Authors:

Phoebe Huang

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About Paper:

Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a powerful approach for treating hematologic malignancies, yet its long-term efficacy is often limited by T cell exhaustion and functional heterogeneity. This study investigates how CAR-T cells engineered with 4-1BB, an FDA- approved co-stimulatory domain used in clinical CAR-T therapy, respond to sustained antigen exposure. CAR-T cells are co-cultured with cancer cells over two weeks to model chronic stimulation. Using dFAST (Digital 34 FluoroSpot Amplification for Spatiotemporal Profiling), our lab's recently developed platform for multidimensional single-cell phenotypic and functional analysis, we performed multiplexed measurements of key effector cytokines granzyme B (GzmB) and interferon-gamma (IFN-γ) to assess cytotoxicity and inflammation. In parallel, cells are classified by CD4/CD8 lineage and differentiation state (naïve, effector terminal, central memory, effector memory), and expression of CD25 and CD69 (activation) and PD-1 (exhaustion) is tracked over time. All parameters are captured simultaneously at single-cell resolution, enabling one-to-one correlation of phenotype, function, and temporal dynamics within the same individual cell. This unified approach allowed us to resolve functional and phenotypic dynamics at single-cell resolution across time. Understanding which subsets retain functionality or are most prone to exhaustion is critical for improving CAR design, guiding cell selection during manufacturing, and enhancing persistence in vivo. These insights are especially relevant as CAR-T therapies expand beyond hematologic cancers into solid tumors, where persistent antigen exposure and immune suppression are more pronounced. Ultimately, identifying markers of potency and durability will help translate CAR-T therapy into more reliable and lasting clinical responses.

Source:

Columbia / Economics-Philosophy / 2028

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Co-authors:

Phoebe Huang