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

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• 作者：D. Van Hemelen^a ; J.N.G. Oude Elberink^b ; B. Bohle^c ; J. Heimweg^a ; M.C. Nawijn^a ; A.J.M. van Oosterhout^a ; ^{a.j.m.van.oosterhout@path.umcg.nl"" rel=""nofollow}
• 关键词：Antigen-specific T_H cells ; Flow cytometry ; Grass-pollen allergy ; CD154 ; CFSE
• 刊名：Journal of Immunological Methods
• 出版年：2011
• 出版时间：31 August 2011
• 年：2011
• 卷：371
• 期：1-2
• 页码：114-121
• 全文大小：539 K

文摘

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.

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.