Image analysis of mitochondria during T lymphocyte and chimeric antigen receptor (CAR) effector responses.

Mitochondria play a central role in cellular metabolism, ATP production, and redox homeostasis, all of which are essential for sustaining T lymphocyte and CAR-T cell effector functions. Mitochondrial dysfunction has been linked to T cell and CAR-T cell exhaustion, reduced cell expansion capacity, and impaired tumor clearance. To investigate the contribution of mitochondria to T lymphocyte and CAR-T cell functionality, this protocol leverages advanced imaging analysis techniques to assess different parameters related to mitochondrial dynamics, structure and function, which may serve as indicators of both metabolic activity and the exhausted state of T cells. Specifically, the protocol focuses on measuring mitochondrial morphology, energization state, and mitochondrial translocation in the three-dimensional space. The image-based methodologies described in this protocol will contribute to a deeper understanding of mitochondrial regulation during T-cell and CAR-T cell responses. This knowledge will facilitate the identification of key metabolic vulnerabilities and support the refinement of therapeutic strategies to enhance T lymphocyte cytotoxic potential, ultimately improving clinical outcomes in T cell-based adoptive cancer immunotherapy.