Characterization of CD8-LV. (A) Schematic representation of the H-αCD8 expression construct and CD8-LV. The MV H protein is truncated at its cytoplasmic tail by 18 amino acids (CTΔ18). Four point mutations (Y418A, R533A, S548L, and F549S) in the ectodomain (ED) marked by asterisks abolish natural receptor recognition. The displayed CD8-scFv is linked to H protein via a glycine-serine linker [(G₄S)₃]. The transmembrane domain (TM) and a histidine tag (6His) are indicated. (B) Expression plasmids encoding H-αCD8 and H-αCD8^opt were transfected into HEK293T cells. Two days later, surface expression of H-αCD8 was quantified by anti-His mAb staining and FACS analysis. The backbone plasmid pCG-1 was used as negative control. Results are expressed as mean ± SEM (n = 3; ***P < .001). (C) Transduction of J76CD8SLAM cells by CD8-LV (black circles), VSV-G-LV (gray diamonds), and MV-LV (gray hexagons) was competed on preincubation of cells with the indicated concentration of OKT8 antibody for 20 minutes at 4°C. After 2 days, transduction efficiency was quantified. Isotype control antibody preincubated cells followed by CD8-LV transduction were included as further control (black squares). (D) To study the specificity of CD8-LV, equal numbers of J76 cells and J76CD8 cells were mixed and transduced with the indicated vectors (MOI = 1) or left untransduced (ut). After 48 hours, the percentages of CD8⁺/GFP⁺ and CD8⁻/GFP⁺ cells were determined by flow cytometry. (E) Freshly isolated human PBMCs were stimulated with 100 IU/mL IL-2 and anti-CD3/CD28 mAbs (1 μg/mL) for 3 days and then transduced with CD8-LV or VSV-G-LV at an MOI of 2. Six days after transduction, PBMCs were analyzed by flow cytometry for GFP expression and CD8 staining.