Th17 Stimulation by DC

To determine the ability of DCs to stimulate naïve CD4+ T cells into T_h17 cells and evaluate the impact of RvE1, we co-cultured naive CD4+ T cells (50,000/200 µl) with CD11c+ DC (1000/200 µl) in U-bottom 96-well plates in 200 μl RPMI containing 10% FBS and 1% Penicillin-Streptomycin. Recent publications show that toll-like receptor (TLR) signaling regulated by DCs was sufficient for T helper cell stimulation and Th17 differentiation (30, 31); Pam₃CSK₄ is a TLR activator necessary for Th17 polarization (32, 33). Thus, Pam₃CSK₄ (100 ng) was used to stimulate cells. Six experimental groups were tested: 1) Unstimulated CD4+ T cells 2) CD4+ T cells+ Pam₃CSK₄ 3) DCs+CD4+ T cells 4) DCs+CD4+ T cells+ RvE1 5) DCs+CD4+ T cells+ Pam₃CSK₄ 6) DCs+ CD4+ T cells+ Pam₃CSK₄+ RvE1. All cells were incubated at 37°C for 5 days. First, cells were treated with 10 nM RvE1 for 24 hours and then stimulated with 100 ng Pam₃CSK₄. Another dose of RvE1 was applied to the cells on day 3. Cells and supernatants were collected for FACS analysis and Multiplex analysis on day 5.