After washing twice with PBS-T as above, 105 MNCs from either EAMG or CFA control rats were added for 24 h at 37°C. Wells were then emptied and incubated with a rabbit antirat IgG (1:400) overnight at 4°C followed by an incubation with a biotinylated antirabbit IgG (1:500; Dakopatts, Copenhagen, Denmark) for 2 h at

RT followed by an incubation with an avidin-biotin peroxidase complex (1:200) for 1 h at RT. After peroxidase staining, the red-brown immunospots corresponding to cells secreting nAChR–IgG antibodies were counted in a blinded fashion using a dissection microscope. The numbers of antibody-secreting cells per 105 MNCs are shown. Lymphocytes from either EAMG or CFA LBH589 in vivo control rats were plated in 96-well round-bottom microtiter plates (Nunc, Copenhagen, Denmark) in triplicate (200 μL containing 4 × 105 cells). The AChR R97–116 peptide (10 μg/mL), myelin basic protein (MBP) 68–86 peptide (10 μg/mL, YGSLPQKSQRSQDENPV, Sangon Ltd, China), Con A (5 μg/mL), or CGS21680 (30 nM, Tocris, UK) were added in triplicate to respective wells. Wells used as negative controls received PBS only. Cells were incubated for 72 h followed by the

addition of 0.5 μCi 3H-thymidine (China Institute of Atomic Energy, Beijing, PR China) during the last 12 h of culture. Cells were harvested onto glass-fiber filters to assay incorporation of radioactivity using a liquid β-scintillation counter (Perkin-Elmer, Wellesley, Nutlin-3a mouse MA, USA). The results were expressed as mean counts per minute

± SD. Rat splenocytes from either EAMG or CFA control rats were harvested and B cells separated using magnetic beads as instructed by the manufacturer (R&D Systems, Minneapolis, MN, USA) or irradiated (750 cGy). Negatively selected cells consisted on average of greater than 90% B cells determined by FACS. A total of 400,000 B cells were cultured in U-bottom 96-well plates wells with 100,000 irradiated splenocytes, AChR R97-116 (10 μg/mL), or lipopolysaccharide (LPS; 5 μg/mL, as positive control) in the presence or absence of CGS21680 (30 nM) for 72 h. Supernatants were collected to detect anti-AChR IgG secretion or 0.5μ Ci/well HAS1 3H-thymidine was added to each well during the last 12 h to measure proliferation as described above. FACS analysis was carried out as described previously [[12]] to detect intracellular cytokines synthesis with some modifications. Lymphocytes from either EAMG or CFA control rats were incubated with AChR R97-116 (10 μg/mL) for 72 h, and during the last 4–5 h, cells were incubated with 50 ng/mL phorbol myristate acetate, 500 ng/mL ionomycin, and Brefeldin A (1:1000). Cells were then stained with antirat CD3 to set the gate and then incubated with FITC-conjugated antirat-CD4 or with PerCP-eFluor710-conjugated anti-rat-CD25 for 20 min at 4 °C.

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