内维持抗病毒免疫， 这是一种有希望的策略，可以完全解决感染或在没有cART治疗的情况下保持长期控制。 然而，通过基于T细胞的免疫疗法清除HIV储库仍然具有挑战性，因为1）T- 细胞衰竭，2）需要终身抗HIV免疫来替代cART，3）保护区，如B细胞滤泡 排除了大多数HIV特异性CD8 T细胞，以及4）LRA的影响不清楚，其中大多数靶向表观遗传 途径，在体内抗病毒CD8 T细胞的功能和分化。我们和其他人最近 在慢性淋巴细胞性脉络丛脑膜炎病毒（LCMV）中， 免疫缺陷病毒（SIV）和HIV感染，以及小鼠和人肿瘤中。相比 终末耗尽的CD8 T细胞，干细胞样CD8 T细胞较少耗尽，介导长期免疫， 在动物和人类的免疫疗法治疗后反应更有效。在慢性LCMV、SIV和 HIV感染后，这些细胞表达CXCR5，迁移到B细胞滤泡并杀死受感染的T滤泡辅助细胞， 艾滋病毒和SIV的主要潜在宿主。此外，这些细胞的频率与病毒血症呈负相关， SIV或HIV。最近，我们发现干细胞样CD8单细胞转录组学和表观遗传学特征， T细胞与急性或慢性LCMV感染后产生的其他CD8亚群不同。另外我们 鉴定了一种涉及转录因子TOX的转录程序，该转录因子TOX对于干细胞样CD8 T细胞至关重要， 分化和慢性病毒感染期间抗病毒CD8 T细胞的长期持久性。来，我来 确定干细胞样CD8 T细胞的转录和表观遗传程序， 免疫疗法对抗HIV。我将使用慢性LCMV和SIV感染的动物模型以及样本 从艾滋病毒患者，并采用尖端技术，包括单细胞分析的T细胞转录组 和表观基因组，CRISPR/Cas9筛选和嵌合抗原受体（CAR）T细胞疗法，以确定 表观遗传修饰LRA的"休克"如何影响干细胞样CD8 T细胞的程序和抗病毒免疫 以及干细胞样CD8 T细胞的转录和表观遗传程序是否可以被采用来增强免疫应答。 通过基于T细胞的免疫疗法"杀死" HIV储库。这项研究的结果将为 新的免疫疗法，实现长期缓解艾滋病毒感染，而不需要cART。

Project Abstract Although combination antiretroviral therapy (cART) effectively suppresses human immunodeficiency virus (HIV) replication, it does not cure HIV infection and requires costly lifelong treatment. A major challenge for curing HIV infection is the long-lived latent HIV reservoir, which evades immune recognition and is responsible for viral rebound shortly after interruption of cART. Substantial research efforts have focused on eliminating latently infected cells through so-called “shock and kill” strategy, which reactivates latent HIV using latency- reversing agents (LRAs) to allow for the “kill” by cytolysis or immune-mediated clearance. Particularly, eradication of theHIV reservoir by anti-HIV T cells, which maintain antiviral immunity in patients as a “living” drug, presents a promising strategy to either fully resolve the infection or maintain long-term control without cART treatment. However, clearance of the HIV reservoir by T-cell based immunotherapy remains challenging because of 1) T- cell exhaustion, 2) the need of lifelong anti-HIV immunity to replace cART, 3) sanctuary sites like B cell follicles that exclude most HIV-specific CD8 T cells, and 4) unclear impact of LRAs, most of which target epigenetic pathways, on the function and differentiation of antiviral CD8 T cells in vivo. We and others have recently characterized a stem-like CD8 T cell subset in chronic lymphocytic choriomeningitis virus (LCMV), simian immunodeficiency virus (SIV), and HIV infections, as well as in mouse and human tumors. Compared to terminally exhausted CD8 T cells, stem-like CD8 T cells are less exhausted, mediate long-term immunity, and respond more potently after treatment of immunotherapies in animals and human. In chronic LCMV, SIV, and HIV infections, these cells express CXCR5, migrate to B cell follicles and kill infected T follicular helper cells, a major latent reservoir of HIV and SIV. In addition, frequency of these cells inversely correlates with viremia of SIV or HIV. Most recently, we showed that the single-cell transcriptomic and epigenetic profiles of stem-like CD8 T cells are distinct from other CD8 subsets generated after acute or chronic LCMV infection. In addition, we identified a transcriptional program involving transcription factor TOX that is essential for stem-like CD8 T cell differentiation and the long-term persistence of antiviral CD8 T cells during chronic viral infection. Here, I will determine the transcriptional and epigenetic programs of stem-like CD8 T cells required for optimal T-cell based immunotherapy against HIV. I will use animal models of chronic LCMV and SIV infections as well as samples from HIV patients, and employ cutting-edge technologies, including single-cell profiling of T-cell transcriptomes and epigenomes, CRISPR/Cas9 screening, and chimeric antigen receptor (CAR) T-cell therapy, to determine how “shock” by epigenetic modifying LRAs affects the program and antiviral immunity of stem-like CD8 T cells and whether transcriptional and epigenetic programs of stem-like CD8 T cells can be adopted to enhance the “kill” of the HIV reservoir by T-cell based immunotherapies. The results from this study will build the foundation for novel immunotherapies that achieve long-term remission from HIV infection without a need for cART.