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

多肽特异性PLZF+类先天T细胞(PIL)是最近发现的一种具有先天功能的T细胞亚群。与不变的自然杀伤T细胞(INKT)相似，PIL T细胞在双阳性(DP)胸腺细胞上呈阳性选择，并在胸腺发育过程中分化为三个功能效应亚群。然而，与识别脂质抗原的iNKT细胞不同，PIL T细胞识别多肽抗原。在最近的一项研究中，我们培育了一种新的转基因小鼠系，允许在稳定条件下特异性地扩增PIL T细胞。这项研究阐明了PIL T细胞选择的机制，但并不是为了深入分析它们在稳态或疾病条件下的T细胞受体(TCR)谱系的多样性或其基本功能(S)。在这个项目中，我们将对PIL T细胞进行高通量的单细胞TCR测序和转录组分析，这将使我们能够识别PIL T细胞的发育轨迹，在转录水平上比较PIL T细胞和iNKT细胞，并探索PIL T细胞TCR谱的多样性。这些数据对于确定PIL T细胞池中存在的特征TCR克隆至关重要。此外，我们将通过产生含有表达从PIL T细胞TCR谱系中选择的TCR序列的单克隆T细胞的逆转录小鼠，来检验确定的TCR特异性是否可以使发育中的胸腺细胞遵循先天类似T细胞的发育途径。此外，我们还将建立一组表达PIL T细胞TCR序列的NFAT-GFP报告细胞系。我们将利用这一系统从不同组织中筛选抗原提呈细胞，以便在体外检测中识别抗原。因此，这些实验将有助于我们阐明参与PIL T细胞选择和激活的全身性多肽抗原的分布和丰度。最后，我们将研究PIL T细胞在外周器官中的分布和定位，并探讨其在黑色素瘤模型中的抗肿瘤作用。通过监测浸润性淋巴细胞的组成和活化状态，我们将深入了解PIL T细胞是否能够侵袭实体瘤组织，以及它们是否能够调节局部的抗肿瘤免疫反应。

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.