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. 2011 Jan 27;23(2):119–128. doi: 10.1093/intimm/dxq461

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

Sourojit Bhowmick 1, Robert B Clark 1, Stefan Brocke 1, Robert E Cone 1,✉
PMCID: PMC3030727  PMID: 21273399

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 (MOG35–55) peptide into the anterior chamber of the eye (AC-MOG), before the induction of or during established experimental autoimmune encephalomyelitis (EAE) induced by MOG35–55, 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.

Keywords: ACAID, EAE, MOG peptide, mouse, Tregs

Introduction

The injection of antigen into the anterior chamber (AC) of the eye (intracameral injection) induces an antigen-specific immune tolerance (immune deviation) termed anterior-chamber-associated immune deviation (ACAID). ACAID is characterized by the suppression of the delayed-type hypersensitivity (DTH) response and the secretion of complement-fixing IgG antibodies and IgE (1–6) to the antigen injected into the AC. Additionally, ACAID induces an antigen-specific suppression of Th2-mediated pulmonary pathology (7). The intracameral injection of antigen differs from intravenous (i.v.) or mucosal administration of antigen (8) and this form of eye-derived tolerance is mediated by at least two populations of AC-induced antigen-specific splenic regulatory cells (Tregs): an antigen-specific CD4+ population that suppresses the antigen-induced proliferation and the induction of DTH (called the afferent regulators) and an antigen-specific CD8+ population that inhibits the expression of DTH (called the efferent regulators) (1, 2,9–11). However, ACAID is independent of CD4+CD25+FoxP3+ Treg cells (11). AC-induced CD8+ regulatory cells suppress IFN-γ production in vitro (12) and in vivo T cells that effect a DTH reaction in immunized mice (1, 9, 13, 14). Further, AC-induced CD8+ regulatory cells are restricted by Qa-1 antigens expressed by effector T cells (13). Since the non-classical MHC class I molecule Qa-1 is known to be expressed only on activated cells (15, 16), AC-induced CD8+ Treg cells specifically suppress activated T cells. Thus, it can be concluded that ACAID suppresses the induction of effector T cells and also the activity of effector T cells by distinct populations of Treg cells. That ACAID may occur in humans is suggested by the demonstration that individuals with acute retinal necrosis develop antibodies but not cell-mediated immunity to Varicella zoster (17).

A goal of induced immune regulation is the specific modulation of the induction (or recurrence) and progression of an active autoimmune disease. Although the injection of the autoantigen interphotoreceptor retinoid binding protein (IRBP) into the AC mitigates the induction of experimental autoimmune uveitis model (18, 19), it is not clear whether the injection of myelin antigens into the AC can mediate experimental autoimmune encephalomyelitis (EAE). In this regard, antigen-presenting cells (APCs) treated in vitro with transforming growth factor (TGF)-β2 behave as similar to APCs induced by an intracameral injection (20–22) and inhibit the induction of Myelin Basic protein (MBP)-specific EAE in C57BL/6 mice, induced by adoptive transfer of lymphocytes (23). However, most investigations on the role of Treg cells in autoimmunity do not discriminate between the induction of an autoimmune response and the regulation of active pathogenic autoimmune immunity.

Because the injection of antigen into the AC induces different phenotypes of Treg cells, we investigated the ability of splenic Treg cells induced by an intracameral injection of MOG35–55 peptide (AC-MOG) to regulate MOG35–55-induced EAE. Here, we show for the first time that EAE can be regulated both at the priming (initiation) phase and at the chronic (effector) phase by different AC-induced Treg cells. We show that AC-induced MOG-specific CD4+ Treg cells suppress EAE at the priming (initiation) phase of the disease but are ineffective in restricting an ongoing disease. In contrast, CD8+ Treg cells induced by an intracameral injection of MOG35–55 restrict disease progression at the effector phase but were ineffective in suppressing EAE initiation. Our results further suggest that the inhibition of active EAE by CD8+ Treg cells requires sensitivity to TGF-β by EAE effector T cells while the CD4+ Treg cells' suppression of the induction of EAE is independent of sensitivity to TGF-β. Thus, these results demonstrate that depending on the stage of EAE, different Treg cell phenotypes could be specifically targeted for therapy.

Methods

Animals

Female C57BL/6 (Ly5.1 and Ly5.2) mice 6–8 weeks old were purchased from Charles River Laboratories (Wilmington, MA, USA). Cbl-b−/− mice (24) are maintained at the University of Connecticut Health Center. All animals were maintained by the Center for Laboratory Animal Care at the University of Connecticut Health Center. The use of animals adhered to the Association for Research in Vision and Ophthalmology (ARVO) resolution on the use of animals in ophthalmic and vision research. All works with animals have been reviewed and approved by the University of Connecticut Health Center Animal Care Committee (ACC 2004-380).

Reagents

MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide was prepared by the W. M. Keck facility at Yale University, New Haven. Incomplete Freund's adjuvant (IFA) and heat-killed Mycobacterium tuberculosis H37Ra were purchased from Difco Laboratory (Detroit, MI, USA). Pertussis toxin (PTX) was purchased from List Biological Labs (Campbell, CA, USA). For flow cytometry, anti-CD45.1, anti-CD8 and anti-CD4 antibodies with appropriate isotype controls were purchased from eBiosciences (San Diego, CA, USA). CD4 and CD8 T-cell isolation kits were purchased from Miltenyi Biotec Inc. (Auburn, CA, USA).

Injection of antigen into the AC (intracameral injection)

For the induction of ACAID, antigen was injected into the AC of mice as described previously (13). Briefly, mice were anesthetized with an intra-peritoneal injection of ketamine (75 mg kg−1)/xylazine (15 mg kg−1) as described. Under a dissecting microscope, a 32-G needle attached to a cannula attached to a manually controlled Hamilton syringe (Stoelting Co., Wood Dale, IL, USA) was inserted into the AC (25). Approximately 3 μl PBS containing 15 μg ovalbumin (OVA) or 15 μg MOG35–55 was injected into the AC. The mice recovered 15–30 min after the intracamera injection and took water and food normally. Mice receiving an intracameral injection of OVA or MOG35–55 were immunized with MOG35-55-CFA at different time points to induceEAE.

Induction of EAE

For induction of active EAE, mice were injected intra-dermally with 200 μg of emulsion containing 200 μg MOG35–55 in IFA supplemented with 500 μg of M. tuberculosis H37Ra. Mice were injected intra-peritoneally with 200 ng of PTX in 100 μl of PBS shortly after and 48 h after the first immunization. Following immunization, animals were kept under observation to score the disease. Observation was done in a blinded fashion. The clinical scale was as follows: 0 = normal, 1 = limp tail, 2 = paraparesis with a clumsy gait, 3 = hind limb paralysis, 4 = quadriplegia and 5 = death.

Preparation of splenic Treg cells (AC-CD4+ or AC-CD8+ cells)

Seven days after intracameral injection, the AC-injected mice were euthanized, spleens recovered, diced and expressed through a 40-mm nylon mesh into PBS (pH 7.2) using the plunger of a 10-ml syringe. The cell suspension was washed two times with PBS and re-suspended in PBS. BD Pharm Lyse (BD Biosciences) was used for lysing erythrocytes according to the manufacturer's protocol. To separate the spleen cells based on the expression of CD4 or CD8, cells suspended in PBS were separated using the CD4+ or CD8+ T-cell isolation kit (negative selection) (Miltenyi Biotec Inc.) according to the manufacturer's protocol. Enrichment was assessed by flow cytometry and was found to be between 92 and 95% for both CD4+ and CD8+ enriched fractions (termed as CD4+ or CD8+ cells). The rest of the splenocytes are termed as CD4− or CD8− cells, which are all the splenocytes except CD4+ or CD8+ cells (according to the enrichment protocol).

Spinal cord mononuclear cell isolation and analysis

Spinal cord mononuclear cells were derived as described previously (26). Cells were counted and then stained with anti-CD45.1, anti-CD4 and anti-CD8 (eBioscience) and analyzed by flow cytometry.

Flow cytometry

To test the purity of isolated CD4 and CD8 cells, cell samples were washed in PBS (pH 7.2) containing 0.2% BSA and 0.1% NaN3 (FACS buffer). Aliquots containing ∼106 cells were incubated with 100 μl of appropriately diluted antibodies for 30 min at 4°C. Flow cytometry analysis was done using FACS Calibur (BD Biosciences) and FLOWJO (Tree Star) software.

Statistics

When comparing more than two groups, repeated measures analysis of variance was used. In all comparisons, P < 0.05 was used to determine statistical significance.

Results

Intracameral injection of MOG35–55 peptide pre- and post-immunization specifically prevents the induction or the progression of MOG35–55-induced EAE, respectively

To investigate whether an intracameral injection of MOG35–55 peptide could modulate MOG35–55 peptide-induced EAE, C57BL/6 mice were immunized with MOG35–55 peptide and CFA to induce EAE and received an intracameral injection of MOG35–55 peptide at different time points with respect to the day of immunization (days −7 pre-immunization and days 1, 8, 10, 13 post immunization (p.i.). EAE was not induced in the group receiving an intracameral injection of MOG35–55 peptide 7 days prior to the day of immunization or 1 day after the day of immunization (Fig. 1a). Moreover, the induction or progression of EAE was not affected by an i.v. injection of 15 μg of MOG35–55 peptide on day 0 (Fig. 1a).

Fig. 1.

Fig. 1.

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Intracameral injection of MOG35–55 peptide prevents the induction and progression of MOG35–55 peptide induced EAE. (a) C57BL/6 mice received an intracameral injection of MOG35–55 peptide at different time points with respect to the day of immunization. One group was given an i.v. injection of MOG35–55 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-MOG35–55 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).

The intracameral injection of MOG35–55 peptide 8 or 10 days after disease induction restricted the progression of EAE (Fig. 1a). In both groups receiving an intracameral, EAE was induced but did not progress after reaching an average score of 1.5 (day 8 AC) and 2 (day 10 AC). An intracameral injection of MOG35–55 peptide on day 13 p.i. did not protect the animals from EAE progression (Fig. 1a).

To determine the antigen specificity of the effect of an intracameral injection of MOG35–55 peptide on EAE, mice received an intracameral injection of OVA on day 0 and day 8 of immunization with MOG35–55 peptide. An injection of OVA into the AC did not suppress EAE induction or progression (Fig. 1a). Injection of a different self-antigen, Myelin Basic Protein (MBP), also did not affect EAE (data not shown).

To show the effectiveness of this treatment, we immunized mice with (MOG35–55) and then only those animals that had a clinical EAE score of 1 or more on day 10 p.i. received AC injections of (MOG35–55). Fig. 1(b) shows only mice that had a clinical EAE score on the day they received an intracameral injection of MOG35–55 peptide (i.e. day 10). All mice in Fig. 1(b) are therefore showing EAE scores of 1 or more on day 10, when they receive an AC injection of (MOG35–55). We found that there was a significant protection from EAE progression in the clinically sick mice after they received an AC injection of MOG35–55 (Fig. 1b).

Intracameral injection of MOG35–55 peptide into CD8−/− mice prevents the induction but not progression of MOG35–55-induced EAE

Antigen-specific splenic CD8+ Treg cells induced by the intracameral injection of an antigen suppress a DTH response in immunized mice (1, 2, 10, 13). To investigate the role of CD8+ regulatory cells induced by the intracameral injection of MOG35–55 peptide, CD8−/− mice (C57BL/6 background) were immunized with MOG35–55 peptide and received an intracameral injection of MOG35–55 7 days prior to the day of immunization or 8 days after immunization. EAE was not induced in mice receiving an intracameral injection of MOG35–55 peptide 7 days prior to the day of immunization. However, intracameral injection of MOG35–55 peptide into CD8−/− mice 8 days p.i. did not prevent disease progression (Fig. 2) as compared with the control wild-type (WT) mice.

Fig. 2.

Fig. 2.

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Intracameral injection of MOG35–55 peptide in CD8−/− mice cannot prevent EAE progression but can suppress the induction of EAE. CD8−/− and wild-type (C57B6) mice were immunized with MOG35–55 peptide and received AC injection of MOG35–55 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 MOG35–55 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).

Transfer of AC-induced CD8+ regulatory cells suppresses EAE progression in the recipient but cannot prevent the induction of EAE

To further clarify the role of AC-induced CD8+ regulatory cells, we recovered MOG-AC-induced splenocytes and separated them into CD8+ and CD8− populations. These cells were then transferred into recipient MOG35–55 peptide-immunized (EAE) mice, i.v., on day 0, day 10 or day 13 with respect to the day of immunization with MOG35–55 peptide. AC-MOG35–55 peptide-induced CD8+ cells suppressed EAE progression when transferred on 10 or 13 days post-immunization but were ineffective when transferred on day 0 (Fig. 3). The AC-induced CD8− splenocytes (all splenocytes except CD8+ cells) prevented the induction of EAE if transferred on the day of immunization but did not reduce the disease score when transferred 10 or 13 days post-immunization (Fig. 3).

Fig. 3.

Fig. 3.

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AC-induced CD8+ regulatory cells can restrict EAE progression but cannot prevent the induction of EAE. Mice were immunized with MOG35–55 peptide to induce EAE. Groups received AC-MOG35–55-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-MOG35–55-CD8+ on day 10, AC-MOG35–55-CD8− on day 0 or AC-MOG35–55-CD8+ on day 13 was statistically significant through the clinical course after peak symptoms (*,  , #, significant difference, P< 0.05).

The MOG-AC-induced CD8+ regulatory cells are antigen specific in their function

To determine the antigen specificity of MOG35–55 peptide-AC-induced CD8+ Treg cells, AC-OVA-induced or AC-MOG-induced CD8+ splenocytes were transferred i.v. into recipient EAE mice on day 10 post-immunization. There was no suppression of disease progression in the group that received CD8+ cells recovered from mice that received an intracameral injection of OVA (Fig. 4), but EAE progression was restricted in the group that received CD8+ cells recovered from mice that received an intracameral injection of MOG35–55 peptide (Fig. 4). CD8 Tregs induced by the AC injection of a different self-antigen, MBP, also did not restrict EAE (data not shown). To confirm that the AC-OVA-induced CD8+ Treg cells were functional, the AC-OVA-induced CD8+ cells suppressed an OVA-specific DTH response when injected into the footpad of OVA-immunized mice challenged with OVA (data not shown).

Fig. 4.

Fig. 4.

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Suppression of EAE by AC-MOG35–55-CD8+ regulatory cells is antigen specific. Mice were immunized with MOG35–55 peptide to induce EAE. Mice received AC-MOG35–55 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-MOG35–55-CD8+ cells was statistically significant through the clinical course after peak symptoms (*, significant difference, P< 0.05).

Transfer of AC-induced CD4+ regulatory cells effectively suppresses EAE induction in the recipient but cannot prevent the progression of EAE

CD4+ spleen cells induced by an intracameral injection of antigen specifically suppress the initiation of a DTH response in a recipient mice when transferred i.v. but do not suppress a DTH response in immunized recipients (1, 2). To investigate the role of AC-induced CD4+ regulatory cells, spleen cells from mice receiving an intracameral injection of MOG35–55 peptide were separated into CD4+ (enriched) and CD4− (all splenocytes except CD4+ cells) populations and these separate populations of cells were transferred i.v. into groups of recipient EAE mice on day 0 or day 10 with respect to the day of immunization. The AC-MOG-induced CD4+ cells suppressed the induction of EAE in the recipients when transferred on day 0 of immunization but were ineffective when transferred on day 10 p.i. (Fig. 5). Although the AC-MOG35–55 peptide-induced CD4− population could prevent disease progression when transferred on day 10, these cells were ineffective when injected on day 0 into recipient EAE mice (Fig. 5).

Fig. 5.

Fig. 5.

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AC-induced CD4+ regulatory cells can prevent EAE induction but cannot restrict the progression of EAE. Mice were immunized with MOG35–55 peptide to induce EAE. Groups received AC-MOG35–55-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-MOG35–55-CD4+ on day 0, AC-MOG35–55-CD4− on day 10 was statistically significant through the clinical course after peak symptoms (*,  , significant difference, P< 0.05).

The suppression of the induction of EAE by AC-MOG-induced CD4+ regulatory cells is antigen specific

To determine the antigen specificity of the AC-MOG-induced CD4+ Treg cells, AC-OVA-induced or AC-MOG-induced CD4+ splenocytes were transferred i.v. into recipient mice on day 0 of immunization with MOG35–55 peptide. There was no suppression of EAE induction in the group that received AC-OVA-induced CD4+ cells (Fig. 6), but EAE induction was restricted in the group that received AC-MOG-induced CD4+ cells (Fig. 6). CD4 Tregs induced by the AC injection of a different self-antigen, MBP, also did not restrict EAE (data not shown). To confirm that the AC-OVA-induced CD4+ Treg cells were functional, the AC-OVA-induced CD4+ cells suppressed an OVA-specific DTH reaction when injected i.v. in an OVA-immunized mice before being challenged with OVA (data not shown).

Fig. 6.

Fig. 6.

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AC-MOG35–55-CD4+ regulatory cells are antigen specific. Different groups of mice were immunized with MOG35–55 peptide to induce EAE. Groups received AC-MOG35–55 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-MOG35–55-CD4+ cells was statistically significant through the clinical course after peak symptoms (*, significant difference, P< 0.05).

The suppression of EAE progression by MOG-AC-induced CD8+ regulatory cells requires functional TGF-β signaling in effector cells

We reported previously that sensitivity to TGF-β by effector T cells is necessary for the suppression of a DTH response by CD8+ regulatory cells induced by an intracameral injection of antigen, although TGF-β sensitivity by T cells is not required for the generation of these cells (14). Further, antibodies to TGF-β inhibit the suppression of a DTH reaction by AC-induced splenic CD8+ regulatory cells (14). Because AC-induced splenic CD8+ regulatory cells can restrict the progression of EAE and not the induction of the disease (Figs 2–4), Cbl-b−/− mice whose T cells are not functionally responsive to TGF-β (27, 28), received an intracameral injection of MOG35–55 peptide. Different experimental groups received either an intracameral injection of MOG35–55 peptide 8 days p.i. or an i.v. injection of AC-MOG-induced splenic CD8+ T cells on day 10 p.i. An intracameral injection of MOG35–55 peptide on day 8 p.i. or the transfer of AC-MOG induced CD8+ Treg cells on day 10 p.i. did not restrict EAE progression in the Cbl-b−/− mice (Fig. 7) although the transfer of AC-MOG-induced CD8+ spleen cells suppressed EAE in the WT mice (Fig. 7).

Fig. 7.

Fig. 7.

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Suppression of EAE by MOG35–55 induced CD8+ regulatory cells requires TGF-β sensitivity by T cells. Cbl-b−/− and C57B/6 (WT) mice were immunized with MOG35–55 peptide. Different groups received an intracameral injection of MOG35–55 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).

Suppression of EAE at the priming phase by MOG-AC-induced CD4+ regulatory cells is independent of functional TGF-β signaling in the effector T cells

TGF-β has been defined as a major suppressive mechanism for Treg cells (29–31). We next tested if AC-induced CD4+ Treg cells also use a TGF-β-dependent mechanism to suppress EAE at the priming phase (Figs 5 and 6). Cbl-b−/− mice whose T cells are not responsive to TGF-β in a context-dependent manner or to FoxP3+ Treg-mediated suppression (27, 28) received AC-MOG-induced splenic CD4+ Treg cells from wild-type C57/B6 mice on the day of immunization. Wild-type C57/B6 control EAE mice received AC-MOG-induced splenic CD4+ Treg cells from wild-type C57/B6. Transfer of AC-MOG-induced CD4+ Treg cells into Cbl-b−/− EAE mice significantly prevented disease development in a similar fashion as in the wild-type C57/B6 mice (Fig. 8). In the same experiment, an intracameral injection of MOG35–55 peptide on day −7 or day 0 of immunization with MOG35–55 peptide restricted EAE induction in Cbl-b−/− as well as wild-type C57/B6 mice (data not shown).

Fig. 8.

Fig. 8.

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Suppression of EAE by MOG35–55-induced CD4+ regulatory cells is independent of TGF-β sensitivity by effector T cells. Cbl-b−/− or WT C57/B6 mice were immunized with MOG35–55 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-MOG35–55-induced CD4+ cells were significant (*, # significant difference, P< 0.05).

ACAID-CD4+ and CD8+ splenocytes migrate to the central nervous system

Because the transfer of AC-induced CD4+ and CD8+ Tregs could suppress EAE at different stages (Figs 3 and 5), we explored the migration of these CD4+ and CD8+ Tregs recovered from mice that received an intracameral injection of MOG to the central nervous system (CNS). Spleen cells from mice (CD 45.1) receiving an intracameral injection of MOG35–55 peptide were transferred i.v. into congenic MOG35–55 peptide-immunized recipient mice (CD45.2), 10 days post-immunization (at disease onset). Twenty-Four hours after transfer, ∼10% of the total mononuclear cells in the spinal cord consisted of splenocytes from donor mice (CD45.1) that received AC-MOG (Fig. 9a and b). Splenocytes from donors, which did not receive any AC injection or received a non-specific antigen (OVA), failed to reach the spinal cord in significant quantity (Fig. 9a and b). Because naive mice (non-immunized/non-EAE) have an intact blood–brain barrier (BBB), ACAID splenocytes were not detected in naive spinal cords after transfer (Fig. 9a and b). We next investigated the cellularity of the migrating donor splenocytes in the spinal cord. Splenocytes from mice receiving AC-MOG, which migrated to the spinal cord, exhibited a significant increase in CD4+ and CD8+ subsets compared with splenocytes from non-AC or OVA-AC mice that were detected in the spinal cord (Fig. 9c). In all cases, actual cell numbers correlated with the percentages.

Fig. 9.

Fig. 9.

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ACAID-CD4+ and CD8+ splenocytes migrate to the CNS. C57B/6 mice (CD45.2) were immunized were immunized with MOG35–55 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).

Discussion

Antigen injected into the AC of the eye exerts a profound and unique suppression of cell-mediated immune responses to the AC-injected antigen in the periphery (2, 10, 32). The injection of antigen into the AC generates circulating F4/80+ cells that migrate to the spleen and thymus. Thymic NKT cells, activated by these F4/80+ cells, then migrate to the spleen where, in concert with F4/80+ cells, marginal zone B cells and splenic NKT cells induce the CD4+ and CD8+ Treg cells specific for the antigen injected into the AC (2, 10, 32–34). That AC-induced CD4+ and CD8+ regulatory cells suppress different phases of a DTH response has been shown by us and others before (1, 2, 9–12). However, the suppression of the induction of an autoimmune response versus the progression of autoimmunity has not yet been clearly dissected. Because an intracameral injection of an antigen induces two different phenotypes of Treg cells, with distinct effector functions, we investigated if AC-MOG-induced splenic regulatory cells can modulate MOG-induced EAE.

To explore how MOG-induced ACAID can modulate MOG-induced EAE, different groups of EAE mice received an intracameral injection of MOG35–55 at different time points during EAE. Intracameral injection of MOG35–55 suppressed both the induction (when given on days −7 or 1 with respect to the day of MOG35–55 immunization) and the progression (when given during the late/chronic phase of EAE) of MOG-induced EAE. In fact, an intracameral injection of MOG35–55 significantly restricted disease progression in clinically sick mice.

AC-induced MOG-specific splenic CD4+ and CD8+ cells have different abilities to suppress either the induction (priming) or the progression of an autoimmune disease (EAE) induced by MOG immunization, respectively. AC-MOG-induced regulatory CD4+ splenic T cells suppressed EAE only when transferred on the day of immunization or within 2 days post-immunization but were ineffective at the late phase (effector/chronic phase) of EAE (days 8–13 p.i.). On the contrary, AC-induced splenic CD8+ T cells restricted active autoimmune disease and protected the mice from neurological symptoms, when transferred around days 8–10 p.i. only but were ineffective when transferred at the early time points (days 0, 1, 2). These CD8+ Tregs injected at the time of immunization may not suppress activated effector T cells 10 days p.i. even if present because we have observed that CD8+ Tregs are no longer effective at suppressing DTH in immunized mice 3 days after in vivo transfer of these CD8+ Tregs (REC and Vella, A unpublished observation). This may be due to a loss of suppressive activity and/or the production of IFN-γ required for suppression (25). The basis for loss in suppressive activity is under investigation. Therefore, CD8+ Tregs can only suppress the chronic/progressive phase of EAE and are ineffective when transferred in the early stage (day 0), unlike the AC-CD4+ Tregs.

The priming of MOG-specific effector T cells in EAE is believed to occur within the first 4–5 days post-immunization. Therefore, transfer of CD4+ cells from donors receiving intracameral MOG35–55 peptide may suppress the priming of EAE effector CD4+ T cells but are unable to suppress an already established disease because they do not suppress the activity of effector MOG-specific CD4+ T cells. This is further supported by the fact that AC-induced CD8+ T cells suppress the ‘expression’ of DTH, when transferred at the site of challenge, while the AC-induced CD4+ T cells are ineffective when transferred to the same site (2, 9, 10, 13). It has been shown extensively that AC-CD4+ Treg cells are not FoxP3+ Tregs and CD4+FoxP3+ Tregs do not have a role in ACAID (11, 35). Also, there was no change in the frequency of CD4+FoxP3+ Tregs in the spleen or lymph nodes following an AC injection (data not shown). Moreover, it is unlikely that the suppression by AC-CD4+ splenic cells is mediated by the FoxP3+ Tregs because we transferred a total of 1.5–2 × 106 splenic CD4+ cells, which should include ∼10% FoxP3+ Tregs, although for an efficient suppression of EAE by FoxP3+ Tregs, at least 2–2.5 × 106 purified FoxP3+ Tregs are required (36, 37). Further, these AC-induced CD4+ Treg cells suppressed the induction of EAE in Cbl-b−/− mice, although Cbl-b−/− effector T cells are resistant to the suppression mediated by both TGF-β and CD4+CD25+ Treg cells (27).

CD8+ Treg cells, unlike CD4+CD25+FoxP3+ Treg cells, suppress activated T cells (38–40). Most CD8+ Treg cells are detected after immunization and CD8+ Tregs are highly antigen specific and their suppression may be directed toward TCR V-region epitopes (39, 41). We and others have previously demonstrated that AC-induced CD8+ regulatory cells are restricted by a Qa-1–antigen complex and they express high levels of CD94/NKG2A (13, 42). Therefore, Qa-1 restricted ACAID-induced CD8+ regulatory cells may only target activated T cells because Qa-1 has been shown to be expressed by activated T cells only (16, 39, 43, 44). Accordingly, we suggest that MOG-AC-induced CD8+ Treg cells only act on MOG-specific activated effector T cells, therefore they are ineffective in suppressing EAE at the priming phase but can restrict disease progression by directly acting on the activated effector cells. Hence, two different populations of Treg cells (CD4+ or CD8+) induced by an intracameral injection of autoantigen can either suppress the priming or the effector phase of an autoimmune response to that antigen.

The suppressive mechanism(s) of AC-induced CD4+ and CD8+ Tregs has not been clearly defined. AC-induced CD8+ Tregs secrete TGF-β, which could be one of their suppressive mechanism(s) (12, 25, 45). In this regard, we showed previously that two strains of mice, Cbl-b−/− and dnTGF-βRII, in which the T cells do not respond to TGF-β functionally, based on different mechanisms, are also resistant to the suppression of the DTH reaction by CD8+ AC-SPL cells (10, 14). These studies also showed that sensitivity to TGF-β is not an obligate requirement for the in vivo induction of AC-induced CD8+ Tregs (14). Further, antibodies to TGF-β inhibit the suppression of DTH reaction by AC-induced splenic CD8+ regulatory cells (14). Therefore, we investigated the role of TGF-β in ACAID-induced-CD4+ and CD8+ splenic Treg cell-mediated suppression of EAE. Since dnTGF-βRII mice do not develop robust EAE, we used the Cbl-b−/− mice for our studies. Mice deficient in Cbl-b have T cells that are CD28 independent in their activation and hyperactive in their responses (27, 28, 46). In addition, Cbl-b−/− mice develop spontaneous autoimmunity and have increased susceptibility to elicited autoimmunity (46). Although Cbl-b−/− mice have normal numbers of functional CD4+CD25+ Foxp3+ regulatory T cells, their effector T cells are resistant to the suppression mediated by CD4+CD25+ Treg cells (27). Cbl-b−/− effector T cells have been shown to express normal levels of TGF-βRII, though an abnormality in Cbl-b−/− T cells in responding to TGF-β has been described previously (27). In the present studies, we further extended these findings and demonstrate that Cbl-b−/− mice are also resistant in vivo to CD8+ Treg cells but unlike CD4+FoxP3 Treg-mediated suppression, Cblb−/− effector T cells are responsive to ACAID-induced CD4+ Treg-mediated suppression. Only the AC-induced CD4+ Treg cells suppressed EAE in Cbl-b−/− mice, while AC-CD8+ regulatory cells failed to suppress EAE. Because Cbl-b−/− T cells do not functionally respond to TGF-β in specific contexts, these results suggest that one mechanism of suppression by AC-induced CD8+ regulatory cells is dependent on the sensitivity of effector T cells to TGF-β but the suppression mediated by the AC-induced CD4+ Treg cells is independent of TGF-β signaling. We show for the first time in vivo that two different populations of AC-induced regulatory cells not only act at different stages of an immune response but also have different mechanisms of suppression. Because splenic AC-CD4+ Treg cells secrete IL-10 (12), it is tempting to speculate that IL-10 might be a suppressive mechanism employed by these AC-CD4+ Treg cells. Experiments are underway to further explore this possibility.

Since AC-CD4 cells suppress EAE when transferred on the day of immunization, and the BBB is intact at that time point, it is presumed that AC-CD4+ T cells do not need to migrate to the CNS for their activity. While AC-CD8 cells are effective in suppressing EAE when transferred at a later time point during EAE, the BBB has already been breached. We therefore investigated whether transferred AC splenocytes migrate to the CNS (spinal cord) during EAE. Interestingly, only splenocytes from mice receiving an intracameral injection of MOG35–55 migrate to the CNS in significant numbers, as compared with splenocytes from non-AC or AC-OVA mice. This suggests that the migration of these cells induced by intracameral injection is antigen specific. Activated T cells can cross the BBB but there is no cognate antigen (OVA) in the spinal cord for AC-OVA-induced OVA-specific Tregs. Another possibility is that since these AC-OVA induced Tregs do not find their cognate antigen (OVA) in the spinal cord, even if they enter the cord, they might not remain there. If these MOG-specific Tregs migrate to the CNS by recognizing their cognate antigen (MOG), then it can be presumed that other CNS-specific antigens like MBP might also induce Tregs that would migrate to the CNS when transferred to a MOG-EAE mouse. But we did not find any remission in EAE when MBP was injected intracamerally into an EAE mouse or when AC-MBP-induced CD4+ or CD8+ Tregs were transferred i.v. into an EAE animal (data not shown). The cellularity of migrating donor splenocytes revealed a significantly higher amount of donor CD4 and CD8 cells in the spinal cord of mice receiving splenocytes from AC-MOG animals. Surprisingly, splenocytes from non-AC or AC-OVA mice that migrate into the CNS of the recipients consisted of mostly non-CD4 and non-CD8 cells. The lack of any specific marker to identify ACAID-induced Tregs limits our ability to experimentally confirm that these migrating CD4 and CD8 cells are ACAID Tregs. However, since these migrating CD4 or CD8 cells are found only in MOG-AC splenocytes supports the antigen specificity of this migration. Further, CD4 or CD8 cells from non-AC or OVA-AC mice did not reach the CNS in a MOG35–55-immunized EAE animal, which indicates that the migrating CD4 and CD8 cells from MOG-AC mice are neither naive T cells nor any AC-induced activated T cells. This in conjunction with the fact that only activated cells migrate to the CNS (47–49) supports the possibility that these migrating donor CD4 and CD8 cells are ACAID-induced Tregs.

In aggregate, we demonstrate that ACAID can be used to suppress both the initiation and progression of an autoimmune response. We show for the first time that modulating an autoimmune disease at the priming stage or the chronic stage requires different types of Treg cells with different suppression mechanism(s). From the perspective of the hope that better immunotherapeutic regimens will eventually be developed to treat autoimmune diseases, this might be of particular significance, as we show a way to effectively ameliorate disease after it has already been expressed. We also propose that, a Treg-cell-based therapy for autoimmune diseases may be successful only if the specific population of Treg cells (CD8+ or CD4+) is used depending on the stage of the disease.

Funding

This work was supported in part by National Institutes of Health (EY017537, EY017289 to R.E.C.); University of Connecticut Health Center Research Advisory Committee.

Acknowledgments

The authors would like to sincerely thank Roshanak Sharafieh and Yen Lemire (Department of Immunology, University of Connecticut Health Center) for their help with the AC injections.

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