The role of Cathepsin L in shaping a functional T cell repertoire

组织蛋白酶 L 在塑造功能性 T 细胞库中的作用

• 批准号: 456882036
• 负责人: Professor Dr. Ludger Klein
• 金额: --
• 依托单位: Forschungsbereich Immunologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/456882036?language=en
• 关键词: role; Cathepsin; shaping; functional; cell

The survival and developmental progression of immature T cells during thymic selection requires productive interactions of their T cell receptor (TCR) with ‘self-peptide’ (p)MHC ligands presented by cortical thymic epithelial cells (cTECs). This process is referred to as positive selection. The traditional view that positive selection supports effective anti-foreign responses by establishing the self-MHC-restriction of the repertoire has recently been challenged. Instead, it was suggested that positive selection mediates the seeding of the periphery with T cells of optimal ‘fitness’. Whether and how the functional competence of mature T cells is lastingly ‘imprinted’ by the pMHC ligands that are encountered during positive selection remains largely enigmatic.The crucial role of cTECs during positive selection is at least in part specified by unique proteolytic machineries that these cells use to generate peptides loaded onto MHC I or II. These proteolytic pathways are likely to endow cTECs with a ‘private’ pMHC ligandome that is different from that on other antigen presenting cells (APCs). For instance, cTECs process substrates of the MHCII loading pathway through Cathepsin L (Ctsl), whereas in other APCs, this occurs mostly through Cathepsin S. Knockout of Ctsl strongly decreases the number of CD4 T cells in the thymus.In preliminary work, we found that the CD4 T cell repertoire of CtslKO mice, irrespective of its reduced cellularity, harbors a ‘normal’ number of cells that recognize a prototypical foreign antigen, Listeriolysin O (LLO). However, these cells failed to undergo proliferative expansion in response to immunization. Similarly, bulk CD4 T cells from Ctsl-deficient mice displayed an impaired response to polyclonal stimulation in vitro and, upon adoptive transfer into lymphopenic hosts, underwent only limited ‘homeostatic’ expansion in vivo. These findings suggest that Ctsl-dependent pMHCII ligands on cTECs not only determine the cellularity, but also the functionality of the CD4 T cell repertoire.In the proposed project, we want to test the hypothesis that there is a substantial overlap in the TCR composition of CD4 T cells in WT or CtslKO mice, but that cells expressing such ‘shared’ TCRs are dysfunctional when selected in the absence of Ctsl. We will employ single-cell TCR sequencing to generate TCR inventories of LLO-specific CD4 T cells from WT and Ctsl-deficient mice to identify TCRs that are found in both genotypes. Representative ‘shared’ TCRs will be re-expressed as TCR transgenes in WT and Ctsl-deficient mice. This will enable us to systematically address the in vivo and in vitro functionality of cells expressing the very same TCR, yet positively selected in either the presence or absence of Ctsl. Furthermore, we will address the biochemical and transcriptional basis of how the functional fitness of CD4 T cells is imprinted through positive selection by Ctsl-dependent pMHC ligands.

在胸腺选择过程中，未成熟T细胞的存活和发育过程需要其T细胞受体（TCR）与胸腺皮质上皮细胞（cTECs）呈现的“自肽”(p)MHC配体的有效相互作用。这个过程被称为积极选择。传统观点认为，积极选择通过建立自我mhc限制来支持有效的抗外源反应，这一观点最近受到了挑战。相反，有人认为，正选择介导了最佳“适应性”T细胞的外围播种。成熟T细胞的功能能力是否以及如何被正选择过程中遇到的pMHC配体持久地“印记”，在很大程度上仍然是一个谜。在阳性选择过程中，ctec的关键作用至少部分是由这些细胞用来产生装载到MHC I或II上的肽的独特蛋白水解机制所指定的。这些蛋白水解途径可能赋予ctec不同于其他抗原呈递细胞（apc）的“私有”pMHC配体。例如，cTECs通过组织蛋白酶L （Ctsl）处理MHCII装载途径的底物，而在其他apc中，这主要通过组织蛋白酶s发生。敲除Ctsl会强烈减少胸腺中CD4 T细胞的数量。在初步工作中，我们发现CtslKO小鼠的CD4 T细胞库，无论其细胞数量减少，都含有“正常”数量的细胞，可以识别一种典型的外来抗原，李斯特菌溶素O （LLO）。然而，这些细胞在免疫应答中未能进行增殖扩增。同样，来自ctsl缺陷小鼠的大量CD4 T细胞在体外对多克隆刺激的反应受损，并且在过继转移到淋巴细胞减少的宿主后，在体内只经历了有限的“稳态”扩张。这些发现表明，ctec上依赖于cctl的pMHCII配体不仅决定细胞结构，还决定CD4 T细胞库的功能。在这个项目中，我们想要验证这样一个假设，即在WT或CtslKO小鼠中，CD4 T细胞的TCR组成存在大量重叠，但是在没有Ctsl的情况下，表达这种“共享”TCR的细胞会出现功能障碍。我们将使用单细胞TCR测序来生成来自WT和ctsl缺陷小鼠的低密度脂蛋白特异性CD4 T细胞的TCR清单，以鉴定在两种基因型中发现的TCR。代表性的“共享”TCR将在WT和ctsl缺陷小鼠中作为TCR转基因重新表达。这将使我们能够系统地解决细胞在体内和体外表达相同的TCR的功能，但在存在或不存在Ctsl的情况下都是阳性选择。此外，我们将探讨CD4 T细胞的功能适应性如何通过ctsl依赖性pMHC配体的正选择而被印记的生化和转录基础。