Peripheral shaping of γδ TCR repertoires

从外周信号如何塑造γδ-T细胞的T细胞受体（TCR）库的问题出发，本项目旨在回答该领域的两个重要问题，即：γδ TCR的同源配体是什么？ γδ T细胞在免疫反应中的作用是什么？除了大量的天然人γδ T细胞（所有这些细胞都携带相似的、低变异的Vγ 9v δ2 TCR，并且所有这些TCR都与相同的亲丁酸蛋白分子结合）外，其他的人γδ T细胞通常使用高度多样化的TCR库，即Vδ1或Vδ3链与6条可变的Vγ链配对。在这些适应性更强的克隆中，独特的tcr池非常大，很少在个体之间共享。据推测，它们高度多样化的tcr与b细胞受体相似，并随机结合表面抗原。然而，近年来，许多独特的Vδ1-或v δ3- tcr已被证明与主要组织相容性复合体（MHC）或MHC相关蛋白结合。在FOR 2799的第一个资助期，我们研究了一组cmv应答的人γδ tcr识别的抗原。我们鉴定了一种Vγ3Vδ1 TCR（命名为TCR04），它对b细胞淋巴瘤细胞系具有特异性反应。用可溶性TCR04染色淋巴瘤细胞，随后进行全基因组CRISPR/Cas9敲除筛选，鉴定出HLA-DR （MHC II）是TCR04的同源抗原。在即将到来的资助期内，我们将进一步确定TCR04和类似的相关γδ-TCR识别HLA-DR的潜在分子决定因素。此外，我们将使用新技术通过同时进行单细胞RNA测序和单细胞TCR测序来跟踪适应性γδ T细胞对病毒感染的反应进展。最后，我们计划鉴定激活适应性γδ T细胞的其他相关抗原。该提案是DFG研究小组FOR 2799“通过γδ T细胞受体接收和翻译信号”的一部分，旨在与网络中的所有其他项目合作。

Starting from the question of how peripheral signals shape the T cell receptor (TCR) repertoire of γδ-T cells, this subproject of FOR 2799 aims to answer two important questions in this field, namely: what are the cognate ligands of γδ TCR and what is the role of γδ T cells during an immune response?Apart from a large population of innate human γδ T cells, all of which carry a similar, low-variance Vγ9Vδ2 TCR and all of which bind to the same butyrophilin molecules, other human γδ T cells typically use a highly diverse TCR repertoire of Vδ1 or Vδ3 chains that pair with six variable Vγ chains. The pool of unique TCRs among these more adaptive clones is very large and rarely shared between individuals. It has been hypothesized that their highly diverse TCRs are similar to B-cell receptors and bind randomly to surface antigens. However, in recent years, a number of unique Vδ1- or Vδ3-TCRs have been shown to bind to the major histocompatibility complex (MHC) or MHC-related proteins. In the first funding period of FOR 2799, we investigated the antigen recognized by a set of CMV-responsive human γδ TCRs. We identified a Vγ3Vδ1 TCR (designated TCR04) that was specifically reactive against a B-cell lymphoma cell line. Staining of lymphoma cells with soluble versions of TCR04 and subsequent genome-wide CRISPR/Cas9 knock-out screening led to the identification of HLA-DR (MHC II) as the cognate antigen for TCR04. In the upcoming funding period, we will now further determine the underlying molecular determinants of HLA-DR recognition by TCR04 and similar related γδ-TCR. In addition, we will use new technologies to track the progression of adaptive γδ T cell responses to viral infections by simultaneous single cell RNA sequencing and single cell TCR sequencing. Finally, we plan to identify additional relevant antigens that activate adaptive γδ T cells. This proposal is part of the DFG research group FOR 2799 "Receiving and Translating Signals via the γδ T Cell Receptor" and is designed to collaborate with all other projects in the network.