title: Refinement of molecular approaches to improve the chance of identification of hematopoietic-restricted minor histocompatibility antigens.
abstract: Minor histocompatibility antigens (mHAgs) constitute the target antigens of the T cell-mediated graft-versus-leukemia response after HLA-identical allogeneic stem cell transplantation (SCT). Several human mHAgs have been identified, but only a few are selectively expressed by hematopoietic cells representing potential targets for specific immunotherapy. Molecular approaches including cDNA library screening and genetic linkage analysis have been successfully applied to identify T cell-defined mHAgs, but each approach has its drawbacks which may lead to mis-identification of the mHAg of interest. We improved both molecular strategies to facilitate more robust identification of hematopoietic-restricted mHAgs. First, we adapted cDNA library cloning by using 293T cells with stable expression of the relevant MHC class I allele, CD80 and CD54. We demonstrated that cDNA library screening using this 293T expression system results in strong activation of cytotoxic T lymphocytes, which significantly contributes to improvement of the assay sensitivity. Second, we refined genetic linkage analysis using single nucleotide polymorphism (SNP) genotyping to narrow down the defined genetic region that holds the mHAg-encoding gene. We showed that SNP marker analysis provides additional information about the genetic position of the antigen-encoding gene. Application of these optimized molecular approaches will lead to more rapid and reliable molecular identification of hematopoietic-restricted mHAgs.
authors.affiliation: Central Hematology Laboratory, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Björn
authors.initials: B
authors.lastname: de Rijke
authors.forename: Agnes
authors.initials: A
authors.lastname: van Horssen-Zoetbrood
authors.forename: Sharon
authors.initials: S
authors.lastname: Veenbergen
authors.forename: Hanny
authors.initials: H
authors.lastname: Fredrix
authors.forename: Theo
authors.initials: T
authors.lastname: de Witte
authors.forename: Elly
authors.initials: E
authors.lastname: van de Wiel-van Kemenade
authors.forename: Harry
authors.initials: H
authors.lastname: Dolstra
mesh_terms: D000818:Animals; D018122:B7-1 Antigen / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005260:Female; D005787:Gene Frequency; D015723:Gene Library; D008040:Genetic Linkage; D015235:HLA-B Antigens / Q000235:genetics; D059948:HLA-B44 Antigen; D015793:HLA-B7 Antigen; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D007371:Interferon-gamma / Q000096:biosynthesis; D020014:K562 Cells; D008213:Lymphocyte Activation / Q000235:genetics*; D008297:Male; D015778:Minor Histocompatibility Antigens / Q000032:analysis / Q000235:genetics*; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection
chemical_list: D018122:B7-1 Antigen; D015235:HLA-B Antigens; C477444:HLA-B*07:02 antigen; C495439:HLA-B*44:03 antigen; D059948:HLA-B44 Antigen; D015793:HLA-B7 Antigen; D015778:Minor Histocompatibility Antigens; D018799:Intercellular Adhesion Molecule-1; D007371:Interferon-gamma