Studies in various models of experimental autoimmune encephalomyelitis (EAE) have provided new insights into autoimmune disease mechanisms. In SJL mice, we and others have shown that proteolipid protein (PLP) 139-151 is the immunodominant encephalitogenic PLP epitope and that it induces severe EAE (1-3). In contrast, B10.S mice are highly resistant, even though both strains carry the H-2 s MHC molecules (3), suggesting that differences in non-MHC genes contribute to susceptibility and resistance to EAE. By using crosses between SJL and B10.S mice, we and others have identified multiple loci that contribute to disease susceptibility (3-6), but the actual genes and cellular mechanisms determining susceptibility have not been elucidated.There is now considerable evidence that CD4 ϩ CD25 ϩ T cells play a critical role in the regulation of autoimmune diseases (7-9). They constitutively express Forkhead box P3 (10, 11). Emerging evidence suggests that CD4 ϩ CD25 ϩ cells are generated in the thymus by a high-affinity interaction of the T cell receptor (TCR) with self-peptides bound to MHC II molecules rendering these self-reactive cells anergic (12)(13)(14) T Cell Proliferation. Single-cell suspensions were obtained from spleens and lymph nodes (LN) of naïve SJL and B10.S mice, and CD4 ϩ CD25 ϩ and CD4 ϩ CD25 Ϫ subsets were fractionated by magnetic separation using LS columns (Miltenyi Biotec, Auburn CA). For proliferation assays, 1.5 to 2.0 ϫ 10 6 cells per ml CD4 ϩ CD25 ϩ or CD4 ϩ CD25 Ϫ cells or both were cultured with anti-CD3 Ab (0-1 g͞ml) in HL-1 medium (BioWhittaker) for 2 days or PLP 139-151 (0-150 g͞ml) for 3 days in the presence of antigen-presenting cells (APC). Sixteen hours after pulsing with 1 Ci of [ 3 H]thymidine (1 Ci ϭ 37 GBq), proliferation was measured as cpm by using a Wallac liquid scintillation counter (PerkinElmer). CD3 ϩ T cells from draining LN were enriched by negative selection (R & D Systems). To determine recall responses to PLP 139-151 in B10.S mice depleted of CD25 ϩ cells, This paper was submitted directly (Track II) to the PNAS office.
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