基于ATAC-Seq技术的肺癌浸润T淋巴细胞表观调控机制研究

Immune checkpoints play a key role in precise control of the immune response. In recent years, immune checkpoint inhibitors were found to significantly increase the recognition of T lymphocytes. However, such inhibitors fail to form a lasting response in most lung cancer patients and appear to progress. It was found that the failure of checkpoint inhibitors may be the result of exhaustion of tumor infiltrating T lymphocytes (TILs). However, there was still lack of comprehensive research on the epigenetic regulation mechanism of TILs. Based on recent advances and our previous research results, we propose the hypothesis: the epigenetic regulation of TILs in lung cancer could cause the exhaustion of T lymphocytes, which leads to be ineffective of inhibitors, so find the key epigenetic targets will be significant to the precise diagnosis and intervention of lung cancer. Therefore, we intend to base on ATAC-Seq technology, and analyze the relationship between the epigenetic regulatory characteristics of infiltrating CD8+T lymphocytes and tumor escape, and find out the epigenetic targets for the exhaustion of the infiltrating CD8+T lymphocytes. Then we would culture T lymphocytes in vitro, and take CRISPR-cas9 technology to knock down target genes, so as to observe changes in chromatin states, transcription factor binding sites and signaling pathways of CD8+ T lymphocytes. Finally we would study the exhaustion mechanism of TILs epigenetic regulation comprehensively.

免疫检查点对于实现免疫反应的精准控制具有关键作用，近年来发现免疫检查点阻断剂可以明显增加T淋巴细胞对肿瘤的识别，但是此类阻断剂在大多数肺癌患者体内最终未能形成持久的反应而出现病情进展。研究发现阻断剂失效可能是肿瘤浸润性T淋巴细胞（TILs）表观调控功能耗竭的结果。然而，目前仍缺乏系统深入对肺癌TILs表观调控机制的研究。我们提出假说：肺癌TILs表观调控的改变，可引起T淋巴细胞功能耗竭，导致免疫逃逸，而发现关键的调控靶点，将对肺癌早期精准诊断及干预有着重要的意义。为此，我们拟采用ATAC-Seq测序技术，分析肺癌浸润CD8+T淋巴细胞表观调控特征，寻找引起肺癌浸润CD8+T淋巴细胞功能耗竭表观调控靶点；在体外培养T淋巴细胞，采用CRISPR-cas9技术敲除靶点基因，寻找CD8+T淋巴细胞染色质状态，转录因子结合位点及信号通路的改变，研究TILs表观调控功能耗竭导致肺癌发生发展的作用机制。

肿瘤免疫治疗在肺癌中的效果不一，肿瘤浸润淋巴T细胞的组成部分决定了肿瘤免疫治疗的效果。肿瘤微环境中的CD8 T细胞由于长期接受抗原刺激，会出现被称为“耗竭”的失能状态。肺癌的浸润CD8T细胞群体还包含两群与耗竭细胞可能存在状态转换关系的“前耗竭”细胞。本研究共纳入50例肺癌手术患者，分别收集肺癌组织，癌旁组织以及术前外周血，并采用流式细胞仪分离肺癌组织，癌旁组织及外周血中的CD45免疫细胞，分别进行ATAC-seq和单细胞RNA（scRNA-seq）建库测序，以期寻找CD8T淋巴细胞功能耗竭的关键调控靶点。本研究中我们鉴定了三类发挥免疫抑制作用的免疫细胞：具有衰竭表型的CD8T细胞、具有促炎作用M2表型的肿瘤相关巨噬细胞（TMA）和具有促肿瘤特性的调节性B细胞（Bregs）。我们发现可能介导CD8T细胞中的转录因子ONECUT2和ETV4，然后结合ATAC-seq测序技术，我们继续针对特定位点进行深入分析，并采用临床病理及预后数据进行验证，重点采用细胞和动物实验，研究CD8T耗竭的发生机制。本研究还采用单细胞转录组测序分析，寻找到耗竭的关键细胞亚群并采用拟时序分析，研究免疫细胞耗竭的路径；并进一步研究细胞间内的相互作用，从而绘制析基因表达调控网络。本文章通过单细胞测序揭示肺癌中肿瘤浸润淋巴T细胞的组成、谱系以及功能状态，为临床肺癌患者的免疫治疗提供新的基础。