Female mice, aged-matched at 8–16 wk, were used in the described experiments. Treatment of animals was in compliance with federal and institutional guidelines, and approved by the TPIMS institute animal care and use committee. T-cell lines reactive to TCR peptides B1, B4 or B5, or MBP Ac1-9 were generated from naive B10.PL mice by stimulating splenocytes with peptide

(40 μg/mL) in RPMI 1640 media containing 10% FBS 6. CD4+ T-cell selleck chemical clones were isolated from peptide-reactive T-cell lines by the technique of two sequential limiting dilution clonings at 0.2 cells per well (as previously described, 6). T-cell line and clone cultures were maintained by the addition of rIL-2 (10 U/mL) every 3 days, and stimulated with TCR peptide and irradiated autologous spleen cells (2–5×106 spleen cells/well) in alternate weekly cycles. L-cell-transfectants expressing-I-Au MHC molecules were used as described earlier 25. TCR peptides were synthesized by S. Horvath (California Institute of Technology, Pasadena,

CA) using Tamoxifen nmr a solid phase technique on a peptide synthesizer (430A; Applied Biosystems) and purified on a reverse phase column by HPLC, as described earlier 46. TCR Vβ8.2 chain peptides are as follows (single-letter amino acid code): B1, aa 1–30(L): EAAVTQSPRNKVAVTGGKVTLSCNQTNNHNL; B4, aa 61–90: PDGYKASRPSQENFSLILELATPSQTSVYF and B5, aa 76–101: LILELATPSQTSVYFCASGDAGGGYE. MBP peptide: MBPAc1-9 (AcASQKRPSQR) was purchased from Macromolecular Resources, Colorado State University. For induction of EAE, mice were immunized s.c. with MBPAc1-9 emulsified very in CFA and i.p. with 0.15 μg of pertussis toxin (PTx; List Biological Laboratories) in PBS. After 48 h mice were injected with 0.15 μg PTx in PBS. Mice were observed daily for the clinical appearance of EAE. Disease severity was scored on a 5-point scale 6: 1, Flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, whole body paralysis; 5, death. Murine DC were derived from tibias and femurs by flushing out the BM with RPMI 1640 medium. Red blood cells were lysed, and BM was cultured in 24-well plates at 1×106 cells/mL in complete medium containing

10 ng/mL IL-4 and 25 ng/mL GM-CSF for 5–7 days 24. The medium was refreshed on day 3 and day 5. For some experiments DC were fixed by suspending the cells at 2×106/mL in PBS containing 0.05% glutaraldehyde for 30 s at 37°C. About 0.2 M of Lysine was added to stop the reaction. Recombinant IL-4 and GM-CSF were both purchased from Peprotech. Subsets of APC were isolated from the spleen and DLN of naïve mice, and mice during active EAE, by positive selection using Microbeads conjugated to antibodies against cell surface markers. For isolation of B cells, anti-CD45R (B220); DC, anti-CD11c (N418); Macrophages, anti-CD11b (Mac-1); Th cells, anti-CD4 (L3T4) conjugated beads were used to manufacturers’ instructions (Miltenyi Biotec). IFN-γ levels in the supernatants from T-cell assays were measured by a sandwich ELISA 19.