γ9δ2TCR expression and functional avidity of transduced T cells expressing γ9δ2TCR G115 with δ2-CDR3 length mutations. (A) γ9δ2TCR expression of indicated transductants was analyzed by flow cytometry using a γδTCR-pan Ab. Shown is the fold change in mean fluorescent intensity (MFI) compared with wild-type controls expressing δ2-G115_wt. (B) IFNγ secretion of δ2-G115_LM–transduced T cells against the tumor target Daudi (E:T, 1:1) was measured by ELISA after 24 hours of incubation in the presence of 100μM pamidronate. Shown is the fold change in IFNγ production compared with reactivity of transductants expressing δ2-G115_wt. (C) Transductants expressing δ2-G115_LM0,1,4,12 were tested in a titration assay against the tumor target Daudi with increasing amounts of pamidronate as indicated. IFNγ production was measured after 24 hours by ELISA. (D) Generated δ2-G115_LMs were matched in a BLAST search with γ9δ2TCRs described in the IMGT database. Shown is the number of citations compared with δ2-G115_LM of similar δCDR3 length. (E) Transductants with δ2-G115_LM2,4,6 were compared side-by-side with transductants expressing individual γ9δ2TCRs of the same δCDR3 length. IFNγ secretion of transduced T cells against the tumor target Daudi (E:T, 1:1) was measured by ELISA after 24 hours in the presence of 100μM pamidronate. Shown is the fold change in IFNγ production compared with reactivity of transductants expressing δ2-G115_wt. Data represent the means ± SD. **P < .01; and **P < .001 by 1-way ANOVA. (F) Crystal structure of γ9δ2TCR G115; the region that was used for alanine stretches within δCDR3 is shown in white, the residual δCDR3 in green, the δ chain in blue, and the γ chain in brown.