Transfer of TCR-4G11 installs BOB1 reactivity on recipient CD8⁺ T cells. CD4⁺ and CD8⁺ T cells were isolated from a healthy HLA-B7⁺ individual using magnetic-activated cell sorting (MACS). T cells were transduced with retroviral supernatant to express TCR-4G11 together with NGF-R. Transduction with an empty vector (mock) containing only the NGF-R marker gene served as control. Transduced T cells were purified based on the expression of marker gene NGF-R using MACS. Level of purity exceeded 98% in all cases. (A) Shown are FACS plots of purified T cells after transduction. CD8⁺ (top row) or CD4⁺ (bottom row) T cells were stained with pMHC tetramer BOB1₄₄:B7 and an antibody against NGF-R. Numbers in corners indicate percentage of cells per quadrant. FACS plots are shown with biexponential axes. (B) T-cell clone 4G11 or purified transduced CD8⁺ or CD4⁺ T cells were coincubated with various HLA-B7⁺ cell lines. Cell lines included K562-B7 transduced to express BOB1 (K562-B7 + BOB1), 2 MM cell lines UM9 and U266, LCL-JY, and 2 ALL cell lines ALL-BV and ALL-VG. IFN-γ concentration was assessed after 18 hours of coculture. Shown are means with standard deviations of 1 experiment carried out in duplicate.