Fibroblasts promote Th17 development from TCs via the stimulation of IL-23 from activated DCs. LPS-preactivated DCs were cocultured with fibroblasts. After 18 hours, fibroblasts were removed from the coculture using anti–Thy-1–coupled magnetic beads. As control, LPS-preactivated DCs (DCact) were cultured alone. Subsequently, CD3⁺ TCs or CD4⁺ were cultured alone (TC) or with either LPS-preactivated DCs (TC + DCact) or LPS-preactivated DCs isolated from the coculture with fibroblasts (TC + [DCact + Fb]) in the presence of respective coculture supernatants. (A,D-E) After 6 days of coculture, IL-17A (A), IL-22 (D), and IFN-γ (E) production was measured by ELISA. *P < .01, compared with TC + DC_act (n = 5 independent experiments). (B-C) After 3 days of coculture, TCs were restimulated with phorbol myristate acetate/ionomycin in the presence of brefeldin A and IL-17A expression (B) and transcription factor RORγt expression (C) were measured by intracellular flow cytometric staining. One representative experiment of 3 is shown. (F) Coculture of CD3⁺ TCs and DC_act (TC + DCact) or with fibroblast-stimulated DC_act (TC + [DCact + Fb]) were performed without antibody (TC + [DCact + Fb]) in the presence of a control antibody (TC + [DCact + Fb + ctr ab]), a neutralizing anti–IL-6 (TC + [DCact + Fb + aIL-6]), or anti–IL-23 antibody (TC + [DCact + Fb + aIL-23]). After 5 days, IL-17A was quantified by ELISA. *#P < .05 (n = 7 independent experiments). (G) IL-17A secretion of CD3⁺ Pan TCs, CD4⁺ TCs, CD4⁺CD45RO⁺ memory TCs, and CD4⁺CD45RA⁺ naive TCs was compared by culturing the different TC types with medium (aTC), supernatants of LPS-preactivated DC (aTC + [DCact]sn), or supernatants of DCact-fibroblast coculture (aTC + [DCact + Fb]sn) in the presence of CD3/CD28 beads for 5 days. *P < .05, compared with aTC. #P < .01, compared with aTC[DCact]sn (n = 4 independent experiments).