Depletion of CD4(+)CD25(+) T cells exacerbates experimental autoimmune encephalomyelitis induced by mouse, but not rat, antigens

Abstract

A key question in the field of autoimmunity concerns the fact that experimental disease is generally induced more easily with closely related, but not completely identical, tissue-restricted antigens. Here, the possibility that naturally occurring regulatory T cells (Tregs) for self-antigens are more potent than those for related antigens was investigated. The self-antigen specificity of naturally occurring Tregs was tested in experimental autoimmune encephalomyelitis (EAE) induced with mouse (self) or closely related (rat) myelin oligodendrocyte glycoproteins (MOGs). Surprisingly, Treg depletion increased EAE severity in mice immunized with mouse, but not rat, MOG. This increase was associated with increased T-cell activation and infiltration of the central nervous system, as well as increased interleukin (IL)-17 production and a higher ratio of interferon-gamma- to IL-10-producing cells. These data suggest that Tregs are specific for self-antigen and do not "cross-protect" against autoimmunity even when disease is induced with closely related foreign antigens.

Fig. 1

PC61 treatment results in an efficient depletion of CD4⁺CD25⁺ T cells. Mice received three i.p. injections of 100 μg/injection of either PC61 or rat IgG every 48hr and were sacrificed 7d post third injection. (A) Absolute cell numbers in pooled spleens and LNs of control IgG (dashed bars) or PC61-treated groups (open bars). (B) Expression levels of CD25 in either spleen or LN of control IgG or PC61-treated mice. Cells were stained with anti-CD4 and anti-CD25 (7D4). All plots are gated on CD4⁺ cells; shaded histograms show isotype control staining.

Fig. 2

Comparison of amino acid sequence of mouse (M) and rat (R) MOG extracellular domain. Amino acid residues which differ from mouse MOG are underlined.

Fig. 3

Treg depletion results in increased EAE severity in mice immunized with mouse MOG but not rat MOG. Mice receiving three i.p. injections of either rat IgG (□) or PC61 (◆) were immunized by standard protocol with mouse MOG (A), rat MOG (B), or with rat MOG omitting boost (C). Animals were monitored and clinical scores were collected daily. ┼, indicates death. Dead animals were noted and omitted from subsequent graphs but were included in all other calculations (see Table I).

Fig. 4

T cells from spleen and LNs of Treg-depleted mice immunized with mouse MOG are hyper-responsive when stimulated with mouse MOG. Mice receiving three i.p. injections of either rat IgG (n=3) or PC61 (n=3) were immunized with mouse MOG and sacrificed on day 20. Spleens or LNs isolated from each group were pooled and CD4⁺ gated cells were stained for expression of (A) CD62L and (B) CD69. (C) Spleen cells were also tested for anti-CD4, anti-CD25, and anti-Foxp3. (D) Cells isolated from mice immunized with mouse or rat MOG were stimulated with mouse MOG and proliferation was measured as described in Materials and Methods. For (D) data are expressed as stimulation indices and represent one of 2 independent experiments with three mice per group. * p<0.0002, § p<0.003 (E) LN cells from mouse or rat MOG immunized mice were stimulated with mouse MOG protein and supernatants were collected for IL-17A detection by ELISA. Dashed bars- control IgG treated; opened bars- PC61-treated animals

Fig. 4

T cells from spleen and LNs of Treg-depleted mice immunized with mouse MOG are hyper-responsive when stimulated with mouse MOG. Mice receiving three i.p. injections of either rat IgG (n=3) or PC61 (n=3) were immunized with mouse MOG and sacrificed on day 20. Spleens or LNs isolated from each group were pooled and CD4⁺ gated cells were stained for expression of (A) CD62L and (B) CD69. (C) Spleen cells were also tested for anti-CD4, anti-CD25, and anti-Foxp3. (D) Cells isolated from mice immunized with mouse or rat MOG were stimulated with mouse MOG and proliferation was measured as described in Materials and Methods. For (D) data are expressed as stimulation indices and represent one of 2 independent experiments with three mice per group. * p<0.0002, § p<0.003 (E) LN cells from mouse or rat MOG immunized mice were stimulated with mouse MOG protein and supernatants were collected for IL-17A detection by ELISA. Dashed bars- control IgG treated; opened bars- PC61-treated animals

Fig. 5

Treg depletion results in increased numbers of infiltrating CD4⁺ cells and increased numbers of IFNγ producing cells in the CNS. Mice treated with either rat IgG (n=3) or PC61 (n=3) were immunized with mouse MOG and sacrificed on day 20. (A) Isolated SC were sectioned and stained for the presence of CD4⁺ T cells. (B) Mononuclear cells from the CNS were gated on CD4⁺ cells and stained for the expression of CD62L or CD69. (C) Cells isolated as in (B) were stained with anti-CD4, anti-CD25, and anti-Foxp3. (D) ELISPOT analysis. CNS tissues from each group were pooled together and 10⁵ cells were plated and stimulated with either mouse MOG or MOG_35-55. Dashed bars- control IgG treated; open bars- PC61-treated animals. Data are expressed as stimulation indices and represent one of 2 independent experiments with three mice per group.

Fig. 6

Depletion of Tregs does not result in the emergence of T cells reactive to new encephalitogenic epitopes but does lead to increased proliferation to MOG_35-55 in the periphery as well as an enrichment of CD4⁺TCRVβ8⁺Foxp3⁻ T cells in the CNS. (A) Spleens or (B) LNs from individual mice treated with either rat IgG (hatched bars, n=3) or PC61 (open bars, n=3) and immunized with mouse MOG were isolated and pooled on day 10 post immunization. Cells were stimulated with different peptides of MOG extracellular domain. Data are expressed as stimulation indices and represent one of two independent experiments with three mice per group. * p<0.0002. (C) Mice treated with either rat IgG or PC61 and immunized with mouse or rat MOG were sacrificed on day 20 post immunization and cells from the CNS were collected. Cells were stained with anti-CD4, anti-TCRVβ8 and anti-Foxp3 antibodies.