耗竭是影响嵌合抗原受体T（CAR-T）细胞功效的关键因素。我们之前的研究表明，溴结构域蛋白 4 (BRD4) 抑制剂可以改变白血病患者耗尽的 T 细胞的表型和功能。本研究旨在利用单细胞 RNA 测序 (scRNA-Seq) 阐明 BRD4 抑制剂减少 CAR-T 细胞耗竭的机制。通过与 CD123 抗原阳性 MV411 共培养来制备耗竭的 CD123 特异性 CAR-T 细胞细胞。消除 MV411 细胞并上调表面抑制性受体后，用 BRD4 抑制剂 (JQ1) 处理耗尽的 CAR-T 细胞 72 小时。随后分离出 CAR-T 细胞，并进行 scRNA-Seq 来表征 JQ1 处理细胞的表型和功能变化。 JQ1 处理后的 CD8 CAR-T 细胞耗竭比例和 CAR-T 细胞耗竭评分均下降与对照处理的细胞相比。此外，与终末耗尽的 CD8 CAR-T 细胞相比，JQ1 治疗导致初始、记忆和祖细胞耗尽的 CD8 CAR-T 细胞比例更高，并伴有增殖、分化和激活能力增强。此外，通过 JQ1 治疗，幼稚型、记忆型和祖细胞耗尽型 CD8 CAR-T 细胞中的 BATF 活性和表达降低，而 EGR1 活性和表达增加。有趣的是，具有较高 EGR1 和 EGR1 靶基因 ssGSEA 评分，加上较低 BATF 和 BATF 靶基因 ssGSEA 评分的 AML 患者预后最好。我们的研究表明，BRD4 抑制剂可以减少 CAR-T 细胞耗竭并阻断耗竭的 T 细胞末端通过下调 BATF 活性和表达以及上调 EGR1 活性和表达来实现分化，提出了一种提高 CAR-T 细胞疗法有效性的方法。© 2024。作者。

Exhaustion is a key factor that influences the efficacy of chimeric antigen receptor T (CAR-T) cells. Our previous study demonstrated that a bromodomain protein 4 (BRD4) inhibitor can revise the phenotype and function of exhausted T cells from leukemia patients. This study aims to elucidate the mechanism by which a BRD4 inhibitor reduces CAR-T cell exhaustion using single-cell RNA sequencing (scRNA-Seq).Exhausted CD123-specific CAR-T cells were prepared by co-culture with CD123 antigen-positive MV411 cells. After elimination of MV411 cells and upregulation of inhibitory receptors on the surface, exhausted CAR-T cells were treated with a BRD4 inhibitor (JQ1) for 72 h. The CAR-T cells were subsequently isolated, and scRNA-Seq was conducted to characterize phenotypic and functional changes in JQ1-treated cells.Both the proportion of exhausted CD8+ CAR-T cells and the exhausted score of CAR-T cells decreased in JQ1-treated compared with control-treated cells. Moreover, JQ1 treatment led to a higher proportion of naïve, memory, and progenitor exhausted CD8+ CAR-T cells as opposed to terminal exhausted CD8+ CAR-T cells accompanied by enhanced proliferation, differentiation, and activation capacities. Additionally, with JQ1 treatment, BATF activity and expression in naïve, memory, and progenitor exhausted CD8+ CAR-T cells decreased, whereas EGR1 activity and expression increased. Interestingly, AML patients with higher EGR1 and EGR1 target gene ssGSEA scores, coupled with lower BATF and BATF target gene ssGSEA scores, had the best prognosis.Our study reveals that a BRD4 inhibitor can reduce CAR-T cell exhaustion and block exhausted T cell terminal differentiation by downregulating BATF activity and expression together with upregulating EGR1 activity and expression, presenting an approach for improving the effectiveness of CAR-T cell therapy.© 2024. The Author(s).