Phenotypic, Functional and Transcriptional Heterogeneity in T Cell Exhaustion

摘要 T细胞耗竭是一种分化状态，以效应器功能丧失和增加为标志 抑制受体如PD-1的表达。尽管通常认为病毒特异性CD8 T细胞 随着时间的推移，同质人群逐渐变得精疲力竭，最近的研究显然已经 证明了CXCR5hi TCF-1hi亚集是一个自我更新的祖细胞群体，可以提供 上升到更耗尽的CXCR5lo TCF-1lo子集。为了更好地剖析“筋疲力尽”的异质性 CD8T细胞，实验室将单细胞RNA-seq(scRNA-seq)应用于慢性LCMV感染并鉴定 病毒特异性CD8T细胞的三个主要亚群，分别是表型、功能和转录 截然不同。该实验室不仅通过实验验证了这些T细胞亚群的存在 细胞学，更高级的计算分析也被用来进一步预测它们的核心 转录网络和发育轨迹。总而言之，研究结果显示，一种TCF-1HI 祖细胞亚群可以产生真正耗尽的PD-1HI亚群或新发现的功能效应器 名为CX3CR1hi子集的种群。这一发现奠定了坚实的框架，允许测试 细胞外信号和内在遗传电路调节这三个子集的形成和功能 CD8T细胞。更重要的是，它提供了前所未有的机会来探索产生 从TCF-1hi祖细胞中获得更多具有功能的CX3CR1hi细胞，以克服T细胞耗竭。这一概念 突破显然适用于控制慢性病毒感染和癌症。有趣的是， 初步研究还表明，CD4辅助T细胞可能通过产生IL-21发挥关键作用 用于Tcf-1hiCX3CR1hi转换。这导致了一种假设，即炎性细胞因子(如CD4- 来源的IL-21)调节病毒特异性CD8 T细胞的细胞、功能和转录多样性 慢性巨细胞病毒感染。封闭抗体、RNA干扰和基因缺失模型将用于 进一步剖析炎性细胞因子和转录网络如何调节T细胞的异质性 疲惫不堪。此外，该实验室建议通过以下方式改变CD8 T细胞分化的方向，使其远离“衰竭” 单独或与PD-1阻断联合使用，提供额外的“CD4帮助”。实验室将测试是否提供 过继转移产生IL-21的CD4T细胞可诱导TCF-1HI祖细胞分化为 CX3CR1hi功能效应细胞。总体而言，从这项研究中获得的知识将提供机械 如何重定向功能性效应T细胞的形成同时限制T细胞 精疲力竭，以改善对慢性感染的病毒控制。

ABSTRACT T cell exhaustion is a differentiation state that is marked by the loss of effector function and increased expression of inhibitory receptors such as PD-1. Although virus-specific CD8 T cells are commonly considered as a homogeneous population that gradually become exhausted over time, recent research has clearly demonstrated that a CXCR5hi TCF-1hi subset is serving as a self-renewing progenitor population that can give rise to a more terminally exhausted CXCR5lo TCF-1lo subset. To better dissect the heterogeneity of “exhausted” CD8 T cells, the lab applied single cell RNA-seq (scRNA-seq) to the chronic LCMV infection and identified three major subsets of virus-specific CD8 T cells that are phenotypically, functionally and transcriptionally distinct. Not only had the lab validated the existence of these subsets of T cells experimentally by flow cytometry, more advanced computational analyses were also performed to further predict their core transcriptional networks and developmental trajectories. Collectively, the findings reveal that a TCF-1hi progenitor subset can give rise to either a truly exhausted PD-1hi subset or a newly identified functional effector population that is named CX3CR1hi subset. This discovery laid a solid framework that allows testing of how extracellular signals and intrinsic genetic circuits regulate the formation and function of these three subsets of CD8 T cells. More importantly, it provides unprecedented opportunities to explore the possibility of generating more functional CX3CR1hi cells from TCF-1hi progenitors to overcome T cell exhaustion. This conceptual breakthrough is obviously applicable to control over chronic viral infection as well as cancer. Intriguingly, the preliminary study has also demonstrated that CD4 helper T cells, possibly through producing IL-21, are critical for TCF-1hi  CX3CR1hi transition. This led to the hypothesis that inflammatory cytokines (such as CD4- derived IL-21) modulate the cellular, functional and transcriptional diversity of virus-specific CD8 T cells during chronic LCMV infection. Blocking antibodies, RNA interference and genetic deletion models will be used to further dissect how inflammatory cytokines and transcriptional networks regulate heterogeneity in T cell exhaustion. Furthermore, the lab proposes to redirect CD8 T cell differentiation away from “exhaustion” by providing additional “CD4 help”, either alone or in combination with PD-1 blockade. The lab will test if providing IL-21 producing CD4 T cells through adoptive transfer could drive TCF-1hi progenitor cell differentiation into functional CX3CR1hi effector cells. Overall, knowledge gained from this research will provide mechanistic insights into how to redirect the formation of functional effector T cells and simultaneously limit T cell exhaustion for improved viral control over chronic infection.