Genetic modification of T-APCs. (A) Schematic of HyMP1 cDNA and plasmid expression vectors. The HyMP1 cDNA consisting of a 5′ hygromycin phosphotransferase segment and a 3′ influenza A matrix protein-1 (HyMP1) is shown with its flanking NheI and BamHI restriction enzyme (RE) sites. This transgene was cloned into the multiple cloning site (MCS) under control of the hEF1α hybrid promoter in the plasmid HyMP1-pEK. Plasmid pMGP̂ac contains the hygromycin phosphotransferase (Hy) cDNA, under control of the human CMV immediate early (IE) promoter. The bovine growth hormone (bGhpA), late SV40 poly A sites (SV40pA), synthetic poly A and pause site (SpAn), Escherichia coli origin of replication (ori ColE1), and unique RE sites are shown. The PacI RE site was used to linearize the plasmids before electroporation. (B) Chemiluminescence Western immunoblot of recombinant HyMP1. Whole-cell protein lysates from T-APCs genetically modified with HyMP1-pEK (lane 1) or pMGP̂ac (lane 2) plasmids, along with molecular weight controls (not shown), were resolved by PAGE under reducing conditions. Western blotting with MP1-specific antibody was used to detect the approximately 176-kDa HyMP1 fusion protein. (C) Phenotype of T-APCs by flow cytometry. Histograms of binding of fluorescence-labeled cell-surface marker–specific mAbs (bold line) relative to isotype control or unstained cells (dotted line) to CD8⁺ T-APCs genetically modified with HyMP1-pEK are shown. T-APCs were stained and analyzed by flow cytometry between days 10 and 14 of a 2-week in vitro OKT3 stimulation cycle. The flow cytometry histograms are almost identical for CD8⁺ Hy⁺MP1neg T-APCs genetically modified with pMGP̂ac (data not shown). The relative percentage of cells in each gate is indicated.