title: MHC class II tetramers made from isolated recombinant α and β chains refolded with affinity-tagged peptides.
abstract: Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses.
authors.affiliation: Laboratory of Experimental Immunology, University of Copenhagen, Copenhagen, Denmark ; Allogeneic Hematopoietic Cell Transplantation Laboratory, Department of Hematology, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Braendstrup
authors.forename: Sune
authors.initials: S
authors.lastname: Justesen
authors.forename: Thomas
authors.initials: T
authors.lastname: Osterbye
authors.forename: Lise Lotte Bruun
authors.initials: LL
authors.lastname: Nielsen
authors.forename: Roberto
authors.initials: R
authors.lastname: Mallone
authors.forename: Lars
authors.initials: L
authors.lastname: Vindeløv
authors.forename: Anette
authors.initials: A
authors.lastname: Stryhn
authors.forename: Søren
authors.initials: S
authors.lastname: Buus
mesh_terms: D000328:Adult; D000345:Affinity Labels / Q000737:chemistry*; D000368:Aged; D000595:Amino Acid Sequence; D001782:Blood Donors; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D055503:Protein Multimerization; D058849:Protein Refolding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D013194:Staining and Labeling; D013696:Temperature; D013997:Time Factors
chemical_list: D000345:Affinity Labels; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins
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