An HLA-E single chain trimer inhibits human NK cell reactivity towards porcine cells.
HLA-E, when expressed by pig cells, could alleviate human natural killer (NK) cell-mediated rejection of porcine xenografts by providing a potent inhibitory ligand for human NK cells expressing CD94/NKG2A. Yet cell-surface expression of HLA-E on porcine epithelial (LLC-PK1) cells was not observed after transfection with an expression vector harboring HLA-E alone or in combination with an expression vector containing human beta2m. A single chain trimer (SCT) of HLA-E consisting of, in the following order (from N- to C-terminus), the leader peptide of human beta2m, VMAPRTLIL (an HLA-E-binding peptide), a 15 amino acid linker, mature human beta2m, a 20 amino acid linker, and mature HLA-E heavy chain was engineered. Cell-surface expression and correct folding of HLA-E SCT was shown by FACS analyses of stably transfected LLC-PK1 cells. Untransfected LLC-PK1 cells were readily lysed by the NK cell lines NKL and NK-92, while LLC-PK1 cells expressing HLA-E SCT were almost completely protected. In addition, the HLA-E SCT recapitulates the peptide dependent properties of normal HLA-E trimeric complexes in that an HLA-E SCT with an hsp60 derived peptide, though expressed at the cell-surface, did not inhibit NK cell-mediated lysis. The HLA-E SCT, which conferred protection against NK cell-mediated killing, also inhibited NK cell IFN-gamma secretion elicited by co-culture of NKL cells with LLC-PK1 cells. Thus, HLA-E SCT, in which all three components of a normal HLA-E protein complex are in one polypeptide chain, is immunologically functional as it is able to modulate NK cell cytotoxicity and cytokine secretion.
Crew
Mark D
MD
Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences 151 Research, 4300 West 7th Street, Little Rock, AR 72205, USA. crewmarkd@uams.edu
Cannon
Martin J
MJ
Phanavanh
Bounleut
B
Garcia-Borges
Carmen N
CN
D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D015478:Antigens, Heterophile / Q000276:immunology*; D002460:Cell Line; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D004847:Epithelial Cells / Q000166:cytology / Q000276:immunology*; D005434:Flow Cytometry; D006680:HLA Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology*; D007700:Kinetics; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics; D013552:Swine; D014162:Transfection; D000097625:HLA-E Antigens
D015478:Antigens, Heterophile; D016207:Cytokines; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D007371:Interferon-gamma; D021382:Protein Sorting Signals; D000097625:HLA-E Antigens