Immunogenicity of HLA class I–restricted CML-associated antigens. (A) Immunogenicity analysis results for the 8 HLA class I–restricted CML-associated peptides with their respective frequencies of preexisting immune recognition by PBMCs from CML patients or HVs in IFN-γ ELISPOT assays (CD8⁺ T-cell response in CML/HVs), as well as the frequencies of peptide-specific CD8⁺ T cells detected after in vitro aAPC-based priming experiments with naive CD8⁺ T cells from HVs and CML patients. (B) Examples of CML-associated ligands evaluated in IFN-γ ELISPOT assays after a 12-day stimulation using PBMCs from CML patients. Results are shown for immunoreactive peptides only. Phytohemagglutinin was used as positive control and the HLA-A*02–restricted DDX5_HUMAN_148-156 peptide YLLPAIVHI served as negative control. Data are expressed as mean ± standard deviation of 2 independent replicates. Naive CD8⁺ T cells from HVs (C) and CML patients (D) were primed in vitro using aAPCs. Graphs show single viable cells stained for CD8 and PE-conjugated multimers of indicated specificity. Tetramer staining was performed after 4 stimulation cycles with peptide-loaded aAPCs. The left panels show P3_A*03-tetramer (C) or P7_B*07-tetramer (D) staining. The middle panels (negative control) depict P3_A*03-tetramer (C) or P7_B*07-tetramer (D) staining of respective T cells primed with an irrelevant peptide. The right panels show T cells from the same donor that were tested for the absence of preexisting memory T cells after a 12-day recall stimulation by tetramer staining (C) or IFN-γ ELISPOT assay (D). (E) Tetramer staining after 4 stimulation cycles with negative control peptide-loaded aAPCs (HLA-A*02, YLLPAIVHI, DDX5_HUMAN_148-156 and HLA-A*03, QIFVKTLTGK, UBC_HUMAN_2-11). ID, identification; neg., negative; n.t., not tested; pos., positive; SFU, spot-forming unit; UPN, uniform patient number.