The loss of mismatched HLA expression on leukemic blasts caused by uniparental disomy on chromosome 6p impaired recognition and killing of donor's alloreactive cytotoxic T lymphocytes. (A) Leukemic blasts at the time of initial diagnosis and at the time of relapse after hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI) were gated by CD34⁺ and CD13⁺, and then the surface expression of mismatched human leukocyte antigen (HLA) alleles was examined with anti–HLA-A2 antibodies. In 3 patients with acute myelogenous leukemia (AML) who experienced relapse after HLA-haploidentical HSCT, HLA-A2 expression was lost in patient 1 at relapse 15 months after HSCT and lost in patient 2 at second relapse 6 months after DLI. (B) Single nucleotide polymorphism (SNP) array analyses of sorted leukemic cells with the loss of an HLA allele revealed that the short arm of chromosome 6 shows copy number-neutral loss of heterozygosity or acquired uniparental disomy as detected by dissociated allele-specific copy number plots (red and blue lines at the bottom), resulting in the total loss of the mismatched HLA haplotype in both patient 1 and patient 2. The presence of acquired uniparental disomy is also indicated by normal total copy numbers with missing heterozygous SNPs (green bars) in the distal part of the short arm. (C) Recipient alloantigen-specific cytotoxic T-lymphocyte (CTL) clones were generated by a conventional cloning method from cytotoxicity-positive wells obtained in the limiting dilution assays using the donor CD8⁺ cells as responders. Donor CTL clones A1, A2, and A3 were specific for HLA-A*0206. Donor CTL clones B1 and B3 were specific for HLA-B*4001, all of which recognize mismatched HLA alleles between the donor and recipient. Those 5 representative CTL clones were tested for HLA specificity and recognition of leukemic blasts obtained at the time of the initial diagnosis and at the time of HLA loss relapse after DLI by a standard ⁵¹Cr-release assay at the effector/target ratio of 30:1. (D) Their interferon-γ production was also assessed against leukemic blasts collected at the time of diagnosis and at the time of HLA-loss relapse.