title: Flow cytometric analysis of cytokine expression in short-term allergen-stimulated T cells mirrors the phenotype of proliferating T cells in long-term cultures.
abstract: BACKGROUND
  Allergen-specific T(H) cells play an important role in IgE-mediated disorders as allergies. Since this T(H) cell-population only accounts for a small percentage of T(H) cells, they are difficult to phenotype without prior selection or expansion.
  
  
  METHODS
  Grass-pollen-specific T(H) cell profiles were evaluated in 5 allergic and 4 non-allergic individuals using three different approaches: CD154 expression on ex vivo grass-pollen-activated PBMCs (i); CFSE-dilution in grass-pollen-restimulated PBMCs (ii) and T cell lines enriched for allergen-specific T cells (iii).
  
  
  RESULTS
  Relatively low numbers of allergen-specific T(H) cells were detected using CD154 expression, limiting the power to detect phenotypic differences between allergic and non-allergic individuals. In contrast, higher frequencies of proliferating T(H) cells were detected by loss-of-CFSE intensity in PBMCs and TCLs after grass-pollen-stimulation, resulting in the detection of significantly more IL-4 producing T(H) cells in allergic vs non-allergic individuals. In addition, higher numbers of IFNγ producing T(H) cells were detected in long-term cultures compared to the CD154 expressing T(H) cells.
  
  
  CONCLUSIONS
  To detect allergen-specific T(H) cells for a common allergen as grass-pollen, expansion is not absolutely necessary, although within 8-day grass-pollen cultures, higher numbers of proliferating T(H) cells resulted in increased statistical power to detect phenotypic differences. However, this approach also detects more bystander activated T(H) cells. TCLs resulted in comparable percentages of cytokine expressing T cells as 8-day cultures. Therefore enrichment can be necessary for detection of T(H) cells specific for a single allergen or allergen-derived peptides, but is dispensable for the detection and phenotyping of allergen-specific T(H) cells using crude extracts.
authors: Laboratory of Allergology and Pulmonary Diseases, Dept. Pathology and Medical Biology, University Medical Center of Groningen, GRIAC Research Institute, University of Groningen, The Netherlands.
authors: D
authors: D
authors: Van Hemelen
authors: J N G
authors: JN
authors: Oude Elberink
authors: B
authors: B
authors: Bohle
authors: J
authors: J
authors: Heimweg
authors: M C
authors: MC
authors: Nawijn
authors: A J M
authors: AJ
authors: van Oosterhout
mesh_terms: D000328:Adult; D000485:Allergens / Q000008:administration & dosage; D023201:CD40 Ligand / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D016207:Cytokines / Q000032:analysis*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D016130:Immunophenotyping / Q000379:methods; D008297:Male; D010641:Phenotype; D011058:Pollen / Q000276:immunology; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology; D006377:T-Lymphocytes, Helper-Inducer / Q000166:cytology / Q000276:immunology*
chemical_list: D000485:Allergens; D016207:Cytokines; D023201:CD40 Ligand