Longitudinal Flow Cytometric Profiling of CAR-T and Endogenous T Cells Following CD19 CAR-T Therapy

Background/Objectives: Longitudinal monitoring after CD19-directed chimeric antigen receptor (CAR) T-cell therapy primarily focuses on CAR-T-cell expansion. However, comprehensive immunophenotypic characterization of both CAR-T and endogenous T cells remains limited. We investigated longitudinal immune dynamics following tisagenlecleucel treatment using multiparameter flow cytometry. Methods: Nineteen patients with relapsed or refractory B-cell malignancies (11 diffuse large B-cell lymphoma and eight B-cell acute lymphoblastic leukemia) receiving tisagenlecleucel were prospectively enrolled. Serial peripheral blood samples collected during the first 28 days after infusion were analyzed for CAR-T-cell kinetics, differentiation status, activation and exhaustion marker expression, longitudinal phenotypic changes, as well as associations with treatment response and immune-related toxicities. Results: CAR-T-cell expansion predominantly peaked on day 7 after infusion. However, in several patients with B-cell acute lymphoblastic leukemia, delayed peak expansion was observed. At peak expansion, CAR-T-cells were enriched for a central memory phenotype and exhibited significantly higher HLA-DR and PD-1 expression than endogenous T cells. During follow-up, the proportion of central memory T-cells progressively declined, whereas the proportion of terminal effector T-cells increased, which was accompanied by reduced expression of HLA-DR, PD-1, and TIGIT. Despite the distinct activation profiles, CAR-T and endogenous T-cells displayed broadly similar differentiation patterns. Exploratory analyses further demonstrated higher absolute CAR-T-cell counts in patients who developed immune effector cell-associated neurotoxicity syndrome. Conclusions: This clinically applicable approach provides immunophenotypic information beyond CAR-T-cell enumeration alone and may facilitate biomarker discovery and longitudinal immune monitoring in patients receiving CAR-T-cell therapy.