Chimeric antigen receptor-engineered (CAR)-T cell therapy represents one of the most promising strategies of cancer treatment, and the function and persistence of CAR-T cells in vivo dictate the therapeutic success. Understanding the heterogeneity of CAR-T cells at single-cell resolution and deciphering the functional sub-populations and their dynamic changes over time post-infusion that contribute to long-term response or resistance has a crucial implication for the next generation of CAR-T cell therapy. We collected 11 samples of pre-infusion CAR-T products (pre-IP) (dubbed as stage T0) as well as 49 samples of peripheral blood at the peak (T1, the peak of CAR-T cells expansion within one month), early (T3 or T4.5) and late (T6 to T15) stages from 26 B-ALL patients, who received CAR19/22 T-cell cocktail therapy. The functional status of CAR-T cells in vivo at the time of sampling was judged as effective if patients were in B-cell aplasia, or ineffective if patients met the criteria of B-cell recovery. Totally, 36 effective samples and 10 ineffective samples were obtained. Single CAR-T cells with immunophenotype of CD3 +CAR + and expression of CAR sequences were subjected to modified STRT-seq (Figure a).