Secretion of Th1- and Th2-type cytokines by polarized CD4⁺cells. (A) Cytokine secretion assays. Control mRNA–transfected (–OX40L) or OX40L mRNA–transfected (+OX40L) immature or cytokine cocktail–matured DCs were used to polarize allogeneic CD4⁺CD45RA⁺ T cells at responder-to-stimulator ratios of 10: 1. After 5 days, T cells were stimulated and analyzed by cytokine secretion assay and double-stained with anti–interferon-γ–phycoerythrin (PE) and anti–CD4–fluorescein isothiocyanate (FITC) antibody. The percentage of CD4⁺/interferon-γ⁺ cells is indicated. (B) Cytometric bead arrays. CD4⁺CD45RA⁺ T cells were polarized with allogeneic control mRNA–transfected and cytokine cocktail–matured DCs (gray histograms) or allogeneic OX40L-transfected CC/PGE2-matured DCs (black overlays) and analyzed via cytometric bead array for secretion of Th1-type cytokines IL-2, IFN-γ, TNF-α (top row) or Th2-type cytokines IL-4, IL-5, IL-10 (bottom row) in cytometric bead arrays or by IFN-γ and IL-4 ELISA (C). (D, left panel) Frequency of tetanus toxoid (TTp30) peptide-specific Th1 (IFN-γ)– and Th2 (IL-4)–type CD4⁺ T-helper cells within the CD4⁺ population of a healthy donor were determined by interferon-γ ELISPOT analysis. (D, right panel) CD45RA⁺naive CD4⁺ T cells isolated from the same donor were stimulated with cytokine cocktail–matured, tetanus toxoid–pulsed DC with (+OX40L) or without (–OX40L) mRNA transfection. After 7 days, cells were restimulated with autologous DCs pulsed with the DR11.5-restricted helper epitope TTp30, and frequencies of TTp30 epitope–specific CD4⁺ T helper cells were determined by IFN-γ and IL-4 ELISPOT assays. Unpulsed, autologous DCs were used as control stimulators (control). Results are presented as mean number of spots with SD calculated from triplicate wells.