Dynamic, single-cell monitoring of CAR T cell identity and activation with Raman spectroscopy

Chimeric antigen receptor (CAR) T cell therapies have reshaped treatment for cancers and immune-mediated diseases, yet their safety and efficacy depend on both the proliferation of engineered cells and their dynamic functional state -- features that remain challenging to monitor in real-time clinical settings. Current methods require labels, extensive processing, and provide only static snapshots of cell identity and activation. Here, we introduce a surface-enhanced Raman spectroscopy and machine learning approach that enables label-free single-cell identification of engineered CAR T cells and time-resolved, semi-continuous monitoring of their functional activation state. Using the intrinsic vibrational signatures from live cells, we detect spectral differences resulting from engineered receptor expression in donor-derived CD19- and GD2-targeted CAR T cells (nine and five donors, respectively) with 81-85% donor-level accuracy and resolve dynamic antigen-specific activation trajectories with temporal precision. These capabilities stem from biochemical signatures consistent with processes such as receptor expression, tonic signalling, and immune synapse formation, demonstrating a single method that reports both cellular identity and activation state with biochemical specificity. Our results extend CAR T cell monitoring beyond static phenotyping and establish the potential of SERS-ML analysis for rapid, point-of-care assessment of engineered immune cells.