Isolation of lymphocytes from lymph nodes, the spleen, and the brain Draining cervical lymph nodes and the spleen were pressed through a steel sieve in 4 ml HBSS and homogenized in a total volume of 13 ml HBSS. After a centrifugation step at 310 g for 10 min the cell pellets were resuspended in an adequate volume of HBSS (approximately 10 7 /cells/ml). Spleen cells were additionally treated with erythrocyte lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA) and washed with HBSS. Brains were pressed through a steel sieve in 5 ml HBSS 3% FCS and homogenized in a total volume of 20 ml HBSS 3% FCS. After a centrifugation step at 170 g for 10 min the cell pellet was resuspended in 1.4 ml dissociation buffer (23 mM CaCl 2 , 50 mM KCl, 42 mM MgCl 2 , 153 mM NaCl) containing 0.4 U collagenase (Serva) and 50 U Benzonase (Novagen) and incubated at 37uC for 1 h. Afterwards the cells were washed with HBSS and applied on a percoll density gradient to separate the lymphocytes from the rest like myelin debris or neuronal cells as described [46] . The lymphocytes were isolated and washed to remove the percoll for subsequent analyzes. mouse CD3 (clone 145-2C11)-, CD4 (clone RM4-5)-, CD8 (clone Ly-2)-, CD19 (clone 1D3)-and CD25 (clone 7D4)-antibodies were purchased from Becton Dickinson. PE-conjugated anti-mouse Foxp3 (clone FJK-16s)-antibody was purchased from NatuTec. Lymphocytes were stained in FACS buffer (PBS containing 0.4% BSA and 0.02% sodium azide) at 4uC for 20 min. Intracellular staining of Foxp3 was performed using the Foxp3 Staining Buffer Set (NatuTec) according to the manufacture's protocol. Briefly, cells were fixed and permeabilized in 500 ml fixation/permeabilization buffer (Concentrate/Diluent 1:4) at RT for 1 h and stained afterwards in permeabilization buffer at RT for 30 min. Flow cytometric analysis was performed on a FACSCalibur (Becton Dickinson).