Figure 7. CD4+ cells in the subarachnoid space proliferate early during the course of EAE.

5×10⁶ MOG TCR tg CD4+ cells isolated from naive 2D2 mice were transferred iv into C57Bl/6 mice and the recipients immunized with MOG35-55. BrdU (120 mg/kg bodyweight) was injected ip two hours before harvest at day 10 and day 18 pi and percentages of BrdU-containing MOG TCR tg Vα3.2+/Vβ11+/CD4+ cells were determined in draining lymph nodes (DLN), leptomeninges (MEN) and spinal cord (SpC) were determined using flow cytometry (A–B). The amount of antigen-non-specific proliferation was analyzed in MOG TCR tg CD4 cells in DLN from animals immunized without MOG35-55 (MOG−). It was not possible to assess proliferation of MOG TCR tg CD4+ cells in the CNS of mice immunized without MOG35-55 due to low numbers of cells present. (C) Two-color immunofluorescence staining of leptomeningeal whole-mount preparations obtained on day 10 pi verifying uptake of BrdU (red) in CD4 cells (green). (D) Leptomeningeal explant prepared from a mouse during the early recovery phase of EAE imaged using confocal time-lapse microscopy directly ex vivo. Images show merged z-stacks demonstrating an initially large CD4+ cell (green) going through cytokinesis during 60 min scanning. Scale bar=5 μm.