CD3/TCRαβ downregulation by PEBL does not affect cell proliferation, but abrogates CD3/TCRαβ signaling. (A) Growth rate of Jurkat transduced with anti-CD3 PEBLs or GFP only (“Control”). Symbols indicate mean (± SD) of triplicate measurements. (B) Survival of PEBL-transduced or control T lymphocytes from 5 donors (7 experiments) cultured with IL-2 (200 IU/mL). Symbols indicate mean of triplicate measurements. (C) CD25 and CD69 mean fluorescence intensity (MFI) in Jurkat after 24 hours with OKT3 or nonreactive mouse IgG2a. Bars indicate mean (±SD) of triplicate measurements. (D) Viable PEBL or control T lymphocytes recovered from cultures with OKT3 compared with cultures without OKT3, all containing IL-2 (200 IU/mL). Symbols represent mean (±SD) of 9 measurements with cells from 3 donors. P values by Student t test are shown for significant differences (****P < .0001). (E) Jurkat cells transduced with either a TCR specific for HBV s183 or a vector containing neomycin-resistant gene only (“NeoR”) were transduced with anti-CD3 PEBL or mCherry only (“Control”) after neomycin selection. CD3, TCRαβ, and TCRVβ3 chain (part of the HBV s183 TCR) expression is shown; TCRVβ3 expression was tested on the cell surface, and intracellularly after cell permeabilization. (F) Transduced Jurkat cells shown in panel E were cocultured with T2 cells loaded with HBV s183 peptide for 24 hours. Shown are CD25 and CD69 MFI minus those measured after culture with T2 cells, but without peptide. Symbols represent mean of triplicate measurements.