Antigen-specific splenic CD4+ and CD8+ regulatory T cells generated via the eye, suppress experimental autoimmune encephalomyelitis either at the priming or at the effector phase

Abstract

The injection of antigen into the ocular anterior chamber (AC) induces the generation of splenic CD4(+) and CD8(+) regulatory T (Treg) cells, specific for the antigen injected into the AC. These Treg cells inhibit the induction (CD4(+)) and also the expression (CD8(+)) of a delayed-type hypersensitivity response. The ability of AC-induced self-antigen-specific Treg cells in modulating autoimmunity is not well defined. Here we show that an injection of encephalitogenic myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide into the anterior chamber of the eye (AC-MOG), before the induction of or during established experimental autoimmune encephalomyelitis (EAE) induced by MOG(35-55), suppresses the induction or progression of EAE, respectively. CD4(+) or CD8(+) splenic Treg cells induced by an injection of AC-MOG prevent EAE either at the inductive (priming) or at the progressive (effector) phase, respectively. This suppression of EAE by an AC-MOG injection or by intravenous transfer of splenic regulatory cells induced by an AC-MOG injection is specific for the antigen injected into the AC. Additionally, our data suggest that splenic CD8(+) Treg cells that suppress active EAE may use a transforming growth factor (TGF)-β-dependent suppression mechanism while the suppression of the induction of EAE by the AC-induced CD4(+) Treg cells is independent of TGF-β. Thus, we show for the first time that regulation of EAE at the priming or the chronic phase requires different phenotypes of Treg cells. Hence, it is important to consider the phenotype of Treg cells while designing effective cell-based therapies against autoimmune disorders.

Fig. 1.

Intracameral injection of MOG_35–55 peptide prevents the induction and progression of MOG_35–55 peptide induced EAE. (a) C57BL/6 mice received an intracameral injection of MOG_35–55 peptide at different time points with respect to the day of immunization. One group was given an i.v. injection of MOG_35–55 peptide on day 0. EAE scores monitored by clinical symptoms (0, normal; 1, limp tail; 2, paraparesis with a clumsy gait; 3, hind limb paralysis; 4, quadriplegia; 5, death) were assessed each day. Data are presented as mean clinical scores of eight mice per group. The difference between the control group and the groups receiving AC-MOG_35–55 peptide 7 days prior immunization or days 1, 8 and 10 p.i. were statistically significant through the clinical course after peak symptoms (*,  , #, %, significant difference, P < 0.02). (b) Data show only those mice that were clinically sick on the day of AC injection (10 days p.i.). Table shows the distribution of mice with different clinical scores on day 10, in the different groups. No disease scores are <1. Data are presented as mean clinical scores of six mice per group ± standard error (*, significant difference, P < 0.05).

Fig. 2.

Intracameral injection of MOG_35–55 peptide in CD8^−/− mice cannot prevent EAE progression but can suppress the induction of EAE. CD8^−/− and wild-type (C57B6) mice were immunized with MOG_35–55 peptide and received AC injection of MOG_35–55 peptide, 7 days before immunization or 8 days p.i. EAE scores monitored by clinical symptoms (as discussed in Fig. 1) were assessed each day. Data are presented as mean clinical scores of eight mice per group. The difference between the control group and the groups receiving intracameral MOG_35–55 peptide 7 days prior immunization (WT and CD8^−/−) or day 8 p.i. (CD8^−/−) was statistically significant through the clinical course after peak symptoms (*,  , #, significant difference, P< 0.05).

Fig. 3.

AC-induced CD8⁺ regulatory cells can restrict EAE progression but cannot prevent the induction of EAE. Mice were immunized with MOG_35–55 peptide to induce EAE. Groups received AC-MOG_35–55-induced splenic CD8⁺ or CD8⁻ cells (2 × 10⁶ cells per mouse) on days 0, 10 and 13 p.i. One group received naive (non-AC) splenic CD8⁺ cells on day 10 p.i. EAE scores monitored by clinical symptoms (as discussed in Fig. 1) were assessed each day. Data are presented as mean clinical scores of eight mice per group. The difference between the control group and the groups receiving AC-MOG_35–55-CD8⁺ on day 10, AC-MOG_35–55-CD8⁻ on day 0 or AC-MOG_35–55-CD8⁺ on day 13 was statistically significant through the clinical course after peak symptoms (*,  , #, significant difference, P< 0.05).

Fig. 4.

Suppression of EAE by AC-MOG_35–55-CD8⁺ regulatory cells is antigen specific. Mice were immunized with MOG_35–55 peptide to induce EAE. Mice received AC-MOG_35–55 or AC-OVA-induced splenic CD8⁺ cells (2 × 10⁶ cells per mouse) on day 10 p.i. EAE scores monitored by clinical symptoms (as discussed in Fig. 1) were assessed each day. Data are presented as mean clinical scores of eight mice per group. Data represents four experiments combined. The difference between the control group and the group receiving AC-MOG_35–55-CD8⁺ cells was statistically significant through the clinical course after peak symptoms (*, significant difference, P< 0.05).

Fig. 5.

AC-induced CD4⁺ regulatory cells can prevent EAE induction but cannot restrict the progression of EAE. Mice were immunized with MOG_35–55 peptide to induce EAE. Groups received AC-MOG_35–55-induced splenic CD4⁺ or CD4⁻ cells (2 × 10⁶ cells per mouse) on days 0 or 10 p.i. One group received naive (non-AC) splenic CD4⁺ cells on day 0 p.i. EAE scores monitored by clinical symptoms (as discussed in Fig. 1) were assessed each day. Data are presented as mean clinical scores of eight mice per group and representative of three experiments. The difference between the control group and the groups receiving AC-MOG_35–55-CD4⁺ on day 0, AC-MOG_35–55-CD4⁻ on day 10 was statistically significant through the clinical course after peak symptoms (*,  , significant difference, P< 0.05).

Fig. 6.

AC-MOG_35–55-CD4⁺ regulatory cells are antigen specific. Different groups of mice were immunized with MOG_35–55 peptide to induce EAE. Groups received AC-MOG_35–55 or AC-OVA-induced splenic CD4⁺ cells (2 × 10⁶ cells per mouse) on day 0 p.i. EAE scores monitored by clinical symptoms (as discussed in Fig. 1) were assessed each day. Data are presented as mean clinical scores of eight mice per group and three experiments. The difference between the control group and the group receiving AC-MOG_35–55-CD4⁺ cells was statistically significant through the clinical course after peak symptoms (*, significant difference, P< 0.05).

Fig. 7.

Suppression of EAE by MOG_35–55 induced CD8⁺ regulatory cells requires TGF-β sensitivity by T cells. Cbl-b^−/− and C57B/6 (WT) mice were immunized with MOG_35–55 peptide. Different groups received an intracameral injection of MOG_35–55 peptide 8 days p.i. or AC-MOG-induced splenic CD8⁺ cells (2 × 10⁶ cells per mouse) on day 10 p.i. The control groups were given PBS. EAE scores monitored by clinical symptoms (as discussed in Fig. 1) were assessed each day. Data are presented as mean clinical scores of eight mice per group ± standard error. Error bars are not shown for some groups to maintain clarity of figure. The differences between the control groups and Cbl-b^−/− groups were not significant (*, significant difference from WT control, P < 0.05).

Fig. 8.

Suppression of EAE by MOG_35–55-induced CD4⁺ regulatory cells is independent of TGF-β sensitivity by effector T cells. Cbl-b^−/− or WT C57/B6 mice were immunized with MOG_35–55 peptide. Different groups received an AC-MOG-induced splenic CD4⁺ cells (2 × 10⁶ cells per mouse) on day 0 p.i. The control groups were given PBS. EAE scores monitored by clinical symptoms (as discussed in Fig. 1) were assessed each day. Data are presented as mean clinical scores of five mice per group ± standard error. The differences between the control groups and the groups receiving AC-MOG_35–55-induced CD4⁺ cells were significant (*, # significant difference, P< 0.05).

Fig. 9.

ACAID-CD4⁺ and CD8⁺ splenocytes migrate to the CNS. C57B/6 mice (CD45.2) were immunized were immunized with MOG_35–55 peptide. Different groups received splenocytes (3 × 10⁶ cells per mouse) from non-AC, AC-MOG or AC-OVA mice, i.v. on day 10 post-immunization. Mononuclear cells from spinal cords were isolated and analyzed 24 h after transfer. (a) Representative FACS dot plots showing percentage of donor splenocytes in the spinal cords. Plots represent gated mononuclear cells from the spinal cord. (b) Bar graph shows mean results from figure (a) (n = 5) (*, significant difference, P < 0.05) (c) Bar graph shows percentage of CD4 and CD8 cells from donor splenocytes detected in the spinal cord, 24 h after transfer ( ,*, significant difference, P < 0.05).