In Situ Tolerance within the Central Nervous System as a Mechanism for Preventing Autoimmunity

Brabb, Thea; von Dassow, Peter; Ordonez, Nadia; Schnabel, Bryan; Duke, Blythe; Goverman, Joan

T Cell Stimulation Assay. Single-cell suspensions of LN or CNS mononuclear cells were plated at 1.5 ϫ 10 4 V ␣ 2 ϩ T cells/ well (MBP TCR1, TEa TCR) or 2 ϫ 10 4 TCR ϩ T cells/well (MBP TCR2, D011.10, and nontransgenic) in 96-well plates in complete media. APCs (10 6 /well) consisted of splenocytes from the appropriate wild-type mouse depleted of TCR ϩ cells by panning with anti-Thy 1.2 (clone 30-H12; BD PharMingen)-coated plates (20) , or by magnetic bead depletion using biotinylated anti-TCR and streptavidin-coated Dynabeads (Dynal). MBP TCR1 and MBP TCR2 T cells were stimulated in the presence or absence of 30 M MBP Ac1-11 peptide (Research Genetics) and T cell-depleted APCs. TEa T cells were stimulated in the presence or absence of 5 g/ml E ␣ 52-68 peptide (a gift from Dr. Alexander Rudensky) and T cell-depleted APCs. D011.10 T cells were stimulated in the presence or absence of 1 M Ova 323-339 peptide (a gift from Dr. Craig Beeson, University of Washington, Seattle, WA) and T cell-depleted APCs. 5-Bromo-2 Јdeoxyuridine (BrdU; 25 g/ml; Sigma-Aldrich) was added after 48 h of culture and cells were harvested 12-16 h later. Proliferation was assessed by staining the cultured cells with PE-labeled anti-V ␣ 2 (for MBP TCR1 and TEa transgenic mice) or anti-TCR monoclonal antibody (for MBP TCR2 and DO11.10 TCR transgenic mice), cell permeabilization, and staining with FITC-labeled anti-BrdU monoclonal antibody (clone 3D4; BD PharMingen) as described previously (21, 22) . For assessment of anergy in CNS T cells, stimulations were performed as described above except in the presence or absence of 50 U IL-2/ml (23) .