The Immunogenicity of a Human Lymphoid Cell-Restricted Minor Histocompatibility Antigen Results from Differential Protein Translation.

Minor histocompatibility antigens (mHA) that are presented selectively on hematopoietic cells, including leukemic cells, but not on nonhematopoietic tissues represent attractive immunotherapeutic targets after HLA-matched allogeneic hematopoietic cell transplantation (HCT) to enhance the graft-versus-leukemia (GVL) effect without instigating graft-versus-host disease. We isolated from an HLA-matched HCT recipient a CD8⁺ cytotoxic T-lymphocyte (CTL) clone, KSN 7A7, which recognized a novel HLA-A3-restricted mHA with lymphoid-restricted presentation. KSN 7A7 demonstrated high-level in vitro cytolysis of a recipient EBV-transformed B lymphoblastoid cell line (BLCL) and low-level cytolysis of recipient PHA-stimulated T cells, but no recognition of recipient dermal fibroblasts or any donor cells. When tested against a panel of allogeneic HLA-A3⁺ leukemic cells, KSN 7A7 showed cytolysis against malignant cells of B-lymphoid origin but not of myeloid origin. To identify the epitope recognized by KSN 7A7 CTL, HLA-A3-associated peptides were extracted from a phenotypically mHA⁺ BLCL, fractionated by reversed-phase HPLC, and tested for the ability to reconstitute CTL recognition of mHA⁻ donor BLCL. After 3 rounds of RP-HPLC, candidate peptide masses within biologically active fractions were selected using nanospray Fourier transform mass spectrometry, and the most abundant candidate peptide ion was sequenced by collision activated dissociation. A synthetic decameric peptide corresponding to this sequence sensitized donor BLCL to CTL lysis, with half-maximal lysis observed at a peptide concentration of 2 nM. A database search revealed that this peptide was encoded by an alternative transcript of the C22orf18 gene (LocusLink 79019), with the epitope derived from an alternative exon that is not known to be utilized by other transcripts of this gene. To identify the basis for differential CTL recognition of donor and recipient BLCL, donor and recipient C22orf18 alleles were sequenced and compared. The mHA⁻ donor was homozygous for a C → T single nucleotide polymorphism (SNP) within the codon for the first residue of the epitope that in turn created a translation termination signal, suggesting that the antigenicity of this locus is a consequence of differential translation of the protein encoded by this transcript in donor and recipient cells. Direct sequencing and PCR-RFLP analysis of the region surrounding this SNP in C22orf18 alleles in 45 additional HLA-A3⁺ individuals revealed that the frequency of the T allele encoding the translation termination signal was 0.25. Phenotyping of BLCL from these 45 individuals for mHA expression confirmed that homozygosity for the T allele was associated with resistance to CTL lysis. RT-PCR analysis revealed that the C22orf18 alternative transcript is almost exclusively expressed in resting and activated CD19⁺ cells, with very low levels of expression detected in CD4⁺, CD8⁺, and CD14⁺ cells, and negligible expression in nonhematopoietic tissues. Thus, therapeutic strategies targeting this mHA could selectively enhance the GVL effect in HCT recipients with B-lymphoid lineage leukemias.