Fig. 3

Long-lived plasma cells persist in the chronically inflamed CNS. Mice were immunized and boosted (day 28) with rhMOG. a The scheme demonstrates the experimental procedure for the EdU pulse-chase experiment, starting after boost. The mice received EdU for 14 days via drinking water. Analysis was performed either directly after stopping EdU-feeding or five to seven weeks after boost. b A representative confocal tile scan of a total spinal cord section from day 77 after boost is shown. Signals after immunofluorescence staining of EdU (red), antibody-secreting cells (κ/λ, green) and DAPI (blue) are shown. Data are representative of six mice from three independent experiments. Scale bar of the tile scan represents 200 μm, scale bar of the magnified inset represents 50 μm. c The frequency of EdU⁺ plasma cells was determined directly after stopping EdU-feeding (pulse) and after a chase period three to five weeks later. 64 to 362 antibody-secreting cells of each mouse were counted and analyzed manually, mice are pooled from three independent experiments. Bars indicate mean, each data point indicates an individual mouse. d Representative confocal microscopy images of inflamed spinal cord of five EAE mice analyzed three to five weeks after stopping EdU-feeding are shown. Antibody-secreting cells (κ/λ, green), EdU (red), DAPI (upper row, blue), IgG (lower panel, left, blue) or IgA (lower panel, right, blue) were stained. Five mice from two independent experiments were analyzed. Scale bars represent 20 μm. e The graph demonstrates the frequency of IgM⁺ and IgG⁺/IgA⁺ EdU⁺ plasma cells after the chase period. 52 to 70 EdU⁺ plasma cells of each mouse were counted manually. Bars indicate mean, each data point indicates one individual mouse pooled from two independent experiments. f Bone marrow lymphocytes of EAE mice were isolated and analyzed by flow cytometry three to five weeks after stopping EdU-feeding. Viable (eFluor780⁻) lymphocytes (CD45.2⁺) were further analyzed for kappa and afterwards for EdU. Bars indicate mean, each data point indicates one individual mouse. Data of two independent experiments are shown. The significance between the groups was tested with the unpaired Mann-Whitney U test (**P < 0.01)