Chemokine expression in CLN of sublingually treated mice and the effect of sublingual administration of pCCL19/pCCL21 with antigen on Th2-mediated allergic responses. (a) Semiquantitative RT-PCR was performed to assess mRNA expression pattern of chemokines, CCL19 and CCL21 in whole cells isolated from CLN of mice sublingually treated with either PBS or OVA. (b) Mice were sublingually administered with either PBS, OVA alone, 100 μg of mock DNA with or without OVA, 100 μg of pCCL19 with or without OVA, and 100 μg of pCCL21 with or without OVA for total three times before systemic sensitization and nasal challenge. OVA-specific IgE levels in serum were assayed by sandwich ELISA. (c) Messenger RNA expression of IL-4, IL-5, and IL-13 in CD4⁺ T cells isolated from spleen of mice sublingually treated with PBS, OVA, OVA plus pCCL19, and OVA plus pCCL21 was determined by quantitative real-time PCR analysis. The expression of each molecule was normalized to the expression of GAPD. Each data was expressed as a ratio relative to mean expression level in PBS-treated control mice. (d) Semiquantitative RT-PCR was performed to assess Foxp3- and IL-10-specific mRNA expression in CD4⁺ T cells isolated from CLN of sublingually treated mice. Data are representative of two separate experiments containing three to five mice in each group. Significance was evaluated by Kruskal-Wallis test (b, c). . N.D. not detected.