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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.

Cytotoxic Efficiency of Human CD8+ T Cell Memory Subtypes