γ9δ2TCR expression and functional avidity of transduced T cells expressing single alanine mutated δ2 chain of clone G115. (A) Peripheral blood T cells were virally transduced with indicated γ9 and δ2 TCR chains and analyzed by flow cytometry using a δ2-chain specific Ab. Shown is the fold change in mean fluorescent intensity (MFI) with wild-type controls expressing δ2-G115_wt. (C) Lytic activity of transductants was tested in a ⁵¹Cr-release assay against the tumor target Daudi (E:T, 10:1). Specific lysis is indicated as the fold change in ⁵¹Cr-release measured in the supernatant after 5 hours compared with reactivity of unmutated wild-type (δ2-G115_wt). Arrows indicate mutations in δ2-G115 that impaired receptor expression (dashed arrows) or functional avidity (solid arrows). (B,D) Crystal structure of γ9δ2TCR G115 indicating relevant amino acids (red arrows), δ-chain (in blue), δCDR3 (in green), and γ-chain (in brown).