Investigating the intracellular signaling mechanisms of coinhibitory receptor PD-1

项目总结和摘要 共抑制性免疫受体程序性细胞死亡-1（PD-1，CD 279）在维持细胞凋亡中起重要作用。 外周耐受性和保护健康组织免受炎性组织损伤。肿瘤利用了 PD-1通路以逃避免疫介导的破坏。尽管PD-1通路抑制剂已经彻底改变了 在癌症护理中，只有一部分患者有反应并显示出长期缓解。尽管临床上取得了成功， PD-1介导的抑制的细胞内信号传导机制仍不清楚。更好的 对PD-1信号传导的理解有可能为单一药物或基本原理确定新的靶点 联合治疗策略，以改善癌症患者的反应。 PD-L1/L2连接PD-1导致下游增殖途径减弱， 效应细胞因子和细胞骨架重排。PD-1通过募集酪氨酸来发挥其抑制功能 磷酸酶SHP 2和/或SHP 1与位于PD-1细胞质尾区的酪氨酸磷酸化基序结合， 去磷酸化TCR相关靶点。鉴于PD-1连接后T细胞中观察到的显著变化， 和膜结合信号传导的复杂性，我们相信额外的蛋白质是介导PD所必需的。 1抑制信号并减弱未鉴定的靶蛋白的功能以影响癌症发病机制。 我的研究使用PD-L1连接后PD-1细胞质尾周围的邻近标记和随后的 蛋白质组学分析已经鉴定机械敏感阳离子通道PIEZO 1为最佳候选命中蛋白。 通过剪切应力或侧膜拉伸激活PIEZO 1在发育中起作用， 各种组织的分化和动力学。最近的数据表明PIEZO 1可以调节免疫细胞 应答TCR接合激活PIEZO 1，导致有助于T细胞引发的钙内流，TCR 信号传导和肌动蛋白细胞骨架调节;所有这些过程都是PD-1介导的抑制的靶向。为 基于这些原因，我们假设PD-1与PIEZO 1共定位以减弱钙内流，从而破坏 TCR介导的信号传导，损害T细胞功能和减弱肿瘤反应。了解 干扰该潜在信号传导轴的结果将提供对PD-1功能的机制性见解， 应该揭示PD-1-PIEZO相互作用如何影响癌症发病机制。 初步数据以及已发表的研究表明，PD-1介导的抑制比 以前的特点。因此，本研究的主要目的是：（1）验证PD-1的作用 在调节候选命中蛋白PIEZO 1和深化PD-1信号转导的理解和（2）确定 PD-1和PIEZO如何调节抗肿瘤免疫。成功完成此提案不仅有助于 表征PD-1介导的信号传导，用于鉴定癌症中的潜在治疗靶点，但也将 产生可应用于许多信号传导仍不清楚的免疫受体的方法。

PROJECT SUMMARY AND ABSTRACT Coinhibitory immunoreceptor programmed cell death-1 (PD-1, CD279) plays a significant role in maintaining peripheral tolerance and protecting healthy tissues from inflammatory tissue damage. Tumors have exploited the PD-1 pathway to evade immune-mediated destruction. Although PD-1 pathway inhibitors have revolutionized cancer care, only a subset of patients respond and show long-lasting remission. Despite this clinical success, the intracellular signaling mechanisms underlying PD-1-mediated inhibition remain unclear. A better understanding of PD-1 signaling has the potential to identify novel targets for single agent or rationale combination therapy strategies to improve patient responses in cancer. Ligation of PD-1 by PD-L1/L2 results in the attenuation of downstream proliferative pathways, decreases in effector cytokines and cytoskeletal rearrangements. PD-1 exerts its inhibitory function by recruiting tyrosine phosphatases SHP2 and/or SHP1 to tyrosine phosphorylation motifs located on the cytoplasmic tail of PD-1 to dephosphorylate TCR-associated targets. Given the dramatic changes observed in T cells upon PD-1 ligation and the complexity of membrane bound signaling, we believe additional proteins are necessary to mediate PD- 1 inhibitory signals and attenuate the function of unidentified target proteins to influence cancer pathogenesis. My studies using proximity labelling around the cytoplasmic tail of PD-1 upon PD-L1 ligation and subsequent proteomic analysis have identified mechanosensitive cation channel PIEZO1 as the top candidate hit protein. The activation of PIEZO1 through shear stress or lateral membrane stretching plays a role in the development, differentiation and dynamics of various tissues. Recent data show that PIEZO1 can regulate immune cell responses. TCR engagement activates PIEZO1 resulting in calcium influx that contributes to T cell priming, TCR signaling and actin cytoskeletal modulation; all of these processes are targeted by PD-1-mediated inhibition. For these reasons, we hypothesize that PD-1 co-localizes with PIEZO1 to attenuate calcium influx thereby disrupting TCR-mediated signaling, impairing T cell function and attenuating tumor responses. Understanding the consequence of perturbing this potential signaling axis will provide mechanistic insights into PD-1 function and should reveal how PD-1-PIEZO interaction influences cancer pathogenesis. Preliminary data as well as published research suggest PD-1-mediated inhibition is more complex than previously characterized. For this reason, the primary goals of this research are (1) to validate the role of PD-1 in regulating candidate hit protein PIEZO1 and deepen the understanding of PD-1 signaling and (2) to determine how PD-1 and PIEZO regulate anti-tumor immunity. Successful completion of this proposal not only will aid in characterizing PD-1-mediated signaling for identification of potential therapeutic targets in cancer, but also will yield methods that can be applied to the many immunoreceptors for which signaling remains unclear.