IL-36β acts as an adjuvant to stimulate Th1 responses in vivo. (A-B) Specific IFN-γ production in lymph node cells from WT mice immunized with mBSA/IL-36β. WT (A) and IL-36R^−/− (B) mice were immunized intradermally with mBSA plus PBS, mBSA plus IL-36β, or mBSA plus CFA. At day 21, draining lymph nodes from each group (n = 4) were collected, pooled, and cultured in the absence or presence of 10 μg/mL of mBSA. Culture supernatants were harvested after 3 days of incubation and then assayed for IFN-γ production in response to mBSA (A-B) by ELISA. Values are the mean ± SD of culture triplicates. *P < .05, compared with the value of mBSA/PBS-treated mice. **P < .005, compared with the value of mBSA/PBS-treated mice. (C) Quantification of T-bet mRNA expression by quantitative RT-PCR in draining lymph node cells. Total mRNA was extracted from pooled lymph node cells of each group (n = 4) for T-bet mRNA expression analysis by quantitative RT-PCR. Results represent T-bet mRNA levels relative to GAPDH. (D) Serum levels of anti-mBSA IgG in WT (gray bars) and IL-36R^−/− (black bars) immunized mice. The levels of anti-mBSA IgG were determined by ELISA. Results are expressed as mean ± SD, optical density (OD) units from each group. *P < .05, compared with the value of mBSA/PBS-treated mice.