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Figure S1. Immunological characterization of BM-DC used for immunization. (a) CD11c expression, measured at days 1, 4, 7, 10 and 14 of BM culture by flow cytometry (FACS) in three independent experiments, demonstrating a peak of 80–90% at day 10 of culture. (b) DC-45D, as prepared for immunization, was characterized for surface marker expression by FACS analysis, as compared to DC-45D activated with LPS (LPS DC-45D). Out of total CD11c+ cells in the cell preparation used for immunization, 8.3% expressed CD83, 33.2% expressed B7-1 (CD80), 23.5% expressed B7-2 (CD86), and 7.8% expressed CD40. Results are expressed as % ± SD. According to the cell surface profile used to characterize the DCs, no significant differences were detected between the DC-45D and DC (data not shown). Secretion levels of pro-inflammatory cytokine in DC-45D, such as interleukin (IL)-12, IL-6, IL-1α and TNFα, measured by ELISA, were significantly lower than those obtained from LPS DC-45D (data not shown). (c and d) DC-45D evoked a specific immune response in vivo, assessed by a lymphocyte proliferation assay using CFSE staining (see Methods). Mice were immunized with either DC-45D or with peptide-free DCs (Muir et al.). After 6 days, lymph nodes were tested ex vivo for proliferation in the presence of either MOG45D (the relevant neuropeptide that was injected in vivo) or Nogo-A (an irrelevant neuropeptide as control). (c) Significant proliferation of CD3+ T cells (= 0.024) and (d) CD19+ B cells (= 0.04) was observed specifically in DC-45D-immunized mice relative to DC-immunized mice in response to MOG45D (index, ± SD). Asterisks indicate statistical significance analyzed by the Student’s t-test: *< 0.05, **< 0.005, ***< 0.0001.

Figure S2. No signs of demyelination were found following DC-immunization in AD-Tg mice. Representative microscope images show two myelin-rich areas in the brain, corpus callosum and striatum, stained with (a–d) anti-MBP, and (e–h) Luxol fast blue, with no detectable demyelination. Scale bar scale indicates 100 μm.

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