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

未成熟T细胞在胸腺选择过程中的生存和发育过程需要它们的T细胞受体(TCR)与胸腺皮质上皮细胞(CTECs)递呈的自肽(P)MHC配体发生有效的相互作用。这个过程被称为正面选择。积极选择通过建立曲目的自我MHC限制来支持有效的反外国反应的传统观点最近受到了挑战。相反，人们认为，正向选择介导了外周T细胞的播种，使其具有最佳的“适合性”。在正向选择过程中，成熟T细胞的功能是否以及如何被pMHC配体持久地“印记”在很大程度上仍然是个谜。cTECs在正向选择过程中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上的多肽的独特的蛋白分解机制所指定的。这些蛋白分解途径可能赋予cTEC一个不同于其他抗原提呈细胞(APC)的“私有”pMHC配体。例如，cTECs通过组织蛋白酶L(CTSL)处理MHCII负载途径的底物，而在其他APC中，这主要通过组织蛋白酶S发生。CTSL的敲除大大减少了胸腺中CD4T细胞的数量。在初步工作中，我们发现CtslKO小鼠的CD4T细胞谱系，无论其细胞密度如何减少，都含有识别典型外来抗原Listeriolysin O(LLO)的“正常”细胞数量。然而，这些细胞未能在免疫后进行增殖扩增。同样，CTSL缺陷小鼠的大量CD4T细胞在体外对多克隆刺激表现出受损的反应，当过继转移到淋巴细胞减少的宿主时，在体内只经历了有限的‘内稳态’扩增。这些发现表明，依赖CTSL的cTECs上的pMHCII配体不仅决定了细胞的数量，而且还决定了CD4T细胞库的功能。在拟议的项目中，我们想要检验一种假设，即在WT或CtslKO小鼠中，CD4T细胞的TCR组成有很大的重叠，但当在没有CTSL的情况下选择时，表达这种共享的TCR的细胞是功能障碍的。我们将使用单细胞TCR测序来生成来自WT和CTSL缺陷小鼠的LLO特异性CD4T细胞的TCR清单，以确定在这两种基因型中发现的TCR。代表性的“共享”TCR将在WT和CTSL缺陷小鼠中以TCR转基因的形式重新表达。这将使我们能够系统地解决在体内和体外表达非常相同的TCR的细胞的功能，但在有或没有CTSL的情况下都是积极选择的。此外，我们还将讨论CD4T细胞的功能适合性如何通过依赖CTSL的pMHC配体的阳性选择来印记的生化和转录基础。

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.