In vitro–exhausted Vγ9Vδ2 T cells have reduced calcium influx and altered metabolism upon CD3 stimulation. (A) Sketch of the calcium influx video-microscopy principle and images recreated before and after UCHT1 injection. (B) Calcium influx measured by video-microscopy of untreated (blue) vs treated (red) Vγ9Vδ2 T cells upon anti-CD3 monoclonal antibody (mAb) UCHT1 and OKT3 injection over time. (C) Calculated peaks of calcium influx after anti-CD3 mAb injection. (D-E) Monitoring of mitochondrial OCR and ECAR at resting state, after anti-CD3 and PMA-ionomycin injection. (F-G) OCR (F) and ECAR (G) peaks calculated as the difference between the values before and after anti-CD3 or PMA-ionomycin injection; n = 4 donors. All statistical analysis were performed using 2-way ANOVA (∗P < .05; ∗∗P < .003; ∗∗∗P < .0005; ∗∗∗∗P < .0001).