Characterization of MAGE-specific CTL response induced by AZA/VPA treatment in patient no. UPIN04. (i) Flow cytometry plots after magnetic enrichment of patient no. UPIN04 before and after treatment. A total of 2 × 10⁶ PBMCs were stimulated with peptide, of which flow cytometric analysis revealed 35.2% to be CD3⁺CD8⁺. From these 594 000 CD8⁺ T cells, 23 cells were isolated in the positively selected fraction, validating the assay. Fifteen peptides derived from a range of the CTAs MAGE-A1/A2/A3, MAGE-C2, BAGE, and RAGE (Alta Bioscience, for CD8 peptides) were chosen from previously described epitopes for CD8⁺, restricted by a variety of human leukocyte antigen alleles (supplemental Table 2). These peptides were chosen on the basis that previous publications have shown expression of MAGE, BAGE, and RAGE in tumor biopsy samples from AML patients by reverse transcription–polymerase chain reaction.¹⁶  In cases in which the patient's human leukocyte antigen type was unknown, PBMCs were screened initially against peptide pools, and the response to single peptides was elicited on subsequent analysis and confirmed by human leukocyte antigen type (http://www.cancerimmunity.org/peptidedatabase/tumorspecific.htm). (ii) Flow cytometry analysis with a MAGE-A2_157-166 tetramer stain of a T-cell line generated from patient no. UPIN04 after magnetic enrichment with anti-PE magnetic beads (Miltenyi Biotec).