Figure 4

CD3/CD28/CpG regulates IL-10 via activation of NF-κB1,pERK, and STAT3. (A) Splenocytes were treated as indicated for 72 hours and IL-10 levels were measured by ELISA. (B) Supernatants from wild type or NF-κB1^-/- splenocytes treated with CD3/CD28, CpG, or TX (CD3/CD28/CpG) for 72 hours were analyzed for IL-10 production by ELISA. (C) Peritoneal macrophages from wild type or NF-κB1^-/- mice were coincubated with purified CD4⁺ T cells from either wild type or NF-κB1^-/- mice, treated with CD3/CD28/CpG for 72 hours and the supernatants were measured for IL-10 expression by ELISA. (D) Splenocytes treated with CD3/CD28, CpG, or TX (CD3/CD28/CpG) for 72 hours were stained for CD4 and pERK or CD4 and pSTAT3. Histograms show pERK or pSTAT3 levels from gated T cells. (E) Quantification of median fluorescence intensity of pERK levels in CD4 cells (N = 3). (F) Splenocytes were treated with ERK inhibitor U0126 at various doses for 72 hours in presence of CD3/CD28/CpG, and IL-10 levels were measure by ELISA. (G) Splenocytes were treated with STAT3 inhibitor NSC 74859 at various doses for 72 hours in presence of CD3/CD28/CpG, and IL-10 levels were measure by ELISA. (H) Wild type or NF-κB1^-/- splenocytes in presence or absence of STAT3 inhibitor (STAT3i, 50 μM) were treated with CD3/CD28/CpG for 72 hours and analyzed for IL-10 production by ELISA. *, P <0.05. N = 3.