Polychromatic profiling of the Vδ2 T-cell subsets. (A) 5-color flow cytometry discriminates between different subsets of naive and memory Vδ2 T cells from human PBMCs. Sequential gating using morphology and TCR expression defines noneffector and effector Vδ2⁺ cell subsets according to CD27 and CD62L expression, which are respectively subdivided according to expression of CD45RA and CD16. (B) After 6 hours of culture of PBMCs with phosphoantigen, intracellular staining for perforin and IFN-γ followed by 7-color flow cytometry as described in “Materials and methods” defines 2 functionally distinct subsets of effector memory Vδ2 T cells by showing that Vδ2T_EMh are IFN-γ–producing lymphocytes with the TCRVδ2⁺CD27^– CD62L^–CD45RA^– CD16^– phenotype, whereas Vδ2T_EMRA are perforin-producing lymphocytes with the TCRVδ2⁺CD27^– CD62L^–CD45RA⁺CD16⁺ phenotype. (C) Frequency of Vδ2T_EMh and Vδ2T_EMRA cells in PBMCs from 4 representative healthy donors and 2 cultured cell lines.