Novel mechanisms regulating PD-1 signaling and function

摘要 免疫检查点疗法阻断T细胞上的抑制性受体以增强抗肿瘤免疫 应答程序性死亡-1（PD-1）途径是T细胞中的关键抑制性检查点， 阻断PD-1的抗体促进免疫介导的恶性肿瘤的鉴定和清除。癌 免疫疗法，如抗PD-1抗体，代表了癌症治疗的范式转变，因为它们确实 而不是直接针对肿瘤细胞。相反，通过利用免疫系统，抗PD-1疗法可以引起 多种肿瘤类型的持久临床反应，包括长期缓解。不幸的是， 抗PD-1治疗仅在一小部分患者中实现。因此，开发额外的治疗方法， 更好地抑制该途径将使该方法的益处扩展到许多其他患者。实现这一 目标，更深入地了解PD-1下游的信号传导事件和分子生物学特征。 PD-1发挥其抑制作用的机制是关键的。为此，我们采取了公正的 方法来确定PD-1检查点途径中的新靶点。我们用了先进的质谱仪 识别PD-1下游的新信号通路。使用这个系统，我们确定了多个候选人， 我们将其分为两类：PD-1功能的启动子或抑制子。令人兴奋的是，我们发现， 用RNAi沉默PD-1启动子而不是抑制子消除了PD-1的抑制作用。我们现在 试图阐明这些蛋白质影响PD-1功能的机制，并确定它们的适用性 作为药物发现的靶标或PD-1治疗的生物标志物，具有三个特定目的： 我们将证实PD-1和新发现的启动子之间的相互作用在初级阶段是直接的， 人类T细胞为了研究物理相互作用，我们将利用高分辨率显微镜。我们将 将体内小鼠癌症模型纳入我们的研究，目的是为我们的研究建立一个角色。 肿瘤进展/消退的候选者。在第二个目标中，我们将进一步描述 在原代人T细胞中检测PD-1抑制因子，并测量细胞因子分泌、增殖和细胞周期。 对刺激作出反应的粘附。在第三个目标中，我们将研究PD-1的亚细胞分布 调节剂并测量其形成成熟免疫突触的能力。总的来说， 这些目标将有助于确定对PD-1功能的机制性理解，以指导 改进的癌症免疫疗法和抗PD-1应答的潜在生物标志物。

ABSTRACT Immune checkpoint therapies block inhibitory receptors on T cells in efforts to augment anti-tumor immune responses. The Programmed death-1 (PD-1) pathway is a critical inhibitory checkpoint in T cells and antibodies blocking PD-1 promote immune-mediated identification and clearance of malignancies. Cancer immunotherapies, like anti-PD-1 antibodies, represent a paradigm shift in cancer treatment because they do not target the tumor cell directly. Instead, by harnessing the immune system, anti-PD-1 therapies can elicit durable clinical responses in multiple tumor types, including long-term remissions. Unfortunately, the benefit of anti-PD-1 therapy is realized in only a fraction of patients. As such, developing additional therapeutics that can better inhibit this pathway will extend the benefit of this approach to many additional patients. To achieve this goal, a deeper insight into the signaling events downstream of PD-1 and characterization of the molecular mechanism(s) by which PD-1 exerts its inhibitory effect is critical. Toward this end, we have taken an unbiased approach to identify novel targets in the PD-1 checkpoint pathway. We used advanced mass spectrometry to identify new signaling pathways downstream of PD-1. Using this system, we identified multiple candidates that we categorized into two groups: promoters or suppressors of PD-1 functions. Excitingly, we found that silencing PD-1 promoters, and not suppressors, with RNAi abrogated the inhibitory effects of PD-1. We now seek to elucidate the mechanisms by which these proteins affect PD-1 function and determine their suitability as either targets for drug discovery or biomarkers for PD-1 treatment with three specific aims: In the first aim we will confirm that the interaction between PD-1 and the newly discovered promoters is direct in primary human T cells. In order to study the physical interactions, we will utilize high-resolution microscopy. We will incorporate an in vivo mouse model of cancer into our studies, with the goal of establishing a role for our candidates in tumor progression/regression. In the second aim we will further characterize the functional role of PD-1 suppressors in primary human T cells and measure cytokine secretion, proliferation, and cellular adhesion in response to stimulation. In the third aim we will investigate the subcellular distribution of PD-1 modulators and measure their ability to form mature immunological synapses. Taken together, the execution of these aims will help define a mechanistic understanding of PD-1 function to guide the development of improved cancer immunotherapies and potential biomarkers of anti-PD-1 responses.