Relationship between CAR spatial organization and CAR T cell function revealed by imaging flow cytometry and transcriptomic profiling 2260438

Abstract

Abstract Introduction 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. BD flow cytometers are Class 1 Laser Products. For Research-Use Only. Not for use in diagnostic or therapeutic procedures. ©2025 BD. All rights reserved. NPM-7267. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)