HMB-PP does not induce down-modulation of surface Vγ9⁺ TCR and sustains cytokine production upon re-stimulation.

(A) MACS-sorted γδ PBL (of which 80–90% Vγ9⁺) were incubated for the indicated times with HMB-PP or OKT3, and stained with anti-Vγ9 mAb for flow cytometry analysis. Bold lines represent treated cells, while shaded are non-stimulated Vγ9⁺ cells (time = 0 hrs). (B) Confocal microscopy photos of γδ T-cells cultured for 24 hrs as in (A) and then stained for Vγ9⁺ TCR. (C–D) Experimental design (C) and CBA analysis (D) of the re-stimulation response of MACS-sorted γδ PBL. After 36 hrs of stimulation, cells were re-plated for secondary activation during 24 hrs, when supernatants were collected and analyzed for Th1 cytokines by CBA. Error bars represent SD and differences refer to IL-2 controls (ns, non-significant; **p<0.01; ***p<0.001). Results shown in this figure are representative of 2–5 independent experiments.