D1- and P4-specific T cells are isolated by tetramer sorting and recognize whole processed and presented Ag. (A) Patient A CD3⁺ T cells were repeatedly stimulated with either D1 or P4 peptide-pulsed T2 cells before staining with peptide-specific tetramer. Tetramer and CD8 double-positive cells were isolated by FACS (representative FACS plots shown). (B) Sorted D1- or P4-positive T cells were tested by ELISpot against K562 cells stably transfected to express HLA-A2 (K562/A2 cells) that were nucleofected to overexpress either DAPK2 or PIM1. As expected, overexpression of DAPK2 induced apoptosis in nucleofected K562 cells.²⁴  P4-specific T cells recognized processed and presented PIM1, an effect that was blocked by the class I–blocking Ab w6/32. (C) Sorted D1- or P4-positive T cells were also tested against the A2-positive MM cell lines MC/CAR and IM9 and the A2-negative MM cell lines OPM2 and NCI 4929. Both D1- and P4-specific T cells showed reactivity to HLA-A2–positive cell lines, but not to HLA-A2–negative cell lines. IFN-γ secretion was blocked by co-incubation with w6/32 Ab.