Flow cytometric analysis of cytokine expression in short-term allergen-stimulated T cells mirrors the phenotype of proliferating T cells in long-term cultures

• Published in: Journal of immunological methods 2011-08, Vol.371 (1-2), p.114-121
• Main Authors: Van Hemelen, D., Oude Elberink, J.N.G., Bohle, B., Heimweg, J., Nawijn, M.C., van Oosterhout, A.J.M.
• Format: Article
• Language: English
• Subjects: Adult; allergens; Allergens - administration & dosage; Antigen-specific TH cells; Biological and medical sciences; CD154; CD40 Ligand - metabolism; Cell Culture Techniques; Cell Proliferation; CFSE; cytokines; Cytokines - analysis; Female; Flow cytometry; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Grass-pollen allergy; Humans; hypersensitivity; Immunoassay - methods; Immunophenotyping - methods; Male; Molecular immunology; peptides; Phenotype; Pollen - immunology; Rhinitis, Allergic, Seasonal - immunology; T-lymphocytes; T-Lymphocytes, Helper-Inducer - cytology; T-Lymphocytes, Helper-Inducer - immunology; Techniques
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2011.06.019

Allergen-specific TH cells play an important role in IgE-mediated disorders as allergies. Since this TH cell-population only accounts for a small percentage of TH cells, they are difficult to phenotype without prior selection or expansion. Grass-pollen-specific TH 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). Relatively low numbers of allergen-specific TH 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 TH 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 TH cells in allergic vs non-allergic individuals. In addition, higher numbers of IFNγ producing TH cells were detected in long-term cultures compared to the CD154 expressing TH cells. To detect allergen-specific TH cells for a common allergen as grass-pollen, expansion is not absolutely necessary, although within 8-day grass-pollen cultures, higher numbers of proliferating TH cells resulted in increased statistical power to detect phenotypic differences. However, this approach also detects more bystander activated TH cells. TCLs resulted in comparable percentages of cytokine expressing T cells as 8-day cultures. Therefore enrichment can be necessary for detection of TH cells specific for a single allergen or allergen-derived peptides, but is dispensable for the detection and phenotyping of allergen-specific TH cells using crude extracts. ► Phenotypes of CD154-expressing T cells mirror long-term cultured T cell phenotypes. ► 8-day cultures show highest statistical power to detect phenotypical differences. ► Phenotypes of enriched T cell lines are similar to those of 8-day cultured PBMCs.