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...

Acute myeloid leukemia (AML) has been thought to be the consequence of two broad complementation classes of mutations: class I and class II. However, overlap-mutations between them or within the same class and the position of TP53 mutation are not fully analyzed. We comprehensively analyzed the FLT3, cKIT, N-RAS, C/EBPA, AML1, MLL, NPM1, and TP53 mutations in 144 newly diagnosed de novo AML. We found 103 of 165 identified mutations were overlapped with other mutations, and most overlap-mutations consisted of class I and class II mutations. Although overlap-mutations within the same class were found in seven patients, five of them additionally had the other class mutation. These results suggest that most overlap-mutations within the same class might be the consequence of acquiring an additional mutation after the completion both of class I and class II mutations. However, mutated genes overlapped with the same class were limited in N-RAS, TP53, MLL-PTD, and NPM1, suggesting the possibility that these irregular overlap-mutations might cooperatively participate in the development of AML. Notably, TP53 mutation was overlapped with both class I and class II mutations, and associated with morphologic multilineage dysplasia and complex karyotype. The genotype consisting of complex karyotype and TP53 mutation was an unfavorable prognostic factor in entire AML patients, indicating this genotype generates a disease entity in de novo AML. These results collectively suggest that TP53 mutation might be a functionally distinguishable class of mutation.

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The interactions between chemokines and their receptors may have an important role in initiating GVHD after allogeneic hematopoietic SCT (allo-HSCT). CCL25 and CCR9 are unique because they are exclusively expressed in epithelial cells and in Peyer's patches of the small intestine. We focused on rs12721497 (G926A), one of the non-synonymous single nucleotide polymorphisms (SNPs) in the CCR9 gene, and analyzed the SNP of donors in 167 consecutive patients who received allo-HSCT from an HLA-identical sibling donor. Genotypes were tested for associations with acute and chronic GVHD in each organ and transplant outcome. Multivariate analyses showed that the genotype 926AG was significantly associated with the incidence of acute stage X2 skin GVHD (hazard ratio: 3.2; 95% confidence interval (95% CI): 1.1-9.1; P ¼ 0.032) and chronic skin GVHD (hazard ratio: 4.1; 95% CI: 1.1-15; P ¼ 0.036), but not with GVHD in other organs or with relapse, non-relapse mortality or OS. To clarify the functional differences between genotypes, each SNP in retroviral vectors was transfected into Jurkat cells. In chemotaxis assays, the 926G transfectant showed greater response to CCL25 than the 926A transfectant. In conclusion, more active homing of CCR9-926AG T cells to Peyer's patches may produce changes in Ag presentation and result in increased incidence of skin GVHD.

Summary Minor histocompatibility antigens (mHags) play crucial roles in the induction of graft versus host disease (GVHD) and/or graft versus leukaemia (GVL) effects following human leucocyte antigen (HLA)‐identical haematopoietic stem cell transplantation (HSCT). Using HLA‐A*3101‐ and ‐A*3303‐restricted cytotoxic T lymphocyte (CTL) clones generated from different post‐HSCT recipients, we identified two novel mHag epitopes encoded by the leader sequence of cathepsin H (CTSH) isoform a. The nonameric sequence ATLPLLCAR was defined as an HLA‐A*3101‐restricted epitope (CTSHR/A31), while a decameric peptide featuring a one N‐terminal amino acid extension, WATLPLLCAR, was presented by HLA‐A*3303 (CTSHR/A33). The immunogenicity of both epitopes was totally dependent on the polymorphic C‐terminal arginine residue and substitution with glycine completely abolished binding to the corresponding HLA molecules. Thus, the immunogenicity of this mHag is exerted by differential HLA binding capacity. CTSH is relatively ubiquitously expressed at protein levels, thus it may be involved in GVHD and anti‐leukaemic/tumour responses. Interestingly, however, CTL clones predominantly lysed targets of haematopoietic cell origin, which could not be explained in terms of the immunoproteasome system. Although the mechanisms involved in the differential susceptibility remain to be determined, these data suggest that CTSH‐encoded mHags could be targets for GVL effects.