Single-cell CAR T atlas reveals type 2 function in 8-year leukaemia remission

Despite a high response rate in chimeric antigen receptor (CAR) T therapy for acute lymphocytic leukemia (ALL), approximately 50% of patients relapse within the first year, representing an urgent question to address in the next stage of cellular immunotherapy. To investigate the molecular determinants of ultra-long CAR T persistence, we obtained single-cell multi-omics sequencing data from 695,819 pre-infusion CAR T cells at the basal level or upon CAR-specific stimulation from 82 pediatric ALL patients enrolled in the first two CAR T ALL clinical trials and 6 healthy donors. We identified that elevated type-2 functionality in CAR T infusion products was significantly associated with patients maintaining a median B-cell aplasia duration of 8.4 years. Analysis of ligand-receptor interactions unveiled that type-2 cells regulate a distinct dysfunctional population, and the addition of IL-4 during antigen-specific activation alleviates CAR T cell dysfunction while enhancing functional fitness at both transcriptomic and epigenomic levels. Serial proteomic profiling of post-infusion sera revealed a higher level of circulating type-2 cytokines in 5-year or 8-year relapse-free responders. In a leukemic mouse model, type-2 high CAR T products demonstrated superior expansion and antitumor activity particularly upon leukemia rechallenge. Restoring antitumor efficacy in type-2 low CAR T cells was attainable by enhancing their type-2 functionality, either through incorporating IL-4 into the manufacturing process or priming manufactured CAR T products with IL-4 prior to infusion. Our findings provide key insights into the mediators of CAR T longevity and suggest potential therapeutic strategies to sustain long-term remission by boosting type-2 functionality in CAR T cells.