Chimeric γδ T Cell Receptors

γδ和αβ T细胞是脊椎动物免疫系统的重要部分，分别表达γδ T细胞受体（TCR）或αβ TCR。我们计划设计嵌合γδ TCR，以揭示γδ T细胞如何响应随时间变化的配体遭遇，并提供改善癌症免疫治疗和对抗病毒感染的新方法的原理证明。为此，我们计划使用我们已成功开发的αβ TCR技术，并首次将其应用于γδ TCR。本论文的工作分为两个部分，其共同点是新型嵌合γδ TCR的应用：首先，我们将探讨γδ T细胞对时间控制的配体相遇的反应。为此，我们将通过向结合PhyB的TCR附加小结构域来工程化γδ TCR以结合Phytochome B（PhyB）作为其配体。蛋白质PhyB来源于植物，并且可以使用红光和远红光在两种构象之间切换，从而结合或不结合工程化的γδ TCR。我们将通过使用不同强度的红光改变γδ TCR-配体相互作用的半衰期并研究T细胞活化。这些实验旨在回答一个重要的未解决的问题：γδ T细胞是否可以使用称为动力学校对的机制来区分不同亲和力的配体。事实上，我们已经使用这种具有αβ TCR的光遗传学系统，以表明半衰期是αβ T细胞活化中的关键参数（Yousefi等人，eLife，2019年）。在生理条件下，T细胞在探索推定的靶细胞时被激活，因此与抗原呈递细胞具有由不同持续时间的暂停分开的时间接触。我们的系统是独特的，以找出是否配体结合的暂停是由γδ T细胞耐受，如果特定的时间模式将导致差异γδ T细胞活化。其次，我们将通过将针对肿瘤抗原的单链Fv片段融合到γδ TCR来使γδ TCR具有新的特异性。虽然γδ T细胞具有识别肿瘤的固有特性，但这通常不足以成功杀死恶性细胞。我们将评估这是否可以用作一种新的基于T细胞的癌症治疗方法。我们已经将该技术与αβ TCR一起使用，并显示重编程的αβ T细胞在体内杀死肿瘤细胞方面比CAR-T细胞优越上级（Baeuerle et al.，Nature Comm，2019）。我们还将探索嵌合γδ TCR是否可用于重定向γδ T细胞以杀死病毒感染的细胞。

γδ and αβ T cells are important parts of the vertebrate immune system, expressing a γδ T cell receptor (TCR) or αβ TCR, respectively. We plan to engineer chimeric γδ TCRs to unravel how γδ T cells respond to temporally varying ligand encounter and provide proof-of-principles of new approaches to improve cancer immunotherapy and to combat virus infection. To this end, we plan to use technologies that we have successfully developed for the αβ TCR, and apply them for the first time to the γδ TCR. The planned work is divided into two parts, whose common denominator is the use of novel chimeric γδ TCRs.Firstly, we will ask how γδ T cells respond to temporally controlled ligand encounter. To this end, we will engineer γδ TCRs to bind to Phytochome B (PhyB) as their ligand by appending a small domain to the TCR that binds to PhyB. The protein PhyB is derived from plants and can be switched between two conformations using red and far-red light, thus either binding or not to the engineered γδ TCR. We will alter the half-life of the γδ TCR-ligand interaction by using different intensities of red light and study T cell activation. These experiments aim to answer an important unsolved question: whether γδ T cells can distinguish between ligands of different affinities using a mechanism known as kinetic proofreading. In fact, we have used this optogenetic system with the αβ TCR, to show that the half-life is the critical parameter in αβ T cell activation (Yousefi et al., eLife, 2019). Under physiological conditions, T cells are activated while exploring the putative target cells, thus having temporal contacts to the antigen-presenting cells separated by pauses of different duration. Our system is unique to find out whether pauses of ligand binding are tolerated by the γδ T cells, and if specific temporal patterns will lead to differential γδ T cell activation. Secondly, we will equip γδ TCRs with new specificities by fusion of a single chain Fv fragment against tumour antigens to the γδ TCR. Although γδ T cells have the inherent property to recognize tumours, this is often not sufficient to successfully kill the malignant cells. We will evaluate whether this could be used as a novel T cell-based therapy against cancer. We have already used this technology with the αβ TCR and shown that the re-programmed αβ T cells are superior in killing tumour cells in vivo than CAR-T cells (Baeuerle et al., Nature Comm, 2019). We will also explore whether chimeric γδ TCRs can be used to redirect γδ T cells to kill virus-infected cells.