Specific recognition of the primary AML blasts by γ9δ2TCR-transduced αβT cells. (A) γ9δ2TCR-transduced αβT cells of healthy donor were incubated with AML blasts without (□) or with 10μM pamidronate () for 48 hours at E:T ratio of 0.3:1. IFNγ secretion was measured by ELISPOT. Daudi and αβT cells were used as positive or negative target control, respectively. Shown is the result of the mean of 2 experiments performed in duplicates for the same donor. Similar results were observed for 2 other healthy donor-derived γ9δ2TCR-transduced αβT cells. *P = .05, **P < .01. (C) γ9δ2TCR or mock-transduced αβT cells were incubated with AML blasts or PBMCs of healthy donors enriched with CD34⁺ cells for 4 hours at E:T ratio 4:1. Then cells were plated in methylcellulose and, after 10 days, colony formation was quantified using an inverted microscope. Shown is the percentage of CFU formed after the incubation with γ9δ2TCR-transduced cells, while CFU formed after the incubation with mock-transduced αβT cells is used as 100%. Shown is 1 of 3 representative experiments, which have been performed with different donors. (D) Microscopic photographs of the representative field of methylcellulose colony formation cultures of AML and healthy donor taken on the same day of the assay. (E-F) αβT cells of AML patient 5 were isolated with CD3-specific beads and transduced with γ9δ2-TCR with standard procedures. After selection and expansion, αβT cells were stained with γδTCR-specific Ab and the expression was assessed by FACS analysis. IFNγ secretion in response to AML cells and the control targets was analyzed by ELISPOT as described in panel B.