title: Discrimination of HLA null and low expression alleles by cytokine-induced secretion of recombinant soluble HLA.
abstract: The disruption of disulfide bridges can decrease or abolish the cell surface expression of HLA class I molecules. Such disulfide bridges are formed by cysteine residues between amino acid (aa) positions 101/164 (alpha(2) domain) and 203/259 (alpha(3) domain). Sequence alterations in codons 101, 164, 203 and 259 have been observed in eleven HLA-A molecules. All of these variants except of A*3014L and A*3211Q have been reported to result in null expression alleles. In the case of HLA-A*3014L, a transversion at nucleotide position 563 replaces cysteine by serine at position 164 of the mature polypeptide. HLA-A*3014L is not detectable by standard microlymphocytotoxicity assay. To verify low or non-expression of this allele, we cloned soluble HLA-A*3014L and the reference allele HLA-A*3001 into a eukaryotic expression vector and transfected K562, C1R and HEK293 cells. Expression of soluble HLA-A*3014L and HLA-A*3001 was measured in the supernatants of transfected and untransfected cells incubated with or without IFN-gamma and/or TNF-alpha using a W6/32 and anti-beta(2)-microglobulin-based sandwich ELISA. Expression of mRNA transcripts of both alleles was determined by real-time RT-PCR. HLA-A*3014L was not detected in the supernatant of unstimulated transfectants. Stimulation with IFN-gamma and/or TNF-alpha led to an increase of HLA-A*3014L secretion to a detectable level and increased HLA-A*3001 expression up to 8-fold, but did not show any difference in the increase of mRNA levels between HLA-A*3014L and A*3001. Because of this lack of any difference in the mRNA transcription, the protein expression defect is most likely caused by the missing disulfide bond formation in the alpha2 domain. Thus, exposing the cells to cytokine stress allows to distinguish between low- and non-expressed alleles and to classify alleles with a questionable expression pattern (Q alleles). Classifying HLA alleles in expressed and non-expressed variants is essential for matching assessments. Additionally, this discrimination between cytokine inducible and non-inducible defect alleles may be important in allotransplant settings in which a cytokine storm usually occurs following pre-transplant myeloablative conditioning or post-transplant immunosuppressive therapy.
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Strasse Strasse 1, 30625 Hannover, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Hinrichs
authors.forename: Constança
authors.initials: C
authors.lastname: Figueiredo
authors.forename: Kaimo
authors.initials: K
authors.lastname: Hirv
authors.forename: Joannis
authors.initials: J
authors.lastname: Mytilineos
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.forename: Peter A
authors.initials: PA
authors.lastname: Horn
authors.forename: Britta
authors.initials: B
authors.lastname: Eiz-Vesper
mesh_terms: D000483:Alleles; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016207:Cytokines / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D015234:HLA-A Antigens / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D020014:K562 Cells; D009154:Mutation / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism*; D055571:Secretory Pathway / Q000187:drug effects* / Q000235:genetics; D012995:Solubility; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D016207:Cytokines; D015234:HLA-A Antigens; C541139:HLA-A*30:01 antigen; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma