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

过继T细胞免疫疗法需要对自体T细胞进行体外基因工程，以便在回输给癌症患者时能够有效地识别和消除癌细胞。这些疗法在治疗血液恶性肿瘤方面取得了显著的临床成功，到目前为止，欧洲药品管理局已经批准了两种嵌合抗原受体(CAR)T细胞疗法。然而，复发仍然是一个重要的问题，部分原因是CAR T细胞的长期持久性较差。在小鼠模型和临床试验中，用记忆T细胞丰富过继免疫疗法已被证明显著改善T细胞的持久性和长期肿瘤控制。T细胞命运的递进分化模型认为，T细胞的分化是通过一个层次树进行的，首先是幼稚的T细胞，然后是潜在的长寿命但弱细胞毒性的记忆性T细胞，最后是短暂但高度细胞毒性的效应T细胞。T细胞因子1(T cell factor1，TCF1)是建立和维持记忆T细胞特性的关键转录因子，近年来在CAR T细胞输注中作为一种可调节增强记忆特性的因子引起了科学家的极大兴趣。在幼稚T细胞中，TCF1促进了与记忆命运相关的染色质部位的可及性，但在效应器T细胞分化过程中，TCF1的表达不可逆转地丢失，许多这些染色质部位变得不可接近。只有那些在T细胞激活后的分裂中保持TCF1表达的细胞才能成为长寿的记忆T细胞，TCF1在记忆T细胞最初命运的决定、维持和功能中的确切功能尚不清楚。阐明这些功能具有很大的临床意义，因为目前CAR T细胞的制造实践已知将细胞命运偏向效应者，了解可以抵消这一过程的机制可能有助于产生具有增强治疗潜力的细胞产品。因此，我们的目标是阐明TCF1直接调控的染色质结构和转录程序。我们将有条件地在蛋白质水平上敲除TCF1，方法是设计一种标记有人工降解物的修饰TCF1，在给予诱导配体的情况下，该降解物引导TCF1通过E3泛素连接酶系统快速降解。这将使我们能够表征急性TCF1耗竭对ATAC-seq染色质可及性的影响，以及通过新生RNA-seq对新生RNA表达的影响。与先前文献中报道的DNA水平的敲除和mRNA水平的敲除模型不同，基于退化的蛋白质水平的敲除作用与核重塑的24小时时间尺度一样迅速。这消除了TCF1基因敲除的长期次要影响的噪音，使我们第一次能够准确地描述TCF1在T细胞分化的选定阶段的主要功能。我们也可以在单细胞水平上进行这些实验，以便能够根据细胞周期中细胞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本。我们将在人类T淋巴细胞株Jurkat上进行这些实验。

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.