title: A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens.
abstract: Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.
authors.affiliation: Laboratory of Biochemistry, DCBDC, NCI, NIH, Bethesda, MD 20892, USA.
authors.forename: X
authors.initials: X
authors.lastname: Zhu
authors.forename: S
authors.initials: S
authors.lastname: Bavari
authors.forename: R
authors.initials: R
authors.lastname: Ulrich
authors.forename: S
authors.initials: S
authors.lastname: Sadegh-Nasseri
authors.forename: S
authors.initials: S
authors.lastname: Ferrone
authors.forename: L
authors.initials: L
authors.lastname: McHugh
authors.forename: M
authors.initials: M
authors.lastname: Mage
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015798:HLA-DR1 Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D018089:Superantigens / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
chemical_list: D000942:Antigens, Bacterial; D017931:DNA Primers; D018076:DNA, Complementary; D000939:Epitopes; D015798:HLA-DR1 Antigen; D011994:Recombinant Proteins; D018089:Superantigens