GSE162975
Single-Cell Decoding of the Clinical Outcome-Associated Diversification and Dynamic  Changes of CAR-T Cells in Patients with B-cell ALL
Cellular evolution and molecular programs of chimeric antigen receptor-engineered  (CAR)-T cells post-infusion are pivotal for developing better treatment strategies. Here,  we constructed a high-precision single-cell transcriptional dynamic landscape of 7,578  CAR-T cells from 26 B-ALL patients and evaluated their long-term therapeutic efficacy.  We demonstrated that the cytotoxic profile rather than memory property was a  favorable biomarker for long-term remission. At the single-cell resolution, we  uncovered the vast heterogeneity of CAR-T cells in vivo and identified eight CAR-T  cell subtypes. Remarkably, the dominance of cytotoxic subtypes was coincident with  long-term remission, while the emergence of subtypes with B-cell transcriptional  features could be detected early and predict relapse. Furthermore, we defined  transcriptional hallmarks of distinct CAR-T cell populations and revealed molecular  changes along computationally-inferred cellular evolution of CAR-T cells in vivo .  Collectively, these results illuminated intrinsic properties for durable response and  provided molecular cues for improving CAR-T immunotherapy. Overall design: Herein, we comprehensively dissected the dynamical changes in the transcriptomic profile of CAR-T cells enriched from IPs and paired peripheral blood samples longitudinally, collected at serial time points post-infusion for up to 15 months, and explored their clinical implications in the long-term response or resistance in 26 B-ALL patients.   In order to obtain highly reliable CAR-T cells, a two-step purification procedure was adopted to purify CAR-T cells from infusion products and peripheral blood samples. Firstly, immunophenotypic CAR-T cells (CD3+ CAR+) were isolated and individually sorted into 96-well plates by Moflo XDP (Beckman Coulter). Then, sorted CAR-T cells were further validated by the amplification of CAR sequence in single-cell polymerase chain reaction (scPCR) before library preparation.