Defining the transcriptional, phenotypic, and functional heterogeneity of virus-specific CD4 T cells during chronic viral infection

通过可溶性因子和细胞表面配体的CD4“帮助”对维持CD8 T细胞反应和体液至关重要 慢性病毒感染期间的免疫力。最近，我们的实验室进一步确定了CD4来源的IL-21的必要性 CX3CR1+CD8 T细胞亚群的形成，具有很强的细胞溶解功能。 然而，尽管它们扮演着重要的角色，但我们对持续暴露于病毒载量和 炎症形态对辅助性T细胞分化的影响尚不完全清楚。使用scRNA-seq，我们的实验室 已经产生了大量的初步数据表明，CD4T细胞对慢性LCMV感染的反应 比之前意识到的更具异质性，有三个转录上不同的亚集主导着 抗病毒反应：CxCR6+Th1、CXCR5+Tfh和Slamf6+记忆样细胞。值得注意的是，我们的数据进一步表明 对慢性而非急性病毒感染有反应的CD4T细胞优先将其分化方向重新定向为 这个类似记忆的子集。综上所述，我们假设长期接触抗原性和炎症性 信号(转化生长因子-β、IL-10等)调节病毒特异性CD4T细胞的转录多样性，而CD4 慢性病毒感染期间的分化被驱动到这个类似记忆的子集，以协调以下内容： 1)减弱Th1介导的免疫病理，2)维持一个能够产生Th1和Tfh的祖细胞池 效应细胞，以及3)促进记忆样CD4T细胞与祖细胞CD8T细胞共定位的利基 将其定向分化为保护性CX3CR1+CD8 T细胞。在目标1中，我们将进行收养转移 (AT)实验，以检查增殖潜力、保护能力和血统关系 病毒特异性CD4T细胞的三个主要亚群。此外，基于基因调控网络 由Scenic(一个R包)制定，我们将使用RNA干扰模型来测试KEY的贡献 转录因子预计在慢性感染过程中起到调节CD4分化的作用。在目标2中， 我们将使用遗传方法和AT实验来操纵抗原驱动的TCR信号，以便 评估抗原信号如何在慢性感染期间调节CD4分化。此外，我们还将阻止 免疫调节性细胞因子在慢性感染期间过度表达以确定其活性 对病毒特异性CD4T细胞命运承诺的影响。最后，在目标3中，我们将确定IL的AT- 单独或与PD-L1阻断联合使用21-产生记忆样CD4T细胞可以增强 祖细胞CX3CR1在CD8T细胞转化中提高T细胞介导性免疫。从这个过程中学到的知识 研究将提供对CD4T细胞在面对 持续感染并帮助确定旨在优化细胞或体液介导的抗病毒的新策略 豁免权。这个职业发展奖提供的额外培训不仅使我能够 我的分子和计算技能，也将使我独一无二地建立一个独立的研究 该计划侧重于解决与CD4T细胞免疫生物学有关的基本问题。

CD4 “help” via soluble factors and cell-surface ligands is critical to sustain CD8 T cell responses and humoral immunity during chronic viral infection. Recently, our lab has further identified a necessity for CD4-derived IL-21 in the formation of a previously unrecognized CX3CR1+CD8 T cell subset that exhibits potent cytolytic function. However, despite their essential role, our understanding of how persistent exposure to viral load and inflammation shapes helper T cell differentiation remains incompletely understood. Using scRNA-seq, our lab has generated substantial preliminary data demonstrating that CD4 T cells responding to chronic LCMV infection are more heterogenous than previously appreciated, with three transcriptionally distinct subsets dominating the antiviral response: Cxcr6+Th1, Cxcr5+Tfh, and Slamf6+ memory-like cells. Notably, our data further indicate that CD4 T cells responding to chronic, but not acute viral infection, preferentially redirect their differentiation towards this memory-like subset. Taken together, we hypothesize that prolonged exposure to antigenic and inflammatory signals (TGF-β, IL-10 etc.) modulate the transcriptional diversity of virus-specific CD4 T cells, and that CD4 differentiation during chronic viral infection is driven towards this memory-like subset to coordinate the following: 1) attenuate Th1-mediated immunopathology, 2) maintain a progenitor pool that can give rise to Th1 and Tfh effector cells, and 3) facilitate a niche wherein memory-like CD4 T cells co-localize with progenitor CD8 T cells to redirect their differentiation towards protective CX3CR1+CD8 T cells. In Aim 1, we will perform adoptive transfer (AT) experiments to examine the proliferative potential, protective capacity, and lineage relationship between the three major subsets of virus-specific CD4 T cells. Additionally, based on the gene regulatory network formulated by SCENIC (an R package), we will use RNA interference models to test the contribution of key transcription factors predicted to play a role in regulating CD4 differentiation during chronic infection. In Aim 2, we will use genetic approaches and AT experiments to manipulate antigen-driven TCR-signaling in order to assess how antigenic signals regulate CD4 differentiation during chronic infection. Moreover, we will block the activity of immunoregulatory cytokines known to be overexpressed during chronic infection to determine their impact on the fate commitment of virus-specific CD4 T cells. Lastly, in Aim 3 we will determine whether AT of IL- 21-producing memory-like CD4 T cells, either alone or in conjunction with PD-L1 blockade, can augment progenitorCX3CR1hi CD8 T cell transition to improve T cell-mediated immunity. Knowledge gained from this research will provide mechanistic insights into the functional adaption process CD4 T cells display in the face of persistent infection and help identify novel strategies aimed at optimizing cellular or humoral-mediated antiviral immunity. The additional training afforded by this career development award will not only enable me to expand my molecular and computational skillsets, but will also uniquely position me to build an independent research program focused on addressing fundamental questions pertaining to CD4 T cell immunobiology.