Live Imaging of CAR T Cell Ca²⁺ Signals in Tumor Slices Using Confocal Microscopy

The immune synapse is a key structure organizing T-cell activation against foreign entities, such as cancer cells expressing neoantigens. One crucial step in this activation cascade is the intracellular Ca²⁺ ([Ca²⁺]_i) response that shapes T cells for proliferation, differentiation, and cytotoxicity. The development of calcium probes coupled to real-time fluorescence microscopy has allowed a close study of this phenomenon in vitro. Such systems have provided valuable insights on the consequences of Ca²⁺ responses on T cells, including cytotoxicity and cytoskeletal remodeling. However, in vitro models do not recapitulate the tissue architecture that T cells come in contact with in vivo. Thus, there is a growing necessity for better understanding the factors influencing Ca²⁺ response in T cells including in genetically modified T cells (e.g., CAR T cells). In this methodology chapter, we describe an experimental system to measure [Ca²⁺]_i signals of CAR T cells loaded with the calcium probe Fluo-4 on fresh tumor slices. Combined with confocal fluorescent imaging, this model offers an approach to image early T-cell activation in a three-dimensional (3D) tissue environment.