Functional expression of γ9δ2 TCR in αβT cells. Surface expression of specific staining for Vδ2 together with either staining for Vγ9, for γδTCR, or for αβTCR as indicated in panel A. Presented representative dot plots have been confirmed in at least 3 independent experiments. (B) Specific lysis of the Daudi cells or autologous PBMCs (Auto-PBMC) tested by ⁵¹Cr-release assay with T cells that were transduced with γ9, δ2, or both chains. Mock-transduced cells served as a negative control in all the experiments. Shown is a representative experiment at the E:T 10:1. TNFα (C) and IFNγ (D) secretion by the γ9δ2TCR or mock-transduced αβT cells in response to Daudi cells was tested. αβT cells were incubated with target cells at the E:T 0.3:1 for 48 hours in absence or presence of 10μM pamidronate,, respectively. Shown are representative results of at least 3 independent experiments. Statistical analysis was determined by 2-way ANOVA analysis; *P < .01; **P < .001. (E) Proliferation of CMFDA-labeled γ9δ2TCR or mock-transduced αβT cells in response to Daudi cells. Autologous and allogeneic PBMCs were tested following 4 days at an E:T ratio of 3:1. CMFDA labeling is diluted in the proliferating cells. (F) γ9δ2TCR-transduced αβT cells were FACS-sorted based on their δ2TCR and αβTCR expression resulting in 2 distinct populations either expressing γ9δ2TCR^high/αβTCR^low or γ9δ2TCR^low/αβTCR^high. The FACS-sorted fractions were cocultured with partially mismatched LCL lines 1-17 for 24 hours, and production of IFNγ was determined via ELISPOT analysis. Data show mean ± SEM of duplicate samples. Comparable results were obtained in 3 independent experiments and by using transduced primary T cells of 2 healthy donors. Statistical analysis was determined by 2-way ANOVA analysis; *P < .001.