(A) CxLN DCs were purified from C57BL/6 mice treated with RMS-fingolimod or -vehicle, or icv fingolimod or vehicle, for 4 weeks. These or acellular vehicle were injected into each pinna of separate C57BL/6 mice prior to MOG DTH assessment (see Methods). Only DCs from fingolimod-treated mice increased MOG DTH responses (RMS-fingolimod, n = 12/group; icv fingolimod, n = 6/group; P = 0.003; 3 independent experiments). (B) Separate C57BL/6 mice were treated with fingolimod or vehicle i.p. for 14 days (n = 10; 3 independent experiments), and CNS DCs were FACS purified and tested in the DTH model, which showed they similarly modified DTH and were not altered by direct fingolimod exposure. (C) 10 mice (5 vehicle; 5 fingolimod) were randomly selected from A, and CxLNs draining the vehicle control– or DC-treated ears were isolated separately for Treg functional assessment. Only the CxLNs draining ears that had RMS-fingolimod DCs delivered had significantly reduced Treg activity (n = 5; P = 0.01). (D) CNS CD45^hiCD3^–CD11c⁺CD11b⁺ and CD45^hiCD3^–CD11c⁺CD11b^– cells were isolated from 10 normal mice, after which each DC subset (2 × 10² cells) was assessed in the DTH model in separate mice. Only CD11b^– CNS DCs significantly reduced DTH to MOG (n = 10; P = 0.011; 3 independent experiments). (E) We repeated the experiment in D, except MOG was replaced by methylated BSA (n = 3/group). There was no significant modulation of methylated BSA response (P > 0.9), which — in contrast to MOG DTH DC–mediated modulation — suggests a CNS-antigen specific mechanism. Data represent mean ± SEM. *P < 0.05.