Vγ9Vδ2 T-cell activation correlates with decreased mobility of BTN3A1 induced by agonist anti-CD277 mAb and NBP. (A) Left: Confocal images of HEK293 cells expressing EmGFP-BTN3A1 molecules after incubation for 30 minutes with anti-CD277 mAb (#20.1; 10 μg/mL), shown before (Pre-bleach), immediately after (T0-bleach), and 80 seconds (Tp-80 seconds) after photobleaching of regions of interest (indicated rectangular areas). Scale bars represent 6 μm. (−) indicates no treatment. Right: Mean FRAP and fit curves in EmGFP-BTN3A1-expressing HEK293 cells (n = 18), after treatment with anti-CD277 mAb (#20.1; 10 μg/mL). Control indicates no treatment (n = 17). The symbols correspond to the mean ± SEM of FRAP collected every 5 seconds. The curves were fitted by 1-phase exponential equations. The average fluorescence before photobleaching was counted as 100% (dashed line). Immobile fractions are indicated for each condition. (B) FRAP analysis of HEK293 cells expressing EmGFP-BTN3A1 or EmGFP-BTN3A2 molecules after incubation for 30 minutes with anti-CD277 mAbs (#20.1; 10 μg/mL). Data are presented as the value for the percentage of immobile fraction, measured as described under “Microscopy.” Control indicates no treatment. BTN3A1: (−), n = 21; 20.1, n = 13; BTN3A2: (−), n = 12; 20.1, n = 8. Bars represent mean values. (C) FRAP analysis of HEK293 cells expressing mCherry-BTN3A1 or mCherry-BTN3A2 molecules after either incubation for 30 minutes with anti-CD277 mAb (#20.1; 10 μg/mL), incubation overnight with NBP (Pam; pamidronate; 250μM) or statin (mevastatin, 50μM) only, or treatment with statin for 6 hours before incubation overnight with both statin and NBP (Pam + Meva). Data are presented as the value for the percentage of immobile fraction. Control indicates no treatment. BTN3A1: (−), n = 21; 20.1, n = 16; Pam, n = 28; Pam + Meva, n = 18; Meva, n = 8. BTN3A2: (−), n = 12; 20.1, n = 14; Pam, n = 14. Bars represent mean values. (B-C) ***P < .005 (Student t test).
