Myelin proteolipid protein-specific CD4⁺CD25⁺regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis

Abstract

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.

Regulatory and Effector T Cell Activation Levels Are Prime Determinants of In Vivo Immune Regulation

“…In this line, depleting endogenous T reg led to inhibition of most but not all T-dependent immune responses in reported studies (15)(16)(17)(18)(19)(20). In addition, Ag-specific T reg activated during an immune response also participate in the immunosuppression (21)(22)(23)(24). Thus, during an immune response to a given Ag, effective T reg -mediated suppression could depend on the additive effect of basal and Ag-induced immunosuppression, the latter being dependent on the frequency of pre-existing Agspecific T reg .…”

Mitigation of Experimental Allergic Encephalomyelitis by TGF-β Induced Foxp3+ Regulatory T Lymphocytes through the Induction of Anergy and Infectious Tolerance

“…Deficiency in Foxp3 precipitates early-onset multiorgan autoimmunity (33,34). The frequency and function of Treg has also been associated with susceptibility to and severity of a number of autoimmune, infectious, and malignant diseases (35)(36)(37)(38)(39). Insight into how manipulation of the Treg compartment influences these diseases is not only biologically but therapeutically significant.…”

An epitope from Acanthamoeba castellanii that cross-react with proteolipid protein 139-151-reactive T cells induces autoimmune encephalomyelitis in SJL mice

“…To determine the frequency of Ag-specific T cells, LNC obtained from the mice immunized with PLP 139-151 or ACA 83-95 were restimulated with the corresponding peptides (20 µg/ml) for four to six days. Viable lymphoblasts were harvested by Ficoll-Hypaque density gradient centrifugation and incubated with phycoerythrin (PE) conjugated IA s tetramers (30 µg/ml) at room temperature (RT) for 3 h followed by staining with anti-CD4 and 7-amino-actinomycin-D (7-AAD) (Eugene, Oregon) (Reddy et al, 2003(Reddy et al, , 2004. The frequency of tetramer + (tet + ) cells was then enumerated by four-color flow cytometric analysis (FACS caliber, BD Pharmingen, San Diego, California) and Flow Jo software (Tree star, Ashland, Oregon) in a live (7-AAD -) CD4 + population.…”

“…After staining with anti-CD4 and 7-AAD, cells were fixed, permeabilized and stained with cytokine antibodies or isotype controls (eBioscience, San Diego, California). The frequency of cytokine-secreting cells was then determined in the live (7-AAD -) CD4 + subset by flow cytometry (FACS Scan, BD Pharmingen) and analyzed by Flow Jo software (Tree star) (Reddy et al, 2004). The clones of cytokine antibodies used were: IL-2 (JES6-5H4), IL-4 (11B11), IL-10 (JES5-16E3), interferon (INF)-γ (XMG1.2), tumor necrosis factor (TNF)-α (MP6XT22), IL-17A (eBio 17B7), IL-17F (eBio 18F10) (all from eBioscience) and IL-22 (140301) (R&D Systems, Minneapolis, Minnesota).…”

“…Cytokine ELISA Supernatants harvested from the above cultures on day two were analyzed for cytokines by ELISA (Nicholson et al, 1995;Reddy et al, 2004). The clones of the capture and detection antibody (Ab) pairs used were: IL-2 (JES6-1A12/JES6-5H4), IL-4 (11B11/BVD6-24G2), IL-10 (JES5-16E3/JES5-2A5), IL-17A (eBio17CK15A5/eBio17B7), INF-γ (AN-18/R4-6A2), and TNF-α (TN3-19.12/rabbit polyclonal) (eBioscience).…”