Role of immune modulating butyrophilins in gamma delta T cell activation

摘要 免疫细胞通过检测和响应外来分子来保护我们免受疾病的侵害。了解 这种反应的分子基础是至关重要的，如果我们要产生新的疗法来治疗或预防 涉及免疫细胞的疾病我们研究的目的是描述一种新发现的抗原 检测影响由γ δ T介导的免疫应答的蛋白质（亲酪蛋白3，BTN 3 细胞γ δ T细胞是细胞毒性T细胞，对外来威胁迅速作出反应，并为各种免疫系统提供服务。 作用，包括感染或恶性细胞的直接裂解，因此，它们的激活为 治疗手法与表达更普遍的α β T细胞受体的T细胞相反， 表达V γ 9 V δ 2 T细胞受体的T细胞对小的肽抗原应答， 含磷化合物称为磷抗原。嗜酪蛋白3A 1（BTN 3A 1）是嗜酪蛋白3A 1的受体。 磷酸化抗原，并通过不清楚的机制介导其活化的T细胞。我们开发了一个图书馆 新的合成磷酸化抗原，以及亲酪蛋白构建体和点突变的文库， 这对于理解亲丁酸蛋白的潜在生物学是有价值的工具。在这里，我们提出的目标是， 测试配体与BTN 3A 1的细胞内结构域结合产生构象变化的潜在假设。 以及与T细胞上的反受体相互作用所需的组织变化。理解 BTN 3A 1和相关的3A 2，3A 3和2A 1亚型在分子水平上的功能是重要的，因为它 1)将有助于优化过去和现在的临床试验，检查磷酸化抗原和磷酸化抗原- 扩增的细胞作为免疫疗法，以及2）将在该复合物中鉴定新的分子靶点或策略 进行治疗性操作我们在目标1中的研究将显示磷抗原如何影响的结构基础 使用多种生物物理学和分子生物学方法测定全长内源性BTN 3A 1。在目标2中， 我们将研究BTN 3A 1在磷酸化抗原诱导的Vgamma 9Vdelta 2 T细胞溶解中的功能， 含磷抗原的细胞和相关的细胞因子产生。这将使我们能够共同建立一个 BTN 3的结构-功能模型，关于其结构域组织、寡聚化状态、蛋白质 蛋白质相互作用以及与BTN 2A 1的关系影响其功能。这将在很大程度上在以下背景下进行： 通过使用一种新的体外膜纳米盘/冷冻EM模型系统，我们的最终 目的是提出一个明确的结构模型，证明磷酸化抗原诱导的构象和/或 BTN 3复合物中的组成变化促进T细胞的效应子功能。这将使临床 开发调节亲丁酸蛋白功能以克服免疫检查点的疗法。这些发现 将在抗原检测的生物学理解远未完成的时候到来，因此具有 影响该领域的潜力。

Abstract Immune cells protect us from disease by detecting and responding to foreign molecules. Understanding the molecular basis of this response is critical if we are to generate new therapies for treatment or prevention of diseases involving immune cells. The objective of our research is to characterize a newly discovered antigen detecting protein (butyrophilin 3, BTN3) which influences the immune response mediated by gamma delta T cells. Gamma delta T cells are cytotoxic T cells that respond quickly to foreign threats and serve a variety of roles, including direct lysis of infected or malignant cells, and as such, their activation holds great promise for therapeutic manipulation. In contrast to T cells that express the more prevalent alpha beta T cell receptor and respond to peptide antigens, T cells that express the Vgamma9Vdelta2 T cell receptor respond to small phosphorous-containing compounds known as phosphoantigens. Butyrophilin 3A1 (BTN3A1) is the receptor for phosphoantigens and mediates their activation of T cells through unclear mechanisms. We developed a library of novel synthetic phosphoantigens, as well as a library of butyrophilin constructs and point mutations, both of which are valuable tools for understanding the underlying biology of butyrophilins. Here, we propose aims that test the underlying hypothesis that ligand binding to the intracellular domain of BTN3A1 produces conformational and organizational changes that are required for interaction with counter receptors on T cells. Understanding how BTN3A1 and the related 3A2, 3A3, and 2A1 isoforms function at the molecular level is important because it 1) will help optimize past and present clinical trials that have examined phosphoantigens and phosphoantigen- expanded cells as immunotherapies, and 2) will identify new molecular targets or strategies within this complex for therapeutic manipulation. Our studies in Aim 1 will show a structural basis for how phosphoantigens affect the full length endogenous BTN3A1 using multiple biophysical and molecular biological approaches. In Aim 2, we will investigate the function of BTN3A1 in phosphoantigen-induced Vgamma9Vdelta2 T cell lysis of phosphoantigen containing cells and associated cytokine production. Together, this will allow us to build a structure-function model of BTN3 with regards to how its domain organization, oligomerization status, protein- protein interactions, and relationship to BTN2A1 influence its function. This will largely be done in the context of biological membranes through use of a novel in vitro membrane nanodisc/cryo-EM model system. Our ultimate goal is to present a clear structural model that demonstrates how phosphoantigen-induced conformational and/or compositional changes in the BTN3 complex promote effector functions of T cells. This will enable clinical development of therapies that modulate butyrophilin function to overcome immune checkpoints. These findings will come at a point when the biological understanding of antigen detection is far from complete, and thus have the potential to impact the field.