IFN-γ release and tetramer binding in unmodified and ubiquitinated pp65-VV IVS cultures. (A) Donors listed in Table 1 were tested for specific CD8⁺ IFN-γ production using ICC before (○) and after (▪) IVS with pp65-VV or UbRpp65-VV (▴). The following peptides were used during ICC: p53_149-157, pp65_495-503 with HLA A^*0201 donors, and pp65_417-426 with HLA B^*0702 donors. Donor 002 was not tested, while for donor 005, ICC was performed only after UbRpp65-VV IVS. For each donor, percent CD8⁺ IFN-γ release to irrelevant peptide was subtracted from specific release. (B) CMV pp65_495-503 tetramer was used with HLA A^*0201 donors, CMV pp65_417-426 with HLA B^*0702 donors, and the nonrelated HIV pol_464-472 as control tetramer for each donor. CD8⁺ binding percentages to control tetramer (0.07%-0.1%) were subtracted in each case. ○ denotes CD8⁺ tetramer binding percentages in donor PBMC before IVS; ▪, after pp65-VV IVS; and ▴, after UbRpp65-VV IVS. (C) Donor 001 tetramer binding fluorescence-activated cell-sorter (FACS) plots are shown. A 2-color FACS was used using anti-CD8 FITC-labeled mAb and tetramer conjugated with phycoerythrin (PE). Numbers on the upper-right quadrant indicate CD8⁺ T-cell percentages to (i) pp65_495-503 tetramer, (ii) pol_464-472 control tetramer after UbRpp65-VV IVS, (iii) pp65_495-503 tetramer, and (iv) pol_464-472 control tetramer after pp65-VV IVS.