Escape of B4002⁻iCD34⁺cells from the attack by autologous CTLs specific to B4002⁺iCD34⁺cells. (A) CTLs were cocultured with iCD34⁺ cells possessing either the 6pLOH⁺ or the WT genotypes in 96-well round-bottom plates for 5 days and visualized using a light microscope. Representative images of experiments are shown; black arrows indicate iCD34⁺ cells; white arrows indicate CTLs. Top panels, original magnification ×60; bottom panels, original magnification ×20. (B) CTLs were cocultured with iCD34⁺ cells possessing either the 6pLOH⁺ or the WT genotypes in 96-well round-bottom plates at a 10:1 effector:target (E:T) ratio for 4 hours and then analyzed by FCM. The analysis was performed by gating cells as 7-aminoactinomycin D–negative (live cells), CD3⁺, CD8⁺, or CD107a⁺ cells. (C) CTLs were cocultured in 96-well round-bottom plates with iCD34⁺ cells with 6pLOH⁺, WT, or B*40:02^mut genotypes at the indicated E:T ratios. The levels of IFN-γ in the supernatants from overnight cocultures were determined by enzyme-linked immunosorbent assay. Summarized data (mean ± SEM) from 3 independent experiments are shown. (D) iCD34⁺ cells possessing the 6pLOH⁺, B*40:02^mut, or WT genotypes were labeled with ⁵¹Cr and then cocultured with autologous CTLs in 96-well round-bottom plates for 4 hours. Summarized data (mean ± SEM) from 3 independent experiments are shown. (E) Clonotype analysis (Vβ cDNA sequencing) of the CTL line and BM T cells of the patient before receiving immunosuppressive therapy using single T-cell RT-PCR indicate that the third-most recurrent TCRVβ CDR3 sequence of the CTL line was identical to that of the most predominant T-cell clone in the BM. (F) CDR3α sequence of T cells with the same CDR3β sequence derived from the CTL line and BM T cells. The T cell expressed 2 TCRα chain mRNAs; 1 was functional and the other was unproductive. *P < .05.