CD5 costimulation requires IL-6 for optimal Th17 differentiation. (A) FACS-sorted naive CD4⁺CD45RA⁺CD45RO⁻ T cells were stimulated via plate-coated CD3/CD28 or CD3/CD5 antibodies under Th17 polarizing conditions (30 ng/mL IL-23, 10 ng/mL IL-1β, 10 ng/mL IL-6, 10 ng/mL TGF-β and 10μg/mL anti-IFN-γ) in the presence of 10 U/mL of IL-2. Intracellular levels of IL-17A were measured at day 11 after 5 hours of restimulation with PMA, ionomycin, and BFA. Data are from 7 independent experiments of individual donors. Statistical analyses were performed with paired t test. (B) Naive T cells were stimulated by plate-bound CD3/CD28 or CD3/CD5 antibodies and cultured in Th17-inducing conditions. Phospho-STAT3 levels were measured by FACS. Data shown are from 6 individual experiments with different donors, with mean + SD. (C-D) Real-time semi-quantitative PCR (ABI PRISM 7000, SYBR green method) of IL23R (C) and RORC (D) mRNA expression of naive T cells stimulated for 3 days via coated CD3/CD28 or CD3/CD5 antibodies under Th17-polarizing conditions. Results were normalized to 18S rRNA. Data shown are mean ± SD of triplicate measurement from 1 representative experiment of 3 independent experiments using different donors.