Phenotypic, Functional and Transcriptional Heterogeneity in T Cell Exhaustion

摘要 T 细胞耗竭是一种分化状态，其特征是效应功能丧失和效应功能增强。 抑制性受体（例如 PD-1）的表达。尽管病毒特异性 CD8 T 细胞通常被认为 作为一个随着时间的推移逐渐变得疲惫的同质群体，最近的研究清楚地表明 证明 CXCR5hi TCF-1hi 子集作为自我更新的祖细胞群体，可以提供 上升到更最终耗尽的 CXCR5lo TCF-1lo 子集。更好地剖析“疲惫”的异质性 CD8 T 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 CD8 T 细胞的三个主要亚群，分别在表型、功能和转录上 独特的。实验室不仅通过流式实验验证了这些 T 细胞亚群的存在 细胞计数术，还进行了更先进的计算分析以进一步预测其核心 转录网络和发育轨迹。总的来说，研究结果表明TCF-1hi 祖细胞子集可以产生真正耗尽的 PD-1hi 子集或新识别的功能效应子 称为 CX3CR1hi 子集的群体。这一发现奠定了一个坚实的框架，可以测试如何 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 CD8 T 细胞。更重要的是，它提供了前所未有的机会来探索生成的可能性 来自 TCF-1hi 祖细胞的功能更强的 CX3CR1hi 细胞可克服 T 细胞耗竭。这个概念性的 这一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）至关重要 用于 TCF-1hi → CX3CR1hi 过渡。这导致了这样的假设：炎症细胞因子（例如 CD4- 衍生的 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 LCMV 感染。阻断抗体、RNA干扰和基因缺失模型将被用来 进一步剖析炎症细胞因子和转录网络如何调节 T 细胞的异质性 精疲力尽。此外，该实验室建议通过以下方式重新引导 CD8 T 细胞分化，使其远离“衰竭”： 单独或与 PD-1 阻断结合提供额外的“CD4 帮助”。实验室将测试是否提供 通过过继转移产生 IL-21 的 CD4 T 细胞可以驱动 TCF-1hi 祖细胞分化为 功能性 CX3CR1hi 效应细胞。总体而言，从这项研究中获得的知识将提供机制 深入了解如何重新定向功能性效应 T 细胞的形成并同时限制 T 细胞 为改善对慢性感染的病毒控制而精疲力尽。

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.