Proliferative response of γδ T cells to ZA + IL-2 identifies 2 subsets of CLL patients. (A) Increase in the absolute number of γδ T cells after 7 days stimulation with ZA + IL-2. Fold increase was calculated for each patient as a ratio between the absolute number of viable γδ T cells in PBMCs stimulated with ZA + IL-2 and that in PBMCs stimulated with IL-2 alone [(ZA + IL-2)/IL-2] on day 7. Median value of fold increase (3.62) was selected as a cut-off value to define R and LR patients. (B) Absolute numbers of γδ T cells in R (n = 53) and LR (n = 53) CLL patients after stimulation with ZA + IL-2 or IL-2 alone. After 7 days of culture in ZA + IL-2 conditioned medium, the total count of γδ T cells was significantly greater in R than in LR patients (*P < .001). (C) γδ T-cell proliferation was evaluated after 7 days' stimulation with ZA + IL-2. Representative dot plots from a LR (top) and from an R (bottom) patient are shown. Percent γδ T cells of total CD3⁺ T cells is indicated. (D) Baseline total counts of Vγ9Vδ2 T cells in HD (n = 24), R (n = 27), and LR (n = 33) patients. LR patients showed significantly greater numbers of circulating Vγ9Vδ2 T cells compared with HD and R patients (*P < .001 and P = .005, respectively). (E) Representative cytofluorimetric analysis of Vγ9Vδ2 T cell subsets according to the expression of CD27 and CD45RA markers. Four Vγ9Vδ2 T-cell subsets are identified: CD45RA⁺CD27⁺ N cells (top right); CD45RA⁻CD27⁺ CM cells (top left); CD45RA⁻CD27⁻ EM cells (bottom left); and CD45RA⁺CD27⁻ TEMRA (bottom right). (F) Vγ9Vδ2 T cells subsets distribution in HD (n = 20), R (n = 23), and LR (n = 28) patients. The absolute number of EM and TEMRA Vγ9Vδ2 T cells was significantly greater in LR CLL patients compared with HD (°P = .009 and *P = .03, respectively) and to R CLL patients (°P = .018 and *P = .004, respectively). In panels A and D, horizontal lines represent median value. In panels B and F, bars represent mean values ± SEM.