Cytotoxic Efficiency of Human CD8⁺ T Cell Memory Subtypes

Immunological memory is important to protect humans against recurring diseases. Memory CD8⁺ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8⁺ T cells (T_EM) and central memory CD8⁺ T cells (T_CM), are well defined, single T_EM and T_CM cell cytotoxicity has not been quantified. To quantify cytotoxic efficiency of T_EM and T_CM, we developed a FRET-based single cell fluorescent assay with NALM6 target cells which allows analysis of target cell apoptosis, secondary necrosis following apoptosis, and primary necrosis after T_EM- or T_CM-target cell contact. Both, single cell and population cytotoxicity assays reveal a higher cytotoxic efficiency of T_EM compared to T_CM, as quantified by target cell apoptosis and secondary necrosis. Perforin, granzyme B, FasL, but not TRAIL expression are higher in T_EM compared to T_CM. Higher perforin levels (likely in combination with higher granzyme levels) mediate higher cytotoxic efficiency of T_EM compared to T_CM. Both, T_EM and T_CM need the same time to find their targets, however contact time between CTL and target, time to induce apoptosis, and time to induce secondary necrosis are all shorter for T_EM. In addition, immune synapse formation in T_EM appears to be slightly more efficient than in T_CM. Defining and quantifying single T_EM and T_CM cytotoxicity and the respective mechanisms is important to optimize future subset-based immune therapies.