Role of immune modulating butyrophilins in gamma delta T cell activation

摘要 免疫细胞通过检测并对外来分子做出反应来保护我们免受疾病侵袭。了解 如果我们要创造新的治疗或预防疾病的方法，这种反应的分子基础是至关重要的 涉及免疫细胞的疾病。我们研究的目的是鉴定一种新发现的抗原 检测影响γ-T介导的免疫应答的蛋白(BTn3，BTn3) 细胞。Gamma Delta T细胞是一种细胞毒性T细胞，能对外来威胁迅速做出反应，并为多种 作用，包括直接裂解感染或恶性细胞，因此，它们的激活具有很大的希望 治疗手法。与T细胞表达更普遍的α-βT细胞受体和 对肽抗原的反应，表达VGamma9Vdelta2T细胞受体的T细胞对小分子 被称为磷抗原的含磷化合物。丁咯菲林3A1(BTN3A1)是一种受体 磷酸化抗原，并通过不清楚的机制介导其激活T细胞。我们开发了一个图书馆 新的合成磷抗原，以及丁亲素结构和点突变的文库，两者都是 它们是了解酪氨酸亲和素潜在生物学的宝贵工具。在这里，我们提出的目标是 测试与BTN3A1细胞内结构域结合的配体产生构象的潜在假设 以及与T细胞上的相反受体相互作用所需的组织变化。理解 BTN3A1和相关的3A2、3A3和2A1亚型如何在分子水平上发挥作用是重要的，因为它 1)将有助于优化过去和现在的临床试验，这些试验检查了磷抗原和磷抗原- 扩大的细胞作为免疫疗法，以及2)将在这个复合体中识别新的分子靶点或策略 用于治疗手法。我们在目标1中的研究将显示磷化抗原如何影响的结构基础 利用多种生物物理和分子生物学方法获得全长内源BTN3A1。在目标2中， 我们将研究BTN3A1在磷抗原诱导的VGamma9Vdelta2T细胞裂解中的作用。 含有磷酸抗原的细胞和相关细胞因子的产生。共同努力，这将使我们能够建立一个 BTn3的结构-功能模型，涉及其结构域组织、寡聚状态、蛋白质结构和功能. 蛋白质的相互作用以及与BTN2A1的关系影响其功能。这在很大程度上将在以下背景下进行 通过使用一种新型的体外膜纳米盘/冷冻-EM模型系统来研究生物膜。我们的终极 目标是提出一个清晰的结构模型，说明磷化抗原如何诱导构象和/或 BTN3复合体的组成变化促进了T细胞的效应器功能。这将使临床 开发调节丁咯菲林功能以克服免疫检查点的疗法。这些发现 将出现在对抗原检测的生物学理解远未完成的时候，因此 影响该领域的潜在因素。

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.