High-dimensional temporal mapping of CAR T cells reveals phenotypic and functional remodeling during manufacturing.

Despite the notable success of chimeric antigen receptor (CAR) T cell therapies in hematological malignancies, clinical outcomes remain variable, making it critical to understand how manufacturing influences product composition and function. We developed a 36-marker spectral flow cytometry panel enabling integrated profiling of phenotypic, metabolic, and functional attributes across CAR T cell production. Mid-expansion products (day 5) retained stem-like, metabolically active CD4 Th1 subsets with high proliferative capacity, whereas prolonged culture (day 10) enriched terminally differentiated CD8 Tc1 cells and NK-like T cell populations. CAR and CAR T cells showed similar differentiation trajectories, suggesting that culture conditions may have a larger impact on phenotypic remodeling than CAR integration. Upon antigen encounter and restimulation, day 5 and day 10 products showed comparable cytotoxicity, while differing in their activation and checkpoint profiles. Cryopreservation modestly affected stem cell memory, activation, and metabolic markers but preserved overall phenotype and cytotoxic function. These findings establish a high-dimensional framework for mapping CAR T cell dynamics to support manufacturing optimization and next-generation cell therapy design.