Three memory subsets of human CD8+ T cells differently expressing three cytolytic effector molecules

Multicolor flow cytometric analysis for the expression of three effector molecules, i.e., perforin (Per), granzyme A (GraA), and granzyme B (GraB), in human CD8(+) T cells demonstrated that they included five subpopulations, implying the following pathway for the differentiation of CD8(+) T cells: Per(-)GraA(-)GraB(-)-> Per(-)GraA(+)GraB(-)-> Per(low)GraA(+)GraB(-)-> Per(low)GraA(+)GraB(+)-> Per(high)GraA(+)GraB(+). The analysis of the expression of these molecules in the subsets classified by the combination of the expression of CCR7 and CD45RA or by that of CD27, CD28, and CD45RA showed that functional CD8(+) T cell subsets could be partially identified by these phenotypic classifications. However, the functional subsets could be precisely identified by the classification using five cell surface markers or three cell surface markers and three cytolytic molecules. Per(-)GraA(-)GraB(-) and Per(-/low)GraA(+)GraB(-) cells were predominantly found in CCR5(-)CCR7(+) and CCR5(high/low)CCR7(-) subsets, respectively, of CD8(+) T cells expressing the CD27(+)CD28(+)CD45RA(-) phenotype, whereas Per(low)GraA(+)GraB(+) cells were found in the CCR5(low)CCR7(-) subset of those expressing this phenotype and in a part of the CCR5(-/low)CCR7(-) subset of those expressing the CD27(-/low)CD28(-)CD45RA(-/+) phenotype. Ex vivo EBV-specific CD8(+) T cells, which were Per(low/-)GraA(+)GraB(-/+) cells, hardly or very weakly killed the target cells, indicating that these were not effector T cells. These findings suggest that the Per(-)GraA(-)GraB(-), Per(-/low)GraA(+)GraB(-), and Per(low)GraA(+)GraB(+) cells were central memory, early effector memory, and late effector memory T cells, respectively. Per(-/low)GraA(+)GraB(-) cells gained GraB expression after TCR stimulation, indicating that early effector memory T cells could differentiate into late effector and effector T cells. The present study showed the existence of three memory subsets and the pathway for their differentiation.