Cr51-release assays for the assessment of cytotoxic activity of CD8+ T lymphocytes of 4 HVs against the peptide NPM1#1. (A) NPM1#1–specific cytotoxic T lymphocytes (CTLs) were generated from 4 HVs and cytotoxic activity was assessed by Cr51-release assays using peptide-pulsed T2 cells as target cells. NPM1#1–specific CTLs showed a specific lysis of 57.1%-71.3% at an E/T ratio of 20:1. The counts were verified in 4 different HVs, as indicated by the error bars. T2 cells pulsed with a wild-type peptide or no peptide served as negative controls. The lytic potential of CTLs specifically recognizing epitope peptides derived from NPM1 mutations was further assessed in an autologous setting using PBMCs from AML patients with a blast percentage > 80% as targets. Only blasts with NPM1 mutations (“NPM1 mut”) #1 (B) and #3 (C) were lysed in a context of HLA-A2. Blasts from patients without NPM1 mutations (wild-type; “NPM1 WT”) or without HLA-A2 expression were not recognized. K562 cells lacking HLA-A2 were used as a negative control. These experiments clearly demonstrate that epitope peptides derived from NPM1 mutations #1 and #3 are naturally processed and recognized in the context of HLA-A2.
