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Allergen-specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression


J. D. Campbell, Dynavax Technologies, 2929 Seventh Street Ste. 100, Berkeley, CA 94710, USA. E-mail:


Background A sensitive measurement of low numbers of intracellular cytokine-expressing antigen-specific T cells from peripheral blood mononuclear cells (PBMC) is possible using CD154 as a marker of recently activated T cells. This technique may have potential for monitoring peripheral blood T cell responses to immunotherapy.

Objective To evaluate the applicability of this method for measuring changes in cytokine production by allergen-specific T cells in a clinical trial setting.

Methods Ex vivo ragweed-specific CD154 and intracellular cytokine expression were evaluated using a subset of subjects in an environmental chamber study of allergic rhinitis immunotherapy. PBMC were collected and cryopreserved from Amb a 1-immunostimulatory oligodeoxynucleotide conjugate (AIC)-treated (n=17) and placebo-treated (n=15) ragweed-allergic subjects both after pre- and post-treatment ragweed exposures. In vitro allergen-stimulated CD3+CD4+CD154+ T cell intracellular IL-4, IL-5, IL-13, and IFN-γ expression were evaluated by flow cytometry.

Results Compared with the T helper type 2 (Th2) cytokine expression measured after pre-treatment ragweed exposures, placebo-treated subjects demonstrated a significantly elevated ragweed- and Amb a 1-specific T cell IL-4 and IL-13 co-expression (P=0.005 and P=0.022, respectively) and a significantly elevated ragweed-specific IL-5 expression (P<0.001) following post-treatment ragweed exposures. In contrast, AIC-treated subjects demonstrated no increases in allergen-specific Th2 cytokine expression following post-treatment ragweed exposures. IFN-γ expression remained low and un-changed in both groups. Subject reported total nasal symptom scores demonstrated modest but significant correlations with Amb a 1- and ragweed-stimulated intracellular Th2 cytokine responses.

Conclusion Combined CD154 and intracellular cytokine staining in PBMC can be used to sensitively monitor changes in antigen-specific T cell subset frequencies in clinical studies. Antigen-specific cytokine expression moderately correlated with the reported levels of allergic symptoms.

Trial Registration NCT00537355

Cite this as: J. D. Campbell, P. Buchmann, S. Kesting, C. R. Cunningham, R. L. Coffman and E. M. Hessel, Clinical & Experimental Allergy, 2010 (40) 1025–1035.