title: Single cell assessment of allergen-specific T cell responses with MHC class II peptide tetramers: methodological aspects.
abstract: BACKGROUND
  We report herein critical methodological principles for assessing, at a single cell level, allergen-specific T cell responses using MHC class II peptide tetramers.
  
  
  METHODS
  We developed MHC class II peptide tetramers to monitor T cell responses against the immunodominant Bet v 1(141-155) peptide in individuals with either an HLA-DRB1*0101, DRB1*0401 or DRB1*1501 background. In vitro stimulation was performed with serially truncated versions of the Bet v 1(141-155) epitope chemically conjugated to the Ii-Key peptide.
  
  
  RESULTS
  Identification of Bet v 1(141-155) as a high-affinity epitope for multiple HLA-DRB1 allotypes led to the development of corresponding tetramers detecting Bet v 1(141-155)-specific T cells with a high specificity and sensitivity. Stimulation with Bet v 1(141-155) Ii-Key conjugate peptides is the most efficient procedure to expand Bet v 1(141-155)-specific CD4+ T cells, allowing to detect such cells in both allergic and healthy individuals. MHC class II Bet v 1(141-155) tetramer-positive T cells produce IFN-gamma and IL-10 in healthy individuals, and IL-5 in allergic patients. Frequencies of Bet v 1-specific CD4+ T cells circulating in the blood of allergic or nonallergic individuals range from approximately 10(-5) to 10(-3) CD4+ T cells, outside or within the pollen season, respectively.
  
  
  CONCLUSIONS
  MHC class II peptide tetramers are valuable tools to assess allergen-specific T cell responses, both qualitatively and quantitatively. Selection of a high-affinity T cell epitope, as well as optimization of in vitro stimulation conditions to expand rare T cell progenitors are critical success factors in those analyses.
authors.affiliation: Stallergènes SA, Antony, France.
authors.forename: Erik
authors.initials: E
authors.lastname: Wambre
authors.forename: Laurence
authors.initials: L
authors.lastname: Van Overtvelt
authors.forename: Bernard
authors.initials: B
authors.lastname: Maillère
authors.forename: Robert
authors.initials: R
authors.lastname: Humphreys
authors.forename: Eric
authors.initials: E
authors.lastname: von Hofe
authors.forename: Lydia
authors.initials: L
authors.lastname: Ferhat
authors.forename: Didier
authors.initials: D
authors.lastname: Ebo
authors.forename: Philippe
authors.initials: P
authors.lastname: Moingeon
mesh_terms: D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006684:HLA-DR Antigens / Q000276:immunology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology; D016130:Immunophenotyping / Q000379:methods*; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D010940:Plant Proteins / Q000138:chemical synthesis / Q000276:immunology; D011485:Protein Binding / Q000276:immunology
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; D006684:HLA-DR Antigens; D016056:Immunodominant Epitopes; D010455:Peptides; D010940:Plant Proteins; C061312:Bet v 1 allergen, Betula