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

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 Tv, 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 IFN gamma producing T-H cells were detected in long-term cultures compared to the CD154 expressing T-H cells. Conclusion: 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. (C) 2011 Elsevier B.V. All rights reserved.