Defining potency of CAR⁺ T cells: Fast and furious or slow and steady

Genetically engineered T cells that express chimeric antigen receptors (CAR⁺) are heterogeneous and thus, understanding the immunotherapeutic efficacy remains a challenge in adoptive cell therapy. We developed a high-throughput single-cell methodology, Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to monitor interactions between immune cells and tumor cells in vitro. Using TIMING we demonstrated that CD4⁺ CAR⁺ T cells participate in multi-killing and benefit from improved resistance to activation induced cell death in comparison to CD8⁺ CAR+ T cells. For both subsets of cells, effector cell fate at the single-cell level was dependent on functional activation through multiple tumor cells.