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

Abstract

Minor histocompatibility (H) antigens are the molecular targets of allo-immunity responsible both for the development of antitumor effects and for graft-versushost disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, despite their potential clinical use, our knowledge of human minor H antigens is largely limited by the lack of efficient methods of their characterization. Here we report a robust and efficient method of minor H gene discovery that combines whole genome association scans (WGASs) with cytotoxic Tlymphocyte (CTL) assays, in which the genetic loci of minor H genes recognized by the CTL clones are precisely identified using pooled-DNA analysis of immortalized lymphoblastoid cell lines with/without susceptibility to those CTLs. Using this method, we have successfully mapped 2 loci: one previously characterized (HMSD encoding ACC-6), and one novel. The novel minor H antigen encoded by BCL2A1 was identified within a 26 kb linkage disequilibrium block on chromosome 15q25, which had been directly mapped by WGAS. The pool size required to identify these regions was no more than 100 individuals. Thus, once CTL clones are generated, this method should substantially facilitate discovery of minor H antigens applicable to targeted allo-immune therapies and also contribute to our understanding of human allo-immunity. (Blood. 2008;111: 3286-3294)

Identification of 4 new HLA-DR–restricted minor histocompatibility antigens as hematopoietic targets in antitumor immunity

“…30,31 Most of these techniques did not allow or were difficult to adapt for identification of HLA-class IIrestricted mHags. By screening a recombinant bacteria cDNA library, 32 we could now identify 4 unknown CD4 ϩ T-cell specificities.…”

“…A 15-mer peptide comprising amino acids shared by the 2 19-mer peptides also stimulated IFN-␥ release, whereas a 15-mer peptide containing the donor variant H at position 39 was not recognized ( Figure 3A). Serial truncations of the 15-mer peptide at the N and C termini led to the identification of a minimal 12-mer YFRLGVSDPIRG peptide (amino acids [29][30][31][32][33][34][35][36][37][38][39][40], designated LB-MR1-1R ( Figure 3A). …”

“…[23][24][25][26] Because under noninflammatory or low-inflammatory conditions HLA-class II molecules are predominantly expressed on hematopoietic cells, stimulating CD4 ϩ T-cell immunity late after alloSCT may be another promising strategy to selectively target residual leukemia without inducing severe GVHD. [27][28][29] Although several HLA-class I-restricted mHags have been identified by various methods, [5][6][7][8]10,11,[13][14][15][16][17]30,31 the discovery of HLA-class II-restricted mHags proved to be more difficult and was hampered by technical limitations. We developed a method by which recombinant bacteria cDNA expression libraries were used to identify antigens as targets for CD4 ϩ T cells.…”

Isolation of Human CD4/CD8 Double-Positive, Graft-Versus-Host Disease–Protective, Minor Histocompatibility Antigen–Specific Regulatory T Cells and of a Novel HLA-DR7–Restricted HY-Specific CD4 Clone

“…Several HLA class I-restricted minor H Ags have been identified in humans by screening of plasmid cDNA libraries, elution of HLA-bound peptides, and genetic linkage analysis (13)(14)(15)(16). Recently, a novel strategy using whole-genome association scanning reported characterization of 10 new HLA class I-restricted minor H Ags (17).…”

Discovery of T Cell Epitopes Implementing HLA-Peptidomics into a Reverse Immunology Approach

“…Over the years, increasing numbers of MiHA have been identified by cDNA library screening (10)(11)(12), genetic linkage analysis (13)(14)(15), peptide elution from HLA (16)(17)(18), and genome-wide association analysis (19)(20)(21). Collectively, these methods use a forward immunology approach characterized by the isolation of activated T cells from patients demonstrating a clinical response to DLI after allo-SCT and subsequent elucidation of the Ags recognized.…”