NFAT, bZIP proteins, and transcriptional programs in lymphocytes

摘要 阻断CTLA4、PD-1和其他在耗尽的T细胞上表达的抑制性表面受体的抗体，或 针对肿瘤和间质细胞表达的PD-1配体PD-L1和PD-L2的封闭抗体已被 在促进长期肿瘤消退方面非常成功。封闭抗体的组合 多个抑制性受体，通常通过激活共刺激受体的抗体而得到加强。 比单独使用封闭抗体治疗更有效。然而，尽管如此， 尽管取得了成功，但许多患者对免疫检查点阻断疗法仍然没有反应，强调 需要在分子水平上理解免疫细胞的“衰竭”，无论是在小鼠模型中还是在人类中都是如此。 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素 潜在的T细胞激活。T细胞活化程序主要依赖于NFAT和NFAT的协同结合 它的转录伙伴AP1(Fos-jun)位于基因启动子和增强子的复合DNA位点。同时， NFAT可以激活第二个转录程序，该程序导致低反应状态，通常称为 ‘精疲力竭’或‘功能障碍’。第二个NFAT介导的程序在CD8+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，其特征是 细胞因子表达减少和细胞因子表达增加的功能受损状态谱 多种抑制受体(PD-1、CTLA4、LAG3、TIM3、TIGIT)。因此，这是联合的有效替代方案 检查点阻断疗法可能是为了调节NFAT介导的程序之间的平衡 激活和耗尽，从而使肿瘤浸润性T细胞远离耗竭，转向 效应器功能。我们将在这里检验这一假设。 我们用经过最小程度修饰以防止其与AP1相互作用的基因NFAT1进行了实验，结果表明 证实了耗竭的转录程序独立于NFAT1-AP1的相互作用。我们 已经确定了NFAT在耗竭计划中的重要靶点，包括Nr4a的转录因子 和毒物家族。此外，我们还表明，Nr4a转录因子在疲惫的肿瘤浸润性T细胞中起作用。 细胞，部分是通过抑制bZIP转录因子的表达或激活，否则会 促进效应器一样的表型。在目标1中，我们将鉴定和鉴定bZIP转录因子。 在一定条件下维持肿瘤浸润性CD8+T细胞的效应功能最有效 这通常会导致精疲力竭；在目标2中，我们将定义两个NFAT家族不同角色 成员，NFAT1和NFAT2，在耗竭的转录程序中；在目标3中，我们将使用 识别与NFAT相互作用的蛋白的蛋白质组学策略 程序。 我们建议的研究将测试CD8+TIL被细胞固有的功能沉默的假设 持续NFAT信号转导结合bZIP转录抑制的转录程序 各种因素。这些结果将有助于从更广泛的机制上理解转录机制。 在小鼠和人类肿瘤浸润性T细胞上进行操作。 好了！

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. !