The role of TCF-1 in early fate decisions of memory T cells

连续性T细胞免疫疗法需要自体T细胞的离体基因工程，以能够在重新输注到癌症患者中时有效地识别和消除癌细胞。这些疗法在血液学恶性肿瘤中显示出显著的临床成功，并且两种嵌合抗原受体（CAR）T细胞疗法迄今已被欧洲药品管理局批准。然而，复发仍然是一个重要的问题，部分原因是长期CAR T细胞持续性差。在小鼠模型和临床试验中，用记忆T细胞富集过继免疫疗法已被证明可显著改善T细胞持久性和长期肿瘤控制。T细胞命运的渐进分化模型认为，T细胞分化通过一个分层树进行，从幼稚T细胞开始，然后是潜在的长寿但弱细胞毒性记忆T细胞，最后是短命但高细胞毒性效应T细胞。T细胞因子1（TCF 1）是建立和维持记忆T细胞特征的关键转录因子，近年来作为可以调节以增加CAR T细胞输注中的记忆特征的因子获得了重大的科学兴趣。在幼稚T细胞中，TCF 1促进与记忆命运相关的染色质位点的可接近性，但在效应T细胞分化过程中，TCF 1表达不可逆地丧失，许多这些染色质位点变得不可接近。只有那些在T细胞活化后的分裂中保持TCF 1表达的细胞才能成为长寿命的记忆T细胞，TCF 1在记忆T细胞的初始命运决定、维持和功能中的确切功能尚不清楚。阐明这些功能具有极大的临床意义，因为已知目前的CAR T细胞制造实践使细胞命运偏向效应子，并且了解可以抵消这一过程的机制可以允许产生具有增强治疗潜力的细胞产物。因此，我们的目标是澄清染色质结构和转录程序直接调控TCF 1。我们将有条件地敲低TCF 1在蛋白质水平上的工程改造的修饰TCF 1标记的人工降解决定子，指导TCF 1对快速降解的E3泛素连接酶系统后，诱导配体的管理。这将使我们能够通过ATAC-seq验证急性TCF 1耗竭对染色质可及性的影响，以及通过新生RNA-seq验证对新生mRNA表达的影响。与先前文献中报道的DNA水平敲除和mRNA水平敲除模型不同，基于degron的蛋白质水平敲除与核重塑的<24小时时间尺度一样快地操作。这消除了TCF 1敲低的长期次级效应的噪音，使我们能够第一次精确地确定TCF 1在T细胞分化的选定阶段的主要功能。我们还可以在单细胞水平上进行这些实验，以便能够根据细胞在细胞周期中的位置对细胞进行排序，因为这可能会影响染色质结构和转录组。我们将在人T淋巴细胞系Jurkat中进行这些实验。

Adoptive T cell immunotherapies entail the ex vivo genetic engineering of autologous T cells to be able to effectively identify and eliminate cancer cells upon reinfusion into cancer patients. These therapies have shown significant clinical success in haematological malignancies and two chimeric antigen receptor (CAR) T cell therapies have thus far been approved by the European Medicines Agency. However, relapse remains a significant issue, partly as a result of poor long-term CAR T cell persistence. Enriching adoptive immunotherapies with memory T cells has been demonstrated to significantly improve T cell persistence and long-term tumour control in both mouse models and clinical trials. The progressive differentiation model of T cell fate holds that T cell differentiation proceeds via a hierarchical tree beginning with naïve T cells, followed by potentially long-lived but weakly cytotoxic memory T cells and finally short-lived but highly cytotoxic effector T cells. T cell factor 1 (TCF1) is a key transcription factor in the establishment and maintenance of memory T cell character and has gained significant scientific interest in recent years as a factor which could be modulated to increase memory character in CAR T cell infusions. In naïve T cells TCF1 promotes the accessibility of chromatin sites associated with memory fate, but during the course of effector T cell differentiation TCF1 expression is irreversibly lost and many of these chromatin sites become inaccessible. Only those cells which maintain TCF1 expression in the divisions following T cell activation become long-lived memory T cells.The precise functions of TCF1 in the initial fate determination, maintenance and function of memory T cells remain unclear. Elucidating these functions is of great clinical interest as current CAR T cell manufacturing practices are known to bias cell fate towards effectors, and understanding the mechanisms which can counteract this process could allow the generation of cell products with enhanced therapeutic potential. Therefore, our aim is to clarify the chromatin structures and transcriptional programs directly regulated by TCF1. We will conditionally knock down TCF1 at the protein level by engineering a modified TCF1 tagged with an artificial degron which directs TCF1 towards rapid degradation by the E3 ubiquitin ligase system upon the administration of an inducing ligand. This will allow us to characterise the effects of acute TCF1 depletion on chromatin accessibility by ATAC-seq, and on nascent mRNA expression by nascent RNA-seq. Unlike DNA-level knockout and mRNA-level knockdown models reported in prior literature, degron-based protein-level knockdowns operate as rapidly as the <24h timescales of nuclear remodelling. This eliminates noise from the longer-term secondary effects of TCF1 knockdown, allowing us for the first time to precisely characterise the primary functions of TCF1 at chosen stages in T cell differentiation. We may also conduct these experiments at the single cell level so as to be able to order cells according to their position in the cell cycle, as this is likely to impact chromatin architectures and transcriptomes. We will conduct these experiments in the human T lymphocyte cell line Jurkat.