title: Comparison between major histocompatibility complex class II tetramer staining and surface expression of activation markers for the detection of allergen-specific CD4⁺ T cells.
abstract: BACKGROUND
  Major histocompatibility complex (MHC) class II tetramers (tetramers) allow to detect allergen-specific CD4(+) T cells at a single-cell level. Limits to this technology include HLA restriction and the need to identify immunodominant T cell epitopes.
  
  
  OBJECTIVE
  Assessing the expression of various activation markers following allergen stimulation to replace tetramer staining.
  
  
  METHODS
  Peripheral blood mononuclear cells (PBMCs) from 25 birch pollen, grass pollen or house dust mite allergic individuals were stimulated with peptide mixes encompassing immunodominant epitopes from corresponding major allergens. After 2 weeks of in vitro amplification, cells were stained with both the appropriate tetramer and antibodies directed to CD25, CD30, CD39, CD69, CD137, CD154, GITR, HLA-DR and ICOS, before FACS analysis.
  
  
  RESULTS
  Following allergen stimulation, percentages of tetramer(+) cells among CD4(+) CD154(+) cells range from 5% to 87%, depending upon donors. As for CD154, a large inter-individual variability is observed in terms of surface expression for all activation markers tested in allergen-stimulated PBMCs. T cells reactive with either tetramers (0.4-10.4% CD4(+) T cells) or anti-marker antibodies (2.2-32.7% CD4(+) T cells), but not both, are observed, reflecting the presence of anergic as well as non-specifically activated cells. Tetramer(+) /marker(+) , tetramer(+) /marker(-) and tetramer(-) /marker(+) cells were compared for their capacity to express cytokines, demonstrating that only the former represent bona fide allergen-specific activated CD4(+) T cells, based upon a higher expression of cytokines or corresponding genes in presence of the allergen.
  
  
  CONCLUSIONS
  No strict correlation exists between tetramer staining and the expression of multiple activation markers in stimulated CD4(+) T cells. Dual staining allows to discriminate functional tetramer(+) /marker(+) vs. anergic (tetramer(+) /marker(-) ) allergen-specific T cells or non-specifically activated (tetramer(-) /marker(+) ) T cells. Combining tetramer staining with the detection of activation markers helps understanding patient heterogeneity regarding specific CD4(+) T cell responses. This approach has immediate relevance for monitoring immune changes induced during specific immunotherapy.
authors.affiliation: Stallergenes SA, Antony, France.
authors.forename: M
authors.initials: M
authors.lastname: Bonvalet
authors.forename: E
authors.initials: E
authors.lastname: Wambre
authors.forename: H
authors.initials: H
authors.lastname: Moussu
authors.forename: S
authors.initials: S
authors.lastname: Horiot
authors.forename: W W
authors.initials: WW
authors.lastname: Kwok
authors.forename: A
authors.initials: A
authors.lastname: Louise
authors.forename: D
authors.initials: D
authors.lastname: Ebo
authors.forename: C
authors.initials: C
authors.lastname: Hoarau
authors.forename: L
authors.initials: L
authors.lastname: Van Overtvelt
authors.forename: V
authors.initials: V
authors.lastname: Baron-Bodo
authors.forename: P
authors.initials: P
authors.lastname: Moingeon
mesh_terms: D000485:Allergens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D023201:CD40 Ligand / Q000276:immunology; D016207:Cytokines / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005786:Gene Expression Regulation / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D012221:Rhinitis, Allergic, Perennial / Q000276:immunology
chemical_list: D000485:Allergens; D016207:Cytokines; D000939:Epitopes; D000949:Histocompatibility Antigens Class II; D023201:CD40 Ligand