Increased CD8 density and enhanced activation of CD8-LV TCR-transduced cells. (A) Activated PBMCs were transduced with CD8-LV-TCR-T58 or VSV-G-LV-TCR-T58 at an MOI of 2. Approximately 2 weeks later, TCR expression levels were determined using HLA-A2-Tyr multimers in the presence of anti-CD8 mAb. Mean fluorescent intensities (MFI) of the TCR specific signal are shown for the transduced effector cells of each vector type. Results are expressed as mean ± SEM (n = 10). ns indicates not significant. (B) Activated PBMCs were transduced with CD8-LV-TCR-T58 (dark gray line) or VSV-G-LV-TCR-T58 (light gray line) at an MOI of 2. Then, CD8⁺/TCR-T58^high cells were gated (supplemental Figure 5), and CD8 surface densities were determined at the indicated days as relative MFI normalized to that of untransduced cells. Results are expressed as mean ± SEM (n = 3). The difference between CD8-LV-TCR-T58 and VSV-G-LV-TCR-T58 treated groups is significant (P < .05). (C-D) Activated PBMCs were left untransduced (white bars), transduced by VSV-G-LV-TCR-T58 (gray bars), or CD8-LV-TCR-T58 (black bars). Cells were then cultivated for 12 days, before they were transferred into IL-2 (10 IU/mL) containing medium for 3 days. Effector cells were incubated with or without Mel-624.38 cells for 4 hours and stained with anti-CD8, anti-mCβ, and anti-CD69 mAbs or appropriate isotype controls. The gate determining CD69⁺ cells was set according to the isotype control antibody stained untransduced cells. Overall percentage of CD69⁺ cells (C), and MFI of CD69⁺ cells (D) among CD8⁺/TCR-T58^high cells are shown. Results are expressed as mean ± SEM (n = 3; *P < .05; ***P < .001).