title: Closing the gap: discrimination of the expression profile of HLA questionable alleles by a cytokine-induced secretion approach using HLA-A*32:11Q.
abstract: Matching of human leukocyte antigen (HLA) alleles between donors and recipients plays a major role in hematopoietic stem cell transplantation (HSCT). Null or questionably expressed HLA allelic variants are a major issue in HLA matching, because the aberrant expression of such alleles can have a major impact on the outcome of HSCT and/or its complications such as graft-versus-host disease. The goal of this study was to investigate the potential of a recently developed cytokine-induced secretion assay to differentiate the expression levels of HLA-A*32:11Q (questionable) into a null (N) or low (L) expression variant. An amino acid mutation at position 164 of HLA-A*32:11Q disrupts the disulfide bridge in the α2 domain. HLA-A*32:11Q is not detectable by standard microlymphocytotoxicity assay. To this end, we cloned soluble HLA-A*32:11Q and a reference allele (HLA-A*32:01) into expression vectors and transfected/transduced HEK293 and K562 cells. Allele-expressing K562 cells were simultaneously transfected/transduced with a β2-microglobulin (B2M)-encoding vector to ensure the intact HLA structure with B2M. After treatment with proinflammatory cytokines, secreted soluble HLA molecules were determined by enzyme-linked immunosorbent assay in the supernatant and intracellular accumulation of the recombinant proteins by flow cytometry. HLA-A*32:11Q was nearly undetectable in untreated transfectants. Cytokine treatment increased the secretion of HLA-A*32:11Q to detectable levels and resulted in intracellular accumulation of the allele. There was no difference in mRNA transcription between the A*32 alleles. On the basis of these results, we recommend reclassification of HLA-A*32:11Q as a low expression (L) variant.
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Föll
authors.forename: J
authors.initials: J
authors.lastname: Hinrichs
authors.forename: S
authors.initials: S
authors.lastname: Tischer
authors.forename: A
authors.initials: A
authors.lastname: Battermann
authors.forename: A
authors.initials: A
authors.lastname: Schambach
authors.forename: C
authors.initials: C
authors.lastname: Figueiredo
authors.forename: S
authors.initials: S
authors.lastname: Immenschuh
authors.forename: R
authors.initials: R
authors.lastname: Blasczyk
authors.forename: B
authors.initials: B
authors.lastname: Eiz-Vesper
mesh_terms: D000483:Alleles; D003001:Cloning, Molecular; D015870:Gene Expression / Q000276:immunology*; D005822:Genetic Vectors; D006086:Graft vs Host Disease / Q000276:immunology / Q000517:prevention & control; D057809:HEK293 Cells; D015234:HLA-A Antigens / Q000235:genetics* / Q000276:immunology; D018380:Hematopoietic Stem Cell Transplantation; D006650:Histocompatibility Testing; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000494:pharmacology; D020014:K562 Cells; D009154:Mutation; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000494:pharmacology; D001613:beta 2-Microglobulin / Q000235:genetics / Q000276:immunology
chemical_list: D015234:HLA-A Antigens; C531148:HLA-A*32 antigen; D020033:Protein Isoforms; D014409:Tumor Necrosis Factor-alpha; D001613:beta 2-Microglobulin; D007371:Interferon-gamma