Fig. 5.
MyD88 is required for IL-23 signaling and Th17 cell differentiation in vivo. (A) CD4+ T cells from Myd88fl/fl ERCreT2 mice were cultured and treated at day 1 with 4-OHT (1 μM) or untreated. MyD88 and β-actin were detected by immunoblot analysis from the whole-cell lysates prepared at day 3 or 5. Densitometry values normalized to β-actin are shown in parentheses. (B and D) CD4+ T cells were activated and cultured initially with TGF-β + IL-6 or TGF-β + IL-6 + IL-1β. 4-OHT (1 μM) was added to all of the cultures at day 2, and IL-23 (20 ng/mL) was added to the indicated culture at day 3. On day 5, IL-17A production in culture supernatants was assessed by ELISA (B), or qRT-PCR was performed to measure the levels of Il17a and Il23r (D). *P < 0.05. NS, not significant. (C) CD4+ T cells were cultured as indicated in B without 4-OHT. IL-17A production was measured by ELISA on day 5. *P < 0.05. NS, not significant. Results are representative of three independent experiments (A) or two independent experiments (B–D). (E) WT or Myd88ΔT mice were immunized with 100 µg of MOG peptide in complete Freund’s adjuvant and given 100 ng of pertussis toxin IV on day 0 and 2 postimmunization. Mice were scored (0–5) daily for evidence of clinical disease (n = 16 for WT; n = 13 for Myd88ΔT). Shown are the combined data of three independent experiments, all of which yielded similar results. Error bars represent SEM.