title: HLA class II peptide tetramers vs allergen-induced proliferation for identification of allergen-specific CD4 T cells.
abstract: BACKGROUND
  Fluorescence-labeled MHC class II/peptide tetramer complexes are considered as optimal tools to characterize allergen-specific CD4(+) T cells, but this technique is restricted to frequently expressed HLA class II molecules and knowledge of immunodominant epitopes. In contrast, allergen-stimulated proliferation assessed by CFSE dilution is less sophisticated and widely applicable. The major mugwort allergen, Art v 1, contains only one single, immunodominant, HLA-DR1-restricted epitope (Art v 125-36 ). Thus, essentially all Art v 1-reactive cells should be identified by a HLA-DRB1*01:01/Art v 119-36 tetramer.
  
  
  METHODS
  We compared specificity and sensitivity of tetramer(+) and allergen-induced proliferating (CFSE(lo) ) CD4(+) T cells by flow cytometry.
  
  
  RESULTS
  The frequency of tetramer(+) CD4(+) T cells determined ex vivo in PBMC of mugwort-allergic individuals ranged from 0 to 0.029%. After 2-3 weeks of in vitro expansion, sufficient tetramer(+) T cells for phenotyping were detected in 83% of Art v 125-36 -reactive T-cell lines (TCL) from mugwort-allergic individuals, but not in TCL from healthy individuals. The tetramers defined bona fide Th2 cells. Notably, Art v 125-36 -reactive TCL depleted of tetramer(+) T cells still reacted to the peptide, and only 44% of Art v 125-36 -specific T-cell clones were detected by the tetramer. CFSE(lo) CD4(+) T cells contained only 0.3-10.7% of tetramer(+) T cells and very low proportions of Th2 cells.
  
  
  CONCLUSIONS
  Allergen-specific T cells can be identified by HLA class II tetramers with high specificity, but unexpected low sensitivity. In contrast, allergen-stimulated CFSE(lo) CD4(+) T cells contain extremely high fractions of bystander cells. Therefore, for T-cell monitoring, either method should be interpreted with caution.
authors.affiliation: Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Van Hemelen
authors.forename: V
authors.initials: V
authors.lastname: Mahler
authors.forename: G
authors.initials: G
authors.lastname: Fischer
authors.forename: I
authors.initials: I
authors.lastname: Fae
authors.forename: V
authors.initials: V
authors.lastname: Reichl-Leb
authors.forename: W
authors.initials: W
authors.lastname: Pickl
authors.forename: M
authors.initials: M
authors.lastname: Jutel
authors.forename: S
authors.initials: S
authors.lastname: Smolinska
authors.forename: C
authors.initials: C
authors.lastname: Ebner
authors.forename: B
authors.initials: B
authors.lastname: Bohle
authors.forename: B
authors.initials: B
authors.lastname: Jahn-Schmid
mesh_terms: D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D010641:Phenotype; D011058:Pollen / Q000009:adverse effects; D055503:Protein Multimerization / Q000276:immunology*; D006255:Rhinitis, Allergic, Seasonal / Q000175:diagnosis / Q000276:immunology; D037182:T-Cell Antigen Receptor Specificity / Q000276:immunology
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D018984:Epitopes, T-Lymphocyte; D000949:Histocompatibility Antigens Class II; D010455:Peptides
keywords: T cells; allergens and epitopes; immunologic tests