Alternative splicing due to an intronic SNP in HMSD generates a novel minor histocompatibility antigen

Abstract

Here we report the identification of a novel human leukocyte antigen (HLA)-B44-restricted minor histocompatibility antigen (mHA) with expression limited to hematopoietic cells. cDNA expression cloning studies demonstrated that the cytotoxic T lymphocyte (CTL) epitope of interest was encoded by a novel allelic splice variant of HMSD, hereafter designated as HMSD-v. The immunogenicity of the epitope was generated by differential protein expression due to alternative splicing, which was completely controlled by 1 intronic single-nucleotide polymorphism located in the consensus 5 splice site adjacent to an exon. Both HMSD-v and HMSD transcripts were selectively expressed at higher levels in mature dendritic cells and primary leukemia cells, especially those of myeloid lineage. Engraftment of mHA ؉ myeloid leukemia stem cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/␥c null mice was completely inhibited by in vitro preincubation with the mHA-specific CTL clone, suggesting that this mHA is expressed on leukemic stem cells. The patient from whom the CTL clone was isolated demonstrated a significant increase of the mHA-specific T cells in posttransplantation peripheral blood, whereas mHA-specific T cells were undetectable in pretransplantation peripheral blood and in peripheral blood from his donor. These findings suggest that the HMSD-v-encoded mHA (designated ACC - IntroductionMinor histocompatibility antigens (mHAs) are major histocompatibility complex (MHC)-bound peptides derived from cellular proteins encoded by polymorphic genes. Following human leukocyte antigen (HLA)-matched allogeneic hematopoietic cell transplantation (HCT), donor-recipient disparities in mHAs can induce a favorable graft-versus-leukemia (GVL) effect that is often associated with graft-versus-host disease (GVHD). 1-3 Significant efforts have been made to identify mHAs, particularly those specific for hematopoietic cells, since such mHAs are speculated to contribute to the GVL effect. The first report on the identification of a hematopoietic lineage-specific mHA, HA-1, was generated by the Goulmy group in 1998 (den Haan et al 4 ) as a result of biochemical analysis of peptides eluted from HLA-A*0201 molecules. The only other mHAs with selective expression in hematopoietic cells described to date are HA-2 5 ; ACC-1 and ACC-2 6 ; and DRN-7, 7 HB-1, 8,9 and PANE1, 10 the latter 2 of which are B-cell lineage-specific. Thus, identification of more mHAs should facilitate a better understanding of the biology of GVL and the development of effective immunotherapy to induce GVL reactions.Immunogenicity of most autosomal mHAs identified to date results from single-nucleotide polymorphisms (SNPs) that cause amino-acid substitutions within epitopes, leading to the differential display/recognition of peptides between HCT donor and recipient via several mechanisms: peptide binding to MHC observed in HA-1/A2-, 4 HA-2-, 5 and CTSH-encoded mHAs 11 ; proteasomal cleavage in HA-3 12 ; peptide transport in HA-8 13 ; and altered recognition o...

Identification of human minor histocompatibility antigens based on genetic association with highly parallel genotyping of pooled DNA

“…Each clone demonstrated specific lysis against the B-LCLs of the recipient but not against donor B-LCLs, indicating recognition of minor H antigen ( Figure 1A and Kawase et al 9 ). The minor H antigen for CTL-2A12 had been previously identified by expression cloning 9 ; on the other hand, the target minor H antigen for the HLA-A24-restricted CTL-1B9 clone, which was apparently hematopoietic lineage-specific (Figure 1A) and present in approximately 80% of the Japanese population (data not shown), had not yet been determined. Using these CTL clones, a panel of B-LCLs expressing the restriction HLA (HLA-B44 for CTL-2A12 and HLA-A24 for CTL-1B9) endogenously or retrovirally transduced, were subjected to "immunophenotyping" for the presence or absence of the minor H antigen by ELISA and, if necessary, by standard chromium release assay (CRA).…”

“…9 According to the above criteria, no false-positive SNPs were reported in any array types (Table 1). Confirmation genotyping of individual B-LCLs from both panels revealed none of the 44 that had been immunophenotyped as CTX Ϫ were misjudged, while 8 of the 44 CTX ϩ B-LCLs were found to actually carry no minor H-positive allele for ACC-6, which was likely due to the inclusion of individual B-LCLs showing borderline cytotoxicity (data not shown).…”

“…We eventually focused on rs1879894, as it showed a much more significant genome-wide P value than SNP rs1842353 ( Table 1). In contrast to the CTL-2A12 case, where many mutually correlated SNPs around the most significant one created a broad peak in the statistic plots ( In all SNP array types, a common association peak is observed at 18q21, to which the minor H antigen for CTL-2A12, encoded by the HMSD gene, had been mapped based on expression cloning 9 (arrows).…”

“…Based on this method, termed WGA/CTL, we were able to map the previously characterized ACC-6 minor H locus to a 115-kb block containing only 4 genes, including HMSD. 9 Moreover, using the same approach, a novel minor H antigen encoded by the BCL2A1 gene was identified within a 26-kb block containing only BCL2A1 on chromosome 15q25. Surprisingly, the pool size required to identify these regions was no more than 100 individuals.…”

“…7 CTL-1B9 was isolated from PBMCs harvested on day 30 after transplantation from a patient receiving a marrow graft from his HLA-identical sibling (HLA A11, A24, B39, B51, Cw7, Cw14), and CTL-2A12 has been described recently. 9 …”