(807) Relationship between CAR spatial organization and CAR T cell function revealed by imaging flow cytometry and transcriptomic profiling

Introduction/Rationale: Chimeric antigen receptor (CAR) T cell therapies have revolutionized treatment for hematologic cancers, yet precise detection and characterization by flow cytometry remain challenging. Using high-sensitivity 1-step CAR detection reagent, we characterized CAR expression in primary T cells.

Methods: Human T cells were isolated from peripheral blood mononuclear cells, stimulated with anti-CD3/CD28 Dynabeads and transduced with a lentiviral vector encoding a B-cell maturation antigen (BCMA)-specific CAR. The transduced cells were further expanded in culture for 2 weeks prior to cryopreservation. Upon thawing, cells were rested overnight in IL-2-supplemented media before being re-stimulated in co-cultures assays with the U266 target cell line and/or subjected to spectral and imaging flow cytometry.

Results: Spectral flow cytometry enabled clear discrimination of CAR T cells spiked into donor PBMCs from endogenous T cells, allowing detailed profiling of activation, exhaustion, and memory phenotypes. Imaging flow cytometry with BD FACSDiscover™ S8 and BD CellView™ Technology revealed two distinct CAR spatial patterns: circular and polarized. Functional assays with BCMA-CAR T cells showed antigen-specific degranulation, with CD107a externalization co-localizing with CAR in cells exhibiting the circular pattern. These cells also displayed G1-quiescent transcriptional signatures, indicating readiness for activation and enrichment of immune response pathways. In contrast, polarized CAR+ subsets expressed higher levels of MKI67, HMGB1, and cell cycle genes, consistent with proliferative activity.

Conclusion: These findings suggest CAR spatial organization correlates with functional states and demonstrate the power of integrating advanced flow and imaging cytometry to resolve CAR T cell phenotypes and spatial dynamics with high precision.
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