NFAT, bZIP proteins, and transcriptional programs in lymphocytes

摘要 针对CTLA 4、PD-1和在耗竭的T细胞上表达的其他抑制性表面受体的阻断抗体，或 肿瘤和基质细胞表达的PD-1配体PD-L1和PD-L2的阻断抗体， 在促进长期肿瘤消退方面非常成功。阻断抗体的组合， 多个抑制性受体，通常用共刺激受体的激活抗体加强，已经被 比单独使用个体阻断抗体的治疗更有效。然而，尽管这些 尽管取得了一些成功，但许多患者仍然未能对“免疫检查点阻断”疗法作出反应， 需要在分子水平上理解小鼠模型和人类中免疫细胞的“耗竭”。 钙和钙调神经磷酸酶调节的转录因子NFAT是转录反应的驱动因子。 潜在的T细胞激活。T细胞活化程序主要依赖于NFAT和NFAT-T的协同结合。 在基因启动子和增强子中的复合DNA位点上的转录伴侣AP 1（Fos-Jun）。同时， NFAT可以激活第二个转录程序，该程序施加低反应状态，通常称为 “衰竭”或“功能障碍”。这第二个NFAT介导的程序在CD 8 + T细胞中变得突出 在慢性病毒感染和癌症期间暴露于持续的抗原刺激，并且其特征在于 一系列功能受损状态，细胞因子表达降低， 多种抑制性受体（PD-1、CTLA 4、LAG 3、TIM 3、TIGIT）。因此，一个有效的替代组合 检查点阻断疗法可能是调节NFAT介导的程序之间的平衡， 激活和耗尽，从而使肿瘤浸润T细胞远离耗尽，并朝向 效应子功能我们将在这里测试这个假设。 我们对工程化的NFAT 1进行了实验，最小限度地修改以防止其与AP 1相互作用， 建立了耗尽的转录程序是独立的NFAT 1-AP 1相互作用。我们 已经确定了NFAT在耗竭程序中的重要靶点，包括Nr 4a的转录因子， 毒理学家族此外，我们已经表明Nr 4a转录因子在耗尽的肿瘤浸润T细胞中起作用， 部分通过抑制bZIP转录因子的表达或激活， 促进效应子样表型。在目标1中，我们将鉴定和表征bZIP转录因子 在一定条件下维持肿瘤浸润性CD 8 + T细胞的效应子功能最有效 在目标2中，我们将定义两个NFAT家族的不同角色 成员，NFAT 1和NFAT 2，在耗尽的转录程序中;在目的3中，我们将使用新的 蛋白质组学策略，以确定与NFAT合作的NFAT相互作用蛋白质，以施加耗竭 程序. 我们提出的研究将检验CD 8 + TILs在功能上被细胞内在的 由持续NFAT信号传导介导的转录程序与bZIP转录的抑制偶联 因素这些结果将有助于对转录机制的广泛理解 在小鼠和人类肿瘤浸润性T细胞中运行。 !

ABSTRACT Blocking antibodies to CTLA4, PD-1, and other inhibitory surface receptors expressed on exhausted T cells, or blocking antibodies to the PD-1 ligands PD-L1 and PD-L2 expressed by tumor and stromal cells, have been remarkably successful at promoting long-term tumor regression. Combinations of blocking antibodies to multiple inhibitory receptors, often reinforced with activating antibodies to costimulatory receptors, have been more effective than treatment with individual blocking antibodies alone. Nevertheless, despite these successes, many patients still fail to respond to `immune checkpoint blockade' therapies, emphasizing the need to understand immune cell `exhaustion' at a molecular level, both in mouse models and in humans. The calcium- and calcineurin-regulated transcription factor NFAT is a driver of the transcriptional responses underlying T cell activation. The T cell activation program mainly depends on cooperative binding of NFAT and its transcriptional partner AP1 (Fos-Jun) at composite DNA sites in gene promoters and enhancers. In parallel, NFAT can activate a second transcriptional program that imposes a hyporesponsive state, typically termed `exhaustion' or `dysfunction'. This second NFAT-mediated program becomes prominent in CD8+ T cells exposed to persistent antigen stimulation during chronic viral infections and cancer, and is characterized by a spectrum of functionally compromised states with decreased cytokine expression and increased expression of multiple inhibitory receptors (PD-1, CTLA4, LAG3, TIM3, TIGIT). Thus an effective alternative to combination checkpoint blockade therapies might be to modulate the balance between the NFAT-mediated programs of activation and exhaustion, and thereby to skew tumor-infiltrating T cells away from exhaustion and towards effector function. We will test this hypothesis here. Our experiments with an engineered NFAT1, minimally modified to prevent its interaction with AP1, have established that the transcriptional program of exhaustion is independent of the NFAT1-AP1 interaction. We have identified important targets of NFAT in the exhaustion program, including transcription factors of the Nr4a and Tox families. Moreover, we have shown that Nr4a transcription factors act in exhausted tumor-infiltrating T cells, in part, by repressing the expression or activation of bZIP transcription factors that would otherwise promote an effector-like phenotype. In Aim 1, we will identify and characterize the bZIP transcription factors that are most effective in maintaining the effector function of tumor-infiltrating CD8+ T cells under conditions that would ordinarily lead to exhaustion; in Aim 2, we will define the differential roles of two NFAT family members, NFAT1 and NFAT2, in the transcriptional program of exhaustion; and in Aim 3, we will use novel proteomic strategies to identify NFAT-interacting proteins that cooperate with NFAT to impose the exhaustion program. Our proposed studies will test the hypothesis that CD8+ TILs are functionally silenced by a cell-intrinsic transcriptional program mediated by persistent NFAT signalling coupled with repression of bZIP transcription factors. The results will contribute to a broad mechanistic understanding of the transcriptional mechanisms operating in mouse and human tumor-infiltrating T cells. !