Investigating the intracellular signaling mechanisms of coinhibitory receptor PD-1

项目摘要和摘要 共抑制性免疫受体程序性细胞死亡-1(PD-1，CD279)在维持 外周耐受性和保护健康组织免受炎症组织损害。肿瘤利用了 PD-1途径可逃避免疫介导的破坏。尽管PD-1途径抑制剂已经彻底改变了 癌症治疗，只有一小部分患者有反应并显示出长期缓解。尽管在临床上取得了成功， PD-1介导的抑制作用背后的细胞内信号机制尚不清楚。更好的 对PD-1信号的理解有可能为单一药物或原理确定新的靶点 改善癌症患者反应的联合治疗策略。 PD-L1/L2对PD-1的结扎导致下游增殖通路的减弱，减少了 效应细胞因子与细胞骨架重排。PD-1通过募集酪氨酸发挥抑制作用 位于PD-1胞质尾部的磷酸酶SHP2和/或SHP1到酪氨酸磷酸化基序 使TCR相关靶去磷酸化。鉴于在PD-1结扎过程中观察到的T细胞的显著变化 以及膜结合信号的复杂性，我们认为额外的蛋白质是必要的，以介导PD- 1抑制信号和减弱未知靶蛋白的功能，从而影响肿瘤的发病机制。 PD-L1结扎前后PD-1胞浆尾部邻近标记的研究 蛋白质组学分析已确定机械敏感阳离子通道PIEZO1是HIT蛋白的首选候选蛋白。 PIEZO1通过剪应力或侧膜拉伸激活在发育中起作用， 不同组织的分化和动力学。最近的数据表明，PIEZO1可以调节免疫细胞 回应。TCR参与激活PIEZO1，导致钙内流，有助于T细胞启动 信号和肌动蛋白的细胞骨架调节；所有这些过程都是PD-1介导的抑制作用的靶点。为 这些原因，我们假设PD-1与PIEZO1共定位，以减弱钙内流，从而扰乱 TCR介导的信号转导，损害T细胞功能和减弱肿瘤反应。了解 干扰这一潜在信号轴的结果将提供对PD-1功能和 应该揭示PD-1-压电相互作用如何影响癌症的发病机制。 初步数据和已发表的研究表明，PD-1介导的抑制比 先前所描述的。因此，本研究的主要目的是(1)验证PD-1的作用 在调节候选HIT蛋白PIEZO1和加深对PD-1信号的理解和(2)确定 PD-1和Piezo如何调节抗肿瘤免疫。成功完成这项提案不仅将有助于 表征PD-1介导的信号以识别癌症的潜在治疗靶点，但也将 产生的方法可以应用于信号转导尚不清楚的许多免疫受体。

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.