Different functional responses of Vδ2T_EMhand Vδ2T_EMRAlymphocytes. (A) Cytometric panels refer to a representative healthy donor. Left panel shows cells gated for Vδ2 expressing double staining for CD16 and perforin, indicating that both CD16⁺perforin⁺ and CD16^–perforin^– effector cells were equally present in the chosen individual. Freshly isolated PBMCs were stimulated with BrHPP or anti-CD16 for 6 hours and intracellular staining for IFN-γ was performed (right panels). (B) Vδ2T_CM,T_EMh, and T_EMRA T-cell clones were obtained as previously described.²⁸  The cells were then stimulated with BrHPP (100 nM) or with anti-CD16 (10 μg/mL). Supernatants were harvested after 24 hours and IFN-γ was measured by ELISA. Anti-HLAI was used as control. The data are representative of 3 independent ELISA experiments on different donors. Data represent means ± SD. (C) NK cell, Vδ2T_EMh, and T_EMRA T-cell subsets, freshly isolated from a healthy donor, were used for cytotoxic assay against Daudi cells. The graph shows a strong lytic activity exerted by the NK cells (up to 90%), an “intermediate” lytic activity of Vδ2T_EMRA cells (up to 40%), and no cytotoxic effect by Vδ2T_EMh cells. The data are representative of 2 independent experiments. (D) Synaptic transfer by Vδ2T_EMh and Vδ2T_EMRA lymphocytes from bulk PBMCs coincubated for 1 hour with PKH67-labeled Daudi target cells. Using FL1 for PKH67 detection, PKH67⁺ Daudi cells were gated out, the Vδ2 cell subsets were defined by 5-color flow cytometry as in Figure 1, after simultaneous labeling for surface TCRVδ2, CD27, CD62L, CD45RA, and CD16. Numbers give the PKH67 MFI of the specified cells prior to and after coincubation.