Mingqi Hou, Wenjun Zhang, Ziyue Qi, Guiming Li, Husheng Mei, Shuang Qi, Rui Jin, Yuanzhang Zhao, Xiaosong Tang, Bing Xiu, Xi Chen, Yuanzhang Zhao, Chen Hu, Changlin Qian, Xiuchun Li, Zhan Xu, Yongfei Chen, Chao Wu, Beilei Wang, Lejin Yan, Dan Li, Yushan Huang, Rui Liang, Aoli Wang, Jing Liu, Wenchao Wang, Bin Li, Jun Long, Ping Li, Aibin Liang, Qingsong Liu, Jing Yang
Chimeric antigen receptor (CAR)-T cell therapy has transformed treatment of relapsed/refractory DLBCL, yet resistance driven by regulatory T cells (Tregs) limits its efficacy. Here we identify Timosaponin AIII (TAIII), a clinical-stage natural product, as an effective modulator of CAR-T function that depletes CAR-Tregs while enhancing effector activity. Mechanistically, TAIII acts as an allosteric A2AR inhibitor by competing with cholesterol, suppressing CREB-dependent FoxP3 transcription and disrupting the A2AR-Treg axis. Ablation of A2AR or Tregs in vitro and in vivo abolishes TAIII activity, confirming specificity. Furthermore, TAIII reduces intratumoral Tregs, increases CD8⁺ T cells infiltration, and potentiates PD-1 blockade in solid tumor models. Importantly, TAIII promotes central memory T-cell formation and enhances CAR-T cytotoxic cytokine secretion. Combining or pretreating CAR-T cells with TAIII markedly improves antitumor efficacy and prevents late relapse across preclinical models. These findings establish TAIII as a combinatorial strategy to deplete CAR-Tregs, enhance CAR-T activity, and extend therapeutic durability. Standard of care CD19-targeting CAR-T therapy for Relapsed-Refractory Diffuse Large B Cell Lymphoma (r/r DLBCL) faces frequent resistance often driven by regulatory T cell (Treg) activation. This study identifies Timosaponin AIII (TAIII) as A2AR inhibitor for enhanced CAR-T activity while depleting CAR-Treg formation.