Ly-6C⁺ cells accumulate in the blood and CNS before the onset of EAE. (A) PBMCs from naive and PLP_139-151-immunized SJL mice were analyzed by flow cytometry. Immunized mice were bled on the day before expected clinical onset and during peak EAE. Dot plots are gated on CD11b⁺CD115⁺ cells; percentages of each subset among total blood leukocytes are indicated. (B) Absolute numbers of circulating monocytes per milliliter of blood in naive and PLP_139-151-immunized SJL mice (*P < .05, **P < .02 by comparison to naive). (C) Ly-6C⁺ monocytes were sorted from immunized SJL mice and analyzed for MHC class II and CD11c expression either immediately ex vivo or after a 48-hour culture with GM-CSF. Data shown are representative of 5 separate experiments. (D) Spinal cord mononuclear cells were harvested from naive and PLP_139-151-immunized SJL mice and subjected to flow cytometric analysis. The lowest panels show CD11c and MHC class II staining of CD11b⁺LyC^hi-gated PBMCs from mice in the preclinical (left) or symptomatic (right) stages of EAE. The gates used are depicted in the middle panels. Data shown are representative of 4 separate experiments.