Type I Interferon Signaling via the EGR2 Transcriptional Regulator Potentiates CAR T cell-intrinsic Dysfunction (ATAC-Seq)

Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable success in the treatment of hematopoietic cancers, but resistance is common, and efficacy is limited in solid tumors. We demonstrate that CAR T-cells autonomously propagate epigenetically-programmed type I interferon signaling through chronic stimulation or antigen-independent tonic activation, which progressively hampers antitumor effector function. EGR2 transcriptional regulator knockoutnot only blocks this type I interferon-mediated inhibitory program, but also independently expands early memory CAR T-cells with improved efficacy against liquid and solid tumors. The protective effect of EGR2 deletion in CAR T-cells against chronic antigen-induced dysfunction can be overridden by interferon-ß exposure, suggesting that EGR2 ablation suppresses CAR T-cell dysfunction through inhibition of type I interferon signaling. Finally, enrichment of an EGR2 gene signature is a clinical biomarker for type I interferon-associated CAR T-cell failure and shorter overall patient survival. These findings conceptually connect prolonged CAR T-cell activation with deleterious immunoinflammatory signaling and point to an EGR2-type I interferon axis as a therapeutically amenable biologic system. Overall design: AAVS1 and EGR2 knockout PSMA CAR T-cells produced from two different healthy subjects were isolated from the in vitro “stress test” after multiple consecutive rounds of antigen stimulation. Single-cell multiome-seq libraries were prepared using Chromium Single Cell Multiome ATAC and Gene Expression Reagent Kits V1 (10X Genomics).

嵌合抗原受体（Chimeric antigen receptor, CAR）T细胞疗法在血液系统恶性肿瘤的治疗中已取得显著成效，但耐药现象频发，且在实体瘤中的疗效仍存在局限。本研究证实，CAR T细胞可通过慢性抗原刺激或抗原非依赖性基础活化，自主激活表观遗传调控的I型干扰素（type I interferon）信号通路，进而逐步削弱其抗肿瘤效应功能。EGR2转录调节因子的敲除，不仅可阻断该I型干扰素介导的抑制性程序，还可独立扩增早期记忆性CAR T细胞，提升其对血液系统肿瘤与实体瘤的治疗效果。干扰素-β（interferon-β）暴露可抵消EGR2缺失在CAR T细胞中对抗慢性抗原诱导功能障碍的保护作用，提示EGR2敲除通过抑制I型干扰素信号通路，缓解CAR T细胞功能异常。最后，EGR2基因特征的富集可作为I型干扰素相关CAR T细胞衰竭及患者总生存期缩短的临床生物标志物。上述研究发现从概念上将持续性CAR T细胞活化与有害免疫炎症信号通路相联系，并指明EGR2-I型干扰素轴可作为可靶向干预的生物学系统。总体实验设计：从2名健康受试者体内制备AAVS1敲除及EGR2敲除的PSMA靶向CAR T细胞，经多轮连续抗原刺激后，从体外“压力测试”体系中分离目标细胞。采用Chromium单细胞多组学ATAC与基因表达试剂盒V1（10X Genomics）构建单细胞多组测序文库。