Successful Use of CD154 Expression and Intracellular Cytokine Staining to Monitor Changes in Allergen-Specific Th2 Cells in Peripheral Blood of Subjects in a Placebo-Controlled Study of Tolamba™, a Novel Immunotherapeutic Vaccine for Ragweed Allergies

Abstract

RATIONALE: Sensitive and accurate measurement of allergen-specific TH cells in PBMC is possible using CD154 expression to identify antigen-reactive T cells (Frentsch et al., Nat. Med. 11:1118). To explore the potential of this method for monitoring allergen therapy, we examined changes in ragweed allergen-specific Th2 cell frequency in Tolamba™ or placebo-treated subjects undergoing controlled ragweed pollen exposure. METHODS: PBMC were collected at the last pre-treatment and post-treatment ragweed exposures from a subset of Tolamba™ (n = 17) and placebo-treated (n = 15) ragweed-allergic rhinitis subjects in an environmental ragweed-exposure chamber study. In vitro allergen-stimulated CD3+CD4+CD154+ T cell intracellular IL-4, IL-5, IL-13 and IFN-γ was evaluated by flow cytometry. RESULTS: Placebo-treated subjects responded to multiple ragweed exposures with significant increases in Amb a 1-specific T cell IL-4 (P = 0.049) and IL-13 (P = 0.048) expression and ragweed-specific T cell IL-5 (P = 0.0006) and IL-13 (P = 0.018) expression. In contrast, Tolamba™-treated subjects demonstrated no significant differences in allergen-specific Th2 cytokine expression from pre- to post-treatment. In comparing pre- to post-treatment changes between Tolamba™ and placebo groups, there was a significant difference in Amb a 1-specific T cell IL-5 expression (P = 0.029) and a trend to significance for changes in Amb a 1-specific T cell IL-4 (P = 0.057) and IL-13 expression (P = 0.061). Allergen-specific T cell IFN-γ expression remained low and un-changed. CONCLUSIONS: Ragweed exposure increased placebo-treated subjects' allergen-specific CD154+ T cell IL-4, IL-5 and IL-13 expression. Tolamba™ treatment prevented these increases. Combined CD154 and intracellular cytokine staining appears to be a sensitive biomarker assay for monitoring changes in specific Th subset frequencies in studies of new clinical interventions.