Functional characterization of CML-associated antigen-specific CD8⁺T cells. (A) Functional characterization of CML-associated antigen-specific CD8⁺ T cells, including their CD107a and cytokine expression profile detected by ICS following aAPC-based priming experiments and their cytotoxic capability (VITAL assay). (B) Representative example of increased IFN-γ and TNF production, as well as CD107a expression, after stimulation with the respective P7_B*07-peptide used for the stimulation with aAPCs compared with the corresponding negative control peptide (HLA-B*07, TPGPGVRYPL, NEF_HV1BR_128-137). Phorbol myristate acetate (PMA) and ionomycin served as positive control. The P7_B*07-specific CD8⁺ T-cell population showed a frequency of 1.01%, as detected by tetramer staining (far left panel). (C-E) Selective cytotoxicity of P5_A*03-specific effector T cells analyzed in a VITAL cytotoxicity assay with in vitro primed CD8⁺ T cells from an HV. Tetramer staining of polyclonal effector cells before performance of the VITAL assay determined the amount of P5_A*03-specific effector cells in the population of successfully P5_A*03-primed CD8⁺ T cells (C) and in the population of control cells (D) from the same donor primed with an HLA-matched irrelevant peptide. (E) At an effector-to-target ratio of 2.5:1, P5_A*03-specific effectors (red) exerted 20.9% (± 0.4%) P5_A*03-specific and significant higher lysis of P5_A*03-loaded autologous target cells in comparison with control peptide-loaded target cells (HLA-A*03, RLRPGGKKK, GAG_HV1BR_20-28). P5_A*03-unspecific effectors (blue) only showed 2.0% (± 0.4%) unspecific lysis of the same targets. Results are shown as mean ± standard error of the mean for 3 independent replicates. ***P < .001. FSC, forward scatter; ID, identification; n.s., not significant; n.t., not tested.