Functional and phenotypical characterization of peptide-specific PLZF+ innate-like T cells

肽特异性PLZF+先天样（PIL） T细胞是最近发现的一种具有先天样特征的T细胞亚群。与不变自然杀伤T细胞（iNKT）类似，PIL T细胞在胸腺双阳性（DP）胸腺细胞上被积极选择，并在胸腺发育过程中分化为三个功能效应亚群。然而，与iNKT细胞识别脂质抗原相反，PIL T细胞识别肽抗原。在最近的一项研究中，我们已经产生了一种新的转基因小鼠系，允许在稳态条件下特异性扩增PIL T细胞。这项研究阐明了PIL T细胞选择的机制，但并没有深入分析它们的T细胞受体（TCR）库多样性，也没有深入分析它们在稳态或疾病条件下的基本功能。在本项目中，我们将对分选的PIL T细胞进行高通量单细胞TCR测序和转录组分析，这将使我们能够确定PIL T细胞的发育轨迹，在转录组水平上比较PIL T细胞和iNKT细胞，并探索PIL T细胞TCR库的多样性。这些数据对于确定PIL T细胞池中存在的特征性TCR克隆至关重要。此外，我们将通过产生从PIL T细胞TCR库中选择表达TCR序列的单克隆T细胞的逆转录小鼠，研究是否一个定义的TCR特异性可以使发育中的胸腺细胞进入先天样T细胞发育途径。此外，我们将建立一组表达定义的PIL T细胞TCR序列的NFAT-GFP报告细胞系。我们将利用该系统筛选抗原呈递细胞从各种组织抗原识别在体外试验。因此，这些实验将有助于我们阐明参与PIL T细胞选择和激活的推定肽抗原的系统分布和丰度。最后，我们将研究PIL T细胞在外周器官的分布和定位，并探讨其在黑色素瘤肿瘤模型中的抗肿瘤功能。通过监测浸润淋巴细胞的组成和激活状态，我们将了解PIL T细胞是否可以浸润实体瘤组织，以及它们是否可以调节局部抗肿瘤免疫反应。

Peptide-specific PLZF+ innate-like (PIL) T cells are a recently described T cell subset exhibiting innate-like features. Reminiscent of invariant natural killer T (iNKT) cells, PIL T cells are positively selected on double positive (DP) thymocytes and differentiate into three functional effector subsets during their development in the thymus. However, in contrast to iNKT cells, which recognize lipid antigens, PIL T cells recognize peptide antigens.In a recent study, we have generated a novel transgenic mouse line allowing specific expansion of PIL T cells under steady state conditions. This study elucidated the mechanisms governing PIL T cell selection but did not aim to provide in depth analysis of their T cell receptor (TCR) repertoire diversity nor of their essential function(s) under steady state or disease conditions.In this project, we will perform high-throughput single cell TCR sequencing and transcriptome analysis of sorted PIL T cells, which will allow us to identify PIL T cell developmental trajectories, compare PIL T cells to iNKT cells at transcriptome level and explore the diversity within the PIL T cell TCR repertoire. These data will be crucial to determine characteristic TCR clones present in the PIL T cell pool. Further, we will examine if a defined TCR specificity can commit developing thymocytes to the innate-like T cell developmental pathway by generating retrogenic mice harboring monoclonal T cells expressing TCR sequences selected from the PIL T cell TCR repertoire. In addition, we will establish a set of NFAT-GFP reporter cell lines expressing defined PIL T cell TCR sequences. We will exploit this system to screen antigen presenting cells from various tissues for antigen recognition in in vitro assays. Therefore, these experiments will help us to elucidate the systemic distribution and abundance of putative peptide antigens involved in PIL T cell selection and activation. Finally, we will investigate PIL T cell distribution and localization in peripheral organs, and inquire for anti-tumor function in a melanoma tumor model. By monitoring the composition and the activation status of infiltrating lymphocytes, we will gain insights on whether PIL T cells can infiltrate solid tumor tissues and if they can modulate local anti-tumor immune responses.