title: Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining.
abstract: HLA class I tetramers have revolutionized the study of Ag-specific CD8+ T cell responses. Technical problems and the rarity of Ag-specific CD4+ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4+ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4+ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4+ cells) in human PBLs analyzed directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4+ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques.
authors.affiliation: The Peter Medawar Building for Pathogen Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Scriba
authors.forename: Marco
authors.initials: M
authors.lastname: Purbhoo
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Day
authors.forename: Nicola
authors.initials: N
authors.lastname: Robinson
authors.forename: Sarah
authors.initials: S
authors.lastname: Fidler
authors.forename: Julie
authors.initials: J
authors.lastname: Fox
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Weber
authors.forename: Paul
authors.initials: P
authors.lastname: Klenerman
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Sewell
authors.forename: Rodney E
authors.initials: RE
authors.lastname: Phillips
mesh_terms: D000595:Amino Acid Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D000939:Epitopes / Q000235:genetics; D016655:HIV Core Protein p24 / Q000235:genetics / Q000276:immunology; D015658:HIV Infections / Q000276:immunology; D015798:HLA-DR1 Antigen / Q000737:chemistry / Q000378:metabolism*; D015804:HLA-DR4 Antigen / Q000737:chemistry / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping / Q000379:methods* / Q000706:statistics & numerical data; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology; D020836:Protein Structure, Quaternary; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013745:Tetanus Toxoid / Q000235:genetics / Q000276:immunology; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000939:Epitopes; D016655:HIV Core Protein p24; D015798:HLA-DR1 Antigen; D015804:HLA-DR4 Antigen; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D010446:Peptide Fragments; D010750:Phosphoproteins; D013745:Tetanus Toxoid; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; C081942:influenza hemagglutinin (307-319); C093151:tetanus toxoid (830-843)