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

Abstract

SJL mice are highly susceptible to experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) peptide 139-151, whereas H-2 congenic B10.S mice are resistant. Immunodominance and susceptibility to EAE are associated with a high precursor frequency of PLP 139-151-specific T cells in the naive repertoire of SJL mice. To understand the mechanism of EAE resistance in B10.S mice, we determined the precursor frequency of PLP 139-151-reactive T cells in both strains by using IAs/PLP 139-151 tetramers. SJL and B10.S mice had similar frequencies of tetramer-reactive T cells in the naive peripheral repertoire. However, in SJL mice, the majority of PLP 139-151 tetramer-positive cells were in the CD4+CD25- population, whereas there were more tetramer-positive cells in the CD4+CD25+ population of B10.S mice. Depletion of CD4+CD25+ cells in vivo facilitated the expansion of PLP 139-151-reactive cells with production of T helper 1 cytokines in EAE-resistant B10.S mice. Furthermore, anti-CD25 Ab treatment before immunization resulted in EAE induction in these otherwise resistant mice. These data indicate an important role for autoantigen-specific CD4+CD25+ cells in genetic resistance to autoimmunity.

Fig. 1.

B10.S mice have a diminished recall response to PLP 139-151. (a) Tetramer staining. LNC from SJL and B10.S mice immunized with PLP 139-151 or TMEV 70-86 peptide were restimulated with the corresponding peptides, and percentages of IA^s tetramer-positive cells were determined in the viable (7-AAD^-) CD4⁺ populations. (b) Intracellular cytokine staining. LNC from SJL and B10.S mice immunized with PLP 139-151 were restimulated with PLP 139-151. After 3 days, frequencies of IL-2- and IFN-γ-secreting cells were determined by flow cytometry. A representative experiment from three or four individual experiments involving two or three mice per group is shown in each plot.

Fig. 2.

Naïve B10.S mice have greater proportions of PLP 139-151-reactive CD4⁺CD25⁺ T cells in their periphery than naïve SJL mice. (a) CD3⁺ T cells from naïve SJL and B10.S mice were stained with IA^s tetramers (PLP 139-151 and TMEV 70-86) after treatment with neuraminidase, and the tetramer-positive cells were enumerated in the live CD4⁺ population. Horizontal bars indicate the average frequency of tetramer-positive CD4⁺ cells. (b) CD3⁺ T cells from naïve SJL and B10.S mice were incubated with IA^s tetramers (PLP 139-151 or TMEV 70-86) after neuraminidase treatment, and frequencies of tetramer-positive cells were determined in the live CD4⁺ population in relation to the CD25 marker. (c) The relative precursor frequencies of PLP 139-151 tetramer-positive cells are expressed as the ratio of tetramer-positive cells in the CD4⁺CD25⁺ compartment to those in the CD4⁺CD25 negative compartment (n = 4 per group).

Fig. 3.

CD4⁺CD25⁺ cells in SJL and B10.S mice are functional. (a) CD4⁺CD25⁺ and CD4⁺CD25^- fractions were enriched from naïve SJL and B10.S mice. The different populations, either alone or together, were stimulated with anti-CD3 Ab in the presence of APC, and proliferative responses were measured as cpm. (b) CD4⁺CD25⁺ and CD4⁺CD25^- populations as in a were stimulated with PLP 139-151 (150 μg/ml) in the presence of APC, and proliferative responses were measured. A representative experiment from three to five individual experiments is shown in each graph.

Fig. 4.

Depletion of CD25⁺ cells in vivo enhances PLP 139-151-specific T cell responses in B10.S mice. (a) Proliferation. CD3⁺ T cells from PLP 139-151-immunized B10.S mice that had been treated with anti-CD25 Ab or rat IgG control were restimulated with PLP 139-151, and proliferative responses were measured. (b) IA^s tetramer staining. CD3⁺ T cells from PLP 139-151-immunized B10.S mice that were pretreated with anti-CD25 Ab or rat IgG were restimulated with PLP 139-151 for 4 days. The percentages of IA^s tetramer (PLP 139-151, TMEV 70-86)-positive cells were determined in the viable (7-AAD^-) CD4 subset. A representative experiment from five individual experiments involving two mice per group is shown.

Fig. 5.

Anti-CD25 Ab treatment alters cytokine production toward a Th1 phenotype. (a) Intracellular staining. LNC from PLP 139-151-immunized B10.S mice that were pretreated with anti-CD25 or rat IgG Ab were restimulated twice with PLP 139-151. Viable lymphoblasts were restimulated with anti-TCR and anti-CD28 Ab for 4-6 h. After staining with anti-CD4 Ab and 7-AAD, the frequency of IL-2-, IL-4-, IL-5-, IL-10-, and IFN-γ-secreting cells was determined in the live (7-AAD^-) CD4⁺ subset by using flow cytometry. A representative sample from six individual experiments is shown. *, Isotype control for IL-2 and IL-10; **, Isotype control for IL-4 and IL-5 and IFN-γ. (b) Cytokine ELISA. Supernatants from the above cultures were analyzed by ELISA for production of IL-2, IL-4, IL-10, and IFN-γ. Each bar represents mean ± SEM values for a group of experiments (anti-CD25 Ab, n = 5; rat IgG, n = 6).