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The impact of gamma-delta TCR sequences on selection and peripheral repertoire shaping of gamma-delta T cells.

γ-δ TCR 序列对γ-δ T 细胞的选择和外周谱形成的影响。

基本信息

批准号：
233956143
负责人：
Professor Dr. Immo Prinz
金额：
--
依托单位：
Institut für Systemimmunologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2016-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/233956143?language=en
关键词：
impact gamma delta TCR sequences

项目摘要

Three conserved lineages of lymphocytes rearrange clonal antigen receptors during their development, namely B cells, alpha-beta T cells and gamma-delta T cells. These gamma-delta T cells are rather oligoclonal T lymphocyte populations, with variable TCR diversity depending on their localization within tissues. Since the ligands of gamma-delta T cell receptor (TCR) remain largely unknown it is currently not established whether the gamma-delta TCR recognizes canonical self or foreign structures. We have previously described that developing gamma-delta T cells undergo TCR quality control selection and can differentiate into mature effector cells in the thymus. However, it is currently unclear whether engagement of specific TCR ligands is required for the differentiation of gamma-delta T cells into effector T cell subsets. Moreover, it is not even clear whether the gamma-delta TCR has any function in mature gamma-delta T cells. Thus, the central aim of this proposal is to define how clonal selection and peripheral homeostasis of gamma-delta T cells is instructed by their individual gamma-delta TCR sequences. To achieve this goal, we need comprehensive gamma-delta TCR sequence information. To overcome the limitations of classical sequencing approaches, our lab has established methods for deep-sequencing of very diverse antigen receptor chains. For example, we recently analyzed the alpha-beta TCR repertoire of CD4+Foxp3+ regulatory T cells. There, we suggested that continuous competition of peripheral regulatory T cells for limited self-antigen shapes an organ-optimized, yet highly diverse, local TCR repertoire. In this research project proposal, we intend to adopt our high-throughput sequencing protocols to gamma-delta T cells. In addition, we are modifying our sequencing approach to include rapid amplification of cDNA ends (RACE) to generate unbiased templates comprising all gamma-delta TCR. Also, we are establishing further protocols to sequence gamma-delta TCR chain amplicons derived from genomic DNA samples. Once we are able to define the complete antigen receptor range of gamma-delta T cells, we will monitor thymic selection and peripheral shaping of their TCR. To this end, we will chose and compare templates from lymphoid and mucosal tissues to clarify how the gamma-delta TCR repertoire is shaped locally, and how it may adapt during ageing and in response to commensal microbiota. At the same time, we will rigorously test the null hypothesis that the gamma-delta TCR has no specific post-thymic function. This question can only be answered by the use of genetic models to conditionally induce a loss of functional gamma-delta TCR in mature gamma-delta T cells. In summary, we expect to draw stringent conclusions how thymic and peripheral shaping of the gamma-delta TCR repertoire impacts on gamma-delta T cell function and homeostasis.

淋巴细胞的三个保守谱系在其发育过程中重排克隆抗原受体，即B细胞、α-β T细胞和γ-δ T细胞。这些γ-δ T细胞是相当寡克隆的T淋巴细胞群体，根据其在组织内的定位具有可变的TCR多样性。由于γ-δ T细胞受体（TCR）的配体在很大程度上仍是未知的，因此目前还不确定γ-δ TCR是否识别典型的自身或外来结构。我们先前已经描述了发育中的γ-δ T细胞经历TCR质量控制选择，并且可以在胸腺中分化成成熟的效应细胞。然而，目前尚不清楚特异性TCR配体的接合是否是γ-δ T细胞分化为效应T细胞亚群所必需的。此外，甚至不清楚γ-δ TCR在成熟的γ-δ T细胞中是否有任何功能。因此，该提议的中心目的是定义γ-δ T细胞的克隆选择和外周稳态如何由其个体γ-δ TCR序列指示。为了实现这一目标，我们需要全面的γ-δ TCR序列信息。为了克服经典测序方法的局限性，我们的实验室已经建立了对非常不同的抗原受体链进行深度测序的方法。例如，我们最近分析了CD 4 + Foxp 3+调节性T细胞的α-β TCR库。在那里，我们认为，外周调节性T细胞对有限的自身抗原的持续竞争形成了器官优化的，但高度多样化的局部TCR库。在本研究项目提案中，我们打算采用我们的高通量测序方案来检测γ-δ T细胞。此外，我们正在修改我们的测序方法，包括cDNA末端快速扩增（RACE），以生成包含所有γ-δ TCR的无偏模板。此外，我们正在建立进一步的协议，从基因组DNA样品的序列γ-δ TCR链扩增子。一旦我们能够确定γ-δ T细胞的完整抗原受体范围，我们将监测其TCR的胸腺选择和外周成形。为此，我们将选择并比较淋巴组织和粘膜组织的模板，以阐明γ-δ TCR库如何在局部形成，以及它如何在衰老过程中适应和响应肠道微生物群。同时，我们将严格检验γ-δ TCR没有特异性胸腺后功能的零假设。这个问题只能通过使用遗传模型来有条件地诱导成熟γ-δ T细胞中功能性γ-δ TCR的丧失来回答。总之，我们期望得出严格的结论，胸腺和外周形成的γ-δ TCR库如何影响γ-δ T细胞的功能和稳态。