In the adaptive immune system, cytotoxic T cells target and kill pathogenic cells through the action of plasma membrane T cell receptors (TCRs). Chimeric antigen receptors (CARs) are engineered to elicit an immune response against tumor-associated antigens through the native TCR signaling pathway. While CAR-T cells have shown the ability to recognize and kill cancer cells, CARs lack the sensitivity of native TCRs. Some clues point toward potential defects in spatiotemporal regulation of CAR vs. TCR signaling. Cell activation through the TCR results in formation of a characteristic bulls-eye pattern of receptors and signaling molecules at the interface between the T cell and antigen-presenting surface. This structure, called the “immune synapse” does not efficiently form when T cells are activated through CARs. Full maturation of the immune synapse occurs after the initial phase of cellular activation, but differences in synapse formation could be reflective of earlier receptor reorganization processes. This study explores the hypothesis that reduced CAR sensitivity at least partially originates in the initial assembly of receptor microclusters when receptors are first engaged with ligand. Here, we employ single-molecule localization microscopy tools to quantify CAR microcluster formation in response to ligand stimulation. Limitations in single-color imaging approaches in fixed cells give rise to overcounting artifacts that dominate measurements of sub-diffraction receptor clusters. We improve upon this using a two-color receptor labeling approach and quantify clustering using pairwise correlation functions. T cells are incubated on antigen-presenting supported lipid bilayers to recapitulate the immune synapse for in vitro imaging. By titrating antigen density, we compare CAR and TCR microcluster formation in response to defined doses of antigen. Ongoing work will connect cluster formation to recruitment of signaling partners to probe differences in CAR and TCR downstream responses.
Pyron et al. (Sun,) studied this question.