T_reg cells accumulate in the CSF of patients with NM. (A) Typical FACS staining of CSF cells (sample obtained from 1 patient with LM) for surface CD25 and intracellular FOXP3. For quantification of T_reg cells, stained CSF cells were first gated according to forward and side light-scattering properties and CD4 expression, and then coexpression of FOXP3 and CD25 was assessed. (B) After staining for CD4, CD25, CD127, and FOXP3, cells were gated for CD4⁺ lymphocytes. (B) R1 shows CD4⁺CD25^highCD127^low cells. (C) R2 shows CD4⁺FOXP3⁺CD127^low cells. (D) In vitro proliferation assay with 10⁴ control-derived T_eff cells and increasing numbers of CD4⁺CD25^high T_reg cells isolated from CSF samples obtained from 2 patients with viral infections. CSF-derived T_reg cells suppress proliferation of activated T_eff cells in a dose-dependent manner. (E) Percentages of CD25^highFOXP3⁺ T_reg cells among CD4⁺ CSF cells in patients with NM and control diseases. Symbols indicate percentages of individual patients. Medians are indicated.