A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of α chain, β chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens

Xiaojie Zhu, Sina Bavari, Robert Ulrich, Scheherazade Sadegh-Nasseri, Soldano Ferrone, Louise McHugh, Michael Mage

Research output: Contribution to journalArticle

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 homogenous 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 β chain. Co-transfection with DR α 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 β chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR α chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, β chain, and α 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 α 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.

Original languageEnglish (US)
Pages (from-to)1933-1941
Number of pages9
JournalEuropean Journal of Immunology
Volume27
Issue number8
DOIs
StatePublished - Aug 1 1997

Keywords

  • Covalent peptide
  • HLA-DR1
  • Single-chain
  • Superantigen

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Fingerprint Dive into the research topics of 'A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of α chain, β chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens'. Together they form a unique fingerprint.

  • Cite this