多发性骨髓瘤治疗中PD-1表达上调致靶向BCMA的CAR-T细胞耗竭的机制研究

CAR-T细胞耗竭是CAR-T治疗多发性骨髓瘤患者无效或复发的原因之一。我们前期发现相应患者出现了靶向BCMA CAR-T细胞扩增减弱的耗竭情况及程序性死亡受体-1（PD-1）的高表达，体外实验证实随着细胞杀伤能力减弱也会出现PD-1的高表达，提示PD-1表达上调参与CAR-T细胞耗竭，但具体机制不明。在耗竭的靶向BCMA CAR-T细胞内我们发现pSTAT1磷酸化水平减低，提示JAK/STAT1信号通路改变可能是导致CAR-T细胞耗竭的原因之一。本项目拟用流式细胞术、转录组测序等技术方法探究JAK/STAT1改变是否通过影响细胞增殖周期、细胞因子分泌等促进CAR-T细胞的耗竭。此外，我们将用ChIP-seq、WGBS测序等技术对耗竭的、PD-1沉默的BCMA CAR-T细胞进行测序和功能分析，并在体内外模型中验证，探究其他信号通路是否参与相应机制，为CAR-T耗竭提供新的理论及实验依据。

Chimeric antigen receptor-modified T cells (CAR-T) exhaustion is a major attribution to relapse or nonresponse to CAR T therapy. Over expression of programmed cell death protein 1 (PD-1) inhibit the anti-tumor capacity of CAR-T cells after the ligation of PD-1 with its ligands. Chimeric antigen receptor-modified T cells (CAR-T) exhaustion is a major attribution to relapse or nonresponse to CAR T therapy in multiple myeloma. We have previously found that patients with multiple myeloma who are ineffective or relapsed have depleted anti-BCMA CAR-T cell expansion and high expression of programmed death receptor-1 (PD-1). With the weakened cell killing ability, high expression of PD-1 will also appear, suggesting that the upregulation of PD-1 expression is involved in CAR-T cell exhaustion, but the specific mechanism is still unclear. In exhausted anti-BCMA CAR-T cells, we found that the phosphorylation level of STAT1 was reduced, suggesting that the JAK / STAT1 signaling pathway may be one of the causes of CAR-T cell exhaustion. This project intends to use flow cytometry, transcriptome sequencing and other technical methods to explore whether JAK / STAT1 changes can promote the exhaustion of CAR-T cells by affecting the cell proliferation cycle and cytokine secretion. In addition, we will use transcriptome sequencing, chromatin immunoprecipitation, and genome-wide methylation sequencing technology to sequence and analyze the exhausted CAR-T cells and PD-1KD BCMA CAR-T cells in vivo and in vitro models, to verify and further explore whether other signaling pathways are involved in CAR-T cell exhaustion, and provide new theoretical and experimental basis for CAR-T exhaustion.