title: Allergen-specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression.
abstract: 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-gamma 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-gamma 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Dynavax Technologies, Berkeley, CA 94710, USA. DCampbell@dynavax.com
authors.forename: J D
authors.initials: JD
authors.lastname: Campbell
authors.forename: P
authors.initials: P
authors.lastname: Buchmann
authors.forename: S
authors.initials: S
authors.lastname: Kesting
authors.forename: C R
authors.initials: CR
authors.lastname: Cunningham
authors.forename: R L
authors.initials: RL
authors.lastname: Coffman
authors.forename: E M
authors.initials: EM
authors.lastname: Hessel
mesh_terms: D000485:Allergens / Q000276:immunology; D031164:Ambrosia / Q000276:immunology*; D023201:CD40 Ligand / Q000097:blood*; D005434:Flow Cytometry; D006801:Humans; D007167:Immunotherapy; D018417:Th1 Cells / Q000276:immunology*; D018418:Th2 Cells / Q000276:immunology*
chemical_list: D000485:Allergens; D023201:CD40 Ligand