Imaging BiTE-Mediated Activation of Primary Human CD8⁺ T Cells

CD8⁺ T cells are a vital branch of the adaptive immune response. They are necessary for an effective antiviral and anticancer immunity, which is mediated through their cytotoxic effector functions. CD8⁺ T cell activation, proliferation, and effector functions are dependent on a complex network of Ca²⁺ signalling pathways involving both Ca²⁺ release and Ca²⁺ entry. Dysregulation of intracellular Ca²⁺ signalling pathways has been linked to immunodeficiencies including severe combined immunodeficiency syndrome (SCID), highlighting the need to study both Ca²⁺ dynamics and cytotoxic effector function in patient-derived CD8⁺ T cells. The following protocol describes a workflow for the isolation of primary human CD8⁺ T cells and their activation using bispecific T cell engagers (BiTEs). This allows for a simultaneous assessment of both Ca²⁺ dynamics and cytotoxicity through a target cell line presenting an epitope recognized by the BiTE.