Cytotoxic efficiency of human CD8+T cell memory subtypes

Immunological memory is an important concept 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 relatively well defined, single T EM and T CM cell cytotoxicity has not been quantified. Here, we analyze human CD8 + subtype distribution following SEA stimulation and quantify the expression of death mediators, including perforin, granzyme B, FasL and TRAIL. We find higher perforin, granzyme B and FasL expression in T EM and compared to T CM . To quantify single T EM and T CM cytotoxicity, we develop a FRET-based fluorescent assay with NALM6 target cells stably transfected with a GFP-RFP FRET construct based on a caspase-cleavage sequence (apoptosis sensor Casper-GR). Applying this assay, T EM or T CM induced target cell apoptosis or necrosis can be quantified. We find that single T EM are much more effective than T CM in killing their targets mainly by apoptosis and secondary necrosis. The reason for this is the higher perforin expression and on a more efficient lytic immunological synapse during T EM -target contact compared to T CM -target contact. Defining and quantifying single T EM and T CM cytotoxicity and the respective mechanism should be helpful to optimize future subset-based immune therapies.