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The Journal of Immunology, 2006, 176: 5950-5957.
Copyright © 2006 by The American Association of Immunologists

Peripheral Tolerance Via the Anterior Chamber of the Eye: Role of B Cells in MHC Class I and II Antigen Presentation1

Hossam M. Ashour* and Jerry Y. Niederkorn2,{dagger}

* Immunology Graduate Program, {dagger} Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ags introduced into the anterior chamber (AC) of the eye induce a form of peripheral immune tolerance termed AC-associated immune deviation (ACAID). ACAID mitigates ocular autoimmune diseases and promotes corneal allograft survival. Ags injected into the AC are processed by F4/80+ APCs, which migrate to the thymus and spleen. In the spleen, ocular APCs induce the development of Ag-specific B cells that act as ancillary APCs and are required for ACAID induction. In this study, we show that ocular-like APCs elicit the generation of Ag-specific splenic B cells that induce ACAID. However, direct cell contact between ocular-like APCs and splenic B cells is not necessary for the induction of ACAID B cells. Peripheral tolerance produced by ACAID requires the participation of ACAID B cells, which induce the generation of both CD4+ regulatory T cells (Tregs) and CD8+ Tregs. Using in vitro and in vivo models of ACAID, we demonstrate that ACAID B cells must express both MHC class I and II molecules for the generation of Tregs. These results suggest that peripheral tolerance induced through the eye requires Ag-presenting B cells that simultaneously present Ags on both MHC class I and II molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunological tolerance to self Ags is crucial for maintaining homeostasis in various organs and for survival itself. Two fundamental processes prevent the induction and expression of autoreactive T lymphocytes: central tolerance and peripheral tolerance (1, 2, 3). Central tolerance occurs during T lymphocyte ontogeny in the thymus where T lymphocytes bearing receptors that recognize self Ags expressed in the thymus are deleted by apoptosis. Other T cells that bear Ag receptors for epitopes that are not expressed in the thymus are not deleted and mature into potentially autoreactive T cells. However, other mechanisms are available to silence autoreactive T cells once they leave the thymus and enter peripheral sites. This process is termed peripheral tolerance and includes anergy and suppression. Although there was a period when the existence of suppressor T cells was questioned, the finding by Sakaguchi et al. (4) that immunosuppressive activity was present in CD4+CD25+ T cells and that animals depleted of this cell population developed inflammatory diseases in multiple organs, restored confidence in the existence and importance of suppressor cells in the maintenance of peripheral immune tolerance.

Peripheral tolerance is believed to be a crucial mechanism for preventing immune-mediated inflammation in various organs, including immune privileged sites. Immune privilege is believed to be an adaptation to protect organs such as the eye and brain from immune-mediated inflammation. Tissues in the eye and the brain have little or no regenerative properties and are vulnerable to immune responses, such as delayed-type hypersensitivity (DTH),3 which produce extensive injury to innocent bystander cells. However, Ags encountered in either the eye or the brain elicit the generation of peripheral tolerance that is maintained by Ag-specific regulatory T cells (Treg) (5, 6, 7). The Tregs induced by intraocular injection of Ag are part of the immunoregulatory process termed anterior chamber-associated immune deviation (ACAID), which is a crucial mechanism for maintaining immune privilege in the eye (5, 6). ACAID is the product of an extraordinarily complex series of cellular interactions that begin in the AC of the eye where Ag is captured and processed by F4/80+ APCs, which, under the influence of aqueous humor cytokines, are imprinted to elaborate a unique array of cytokines and cell surface molecules (5, 6). The F4/80+ ocular APCs then migrate to the thymus and spleen where they promote the generation of CD4+CD25+ Tregs and CD8+ Tregs (8, 9, 10, 11, 12, 13, 14).

There is compelling evidence that B cells act as ancillary APCs in the induction of ACAID. A combination of in vitro and in vivo studies has shown that the induction of ACAID begins when F4/80+ ocular APCs migrate to the spleen where they release Ag, which is captured and processed by splenic B cells (15, 16). These studies also demonstrated that antigenic moieties are released from F4/80+ ocular APCs and captured via the BCR on the splenic B cells, internalized, and processed in acidified lysosomes before being presented to T cells (16). B cells are needed for the generation of both CD4+CD25+ Tregs and CD8+ Tregs in ACAID and for maintaining ocular immune privilege (13, 15, 17, 18). The time-honored principle that Ag is presented to CD4+ T cells via MHC class II (MHC-II) and that presentation to CD8+ T cells occurs through MHC-I molecules led us to test the hypothesis that ACAID B cells present Ags on both MHC-I and MHC-II molecules during the establishment of peripheral tolerance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 (H-2b) mice; C57BL/6 BCR-transgenic (BCR-Tg) mice (H-2b), C57BL/6-Tg (IghelMD4)4Ccg/J; B6.129P2-B2mtm1Unc/J (beta2 microglobulin (beta2m) knockout (KO), or MHC-I-deficient) mice; and B6.129S2-Igh-6tm1Cgn/J B cell-deficient (B cell KO) mice were purchased from The Jackson Laboratory. B6.129-H2-Ab1tm1Gru N12 (MHC-II-deficient) mice were purchased from Taconic Farms. All animals were housed and cared for in accordance with the guidelines of the University Committee for the Humane Care of Laboratory Animals, the National Institutes of Health Guidelines on Laboratory Animal Welfare, and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

Screening BCR-Tg mice

BCR-Tg mice (C57BL/6-Tg(IghelMD4)4Ccg/J) are hemizygotic for the hen egg lysozyme (HEL) transgene expressed on BCRs (19). More than 90% of the splenic B cells display the proper allotype, and 60–90% can bind HEL (19, 20). PCR was used to screen DNA isolated from tails of their offspring for expressing the BCR transgene according to The Jackson Laboratory protocol. The PCR products were visualized on a 1.5% agarose gel containing ethidium bromide. A 430-bp band indicated a BCR transgene-positive animal.

Subcutaneous immunization

Mice were immunized by s.c. injection of 250 µg of OVA (OVA; Sigma-Aldrich) in PBS. OVA was emulsified 1:1 in complete Freund’s adjuvant (CFA; Sigma-Aldrich). Each animal received a total volume of 200 µl.

AC injection

A Hamilton automatic dispensing apparatus was used to inject 100 µg (in 5 µl) of either OVA or HEL into the AC as described previously (16).

DTH assay

An ear-swelling assay was used to measure DTH to either OVA or HEL as described previously (13). The results are expressed as follows: specific ear swelling = (24 h measurement – 0 h measurement) for experimental ear – (24 h measurement – 0 h measurement) for negative control ear.

Generation of ocular-like APCs

ACAID-inducing APCs were generated in vitro with a previously described protocol that has been used extensively for analyzing regulatory cells in ACAID (15, 16, 17, 18, 20, 21, 22, 23). Peritoneal exudate cells were collected from C57BL/6 mice and cultured overnight (2 x 106 cells/ml) in complete RPMI 1640 medium supplemented with 10 mg/ml OVA and 2 ng/ml human TGF-beta2 (R&D Systems). These ocular-like APCs induce a form of peripheral tolerance that is identical with ACAID.

Generation of ACAID B cells

We have used an in vitro culture system to generate B cells that are capable of inducing the generation of Tregs that express the same phenotype as those induced by AC injection of Ag (16, 24, 25). OVA-pulsed ocular-like APCs were generated in vitro as described above and cocultured for 48 h with B cells isolated from the spleens of normal C57BL/6 mice using CD45R (B220) microbeads (Miltenyi Biotec). The nonadherent B cell population was collected and treated with anti F4/80+ (Accurate Chem) plus complement (Cedarlane Laboratories) to kill any residual macrophages. These ACAID-inducing B cells were then injected i.v. (4 x 106 cells/mouse) into normal C57BL/6 mice. In some experiments, B cells were treated with mitomycin C (Sigma-Aldrich) or gamma irradiation (2400 rad) before incubation with OVA-pulsed ocular-like APCs. The viability of B cells was determined by trypan blue exclusion immediately before adoptive transfer and was always >95%.

Flow cytometric analysis

BCR-Tg mice were AC injected with OVA (Sigma-Aldrich), HEL (Sigma-Aldrich), or PBS. Seven days later, spleen cells were harvested and B cells purified using CD45R (B220) microbeads (Miltenyi Biotec). B cells were then stained with HEL-biotin (Abcam) followed by streptavidin FITC (BD Biosciences). A single-color flow cytometry was performed to assess proportions of B cells bearing the transgene in each group. To analyze the data, CellQuest Pro software (BD Biosciences) was used on a FACSCalibur instrument (BD Biosciences).

In vitro ACAID model of Treg cell generation

We used an in vitro spleen cell culture system that generates Tregs that express the same properties and surface markers as Tregs produced by AC injection (8, 11, 13, 16, 18, 26). Moreover, the in vitro-generated T regs directly inhibit DTH and are Ag-specific CD8+ T cells (16).

Briefly, ocular-like APCs (5 x 106) were added to a large petri dish (Falcon 3003; BD Biosciences) containing 5 x 107 spleen cells harvested from naive C57BL/6. In some experiments, cell populations were cultured in Transwell chambers separated by a semipermeable membrane (0.4-µm pore size; Costar). All spleen cell cultures were incubated at 37°C for 5–7 days before being tested for Tregs generation. In other experiments, ocular-like APCs and spleen cells from C57BL/6 mice (depleted of B cells and APCs) were incubated with or without B cells (from C57BL/6 mice, MHC-II-deficient mice, or beta2m KO mice). To ensure depletion of APCs from spleen cells, unattached cells were further treated with anti-F4/80+ Ab (Accurate) plus complement (Cedarlane Laboratories). In other experiments, 10 ng/ml recombinant mouse IL-10 (R&D Systems) was added to the culture medium. Viability, as determined by trypan blue exclusion, confirmed that >95% of the in vitro-generated T regs used in the local adoptive transfer (LAT) assay (see below) were viable.

LAT assay

This assay was used to test for Tregs in ACAID (15, 16). Putative Tregs, generated either in vivo or in vitro, were injected (1 x 106 cells in 10 µl) with spleen cells (1 x 106 cells in 10 µl) collected from s.c. immunized donors and 10 mg/ml either OVA or HEL into the left ear pinna of a naive mouse. The presence of Tregs was demonstrated by the inhibition of the ear-swelling responses mediated by immune spleen cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ocular-like APCs deliver an Ag-specific signal to B cells, which then induce suppression of DTH

We and others have used an in vitro spleen cell culture system to duplicate the generation of Tregs that occurs when Ag is introduced into the AC of the eye (8, 11, 13, 16, 18, 26). In this system, ocular-like APCs are produced by culturing F4/80+ macrophages for 24 h in the presence of OVA and the ocular cytokine, TGF-beta. The ocular-like APCs are then cocultured with spleen cells for 5–7 days. The CD4+ Tregs and CD8+ Tregs generated in this system display all of the properties of the splenic Tregs that are elicited when Ag is introduced into the AC (8, 11, 13, 16, 18, 26). Moreover, the CD4+ Tregs and CD8+ Tregs generated in vitro and in vivo are Ag specific (13). We have shown that the in vitro-generated T regs that directly inhibit DTH are Ag-specific CD8+ T cells (16).

Previous studies have suggested that, during the induction of ACAID, ocular-like APCs do not directly present Ag to T cells but instead release antigenic peptides to splenic B cells, which in turn present Ag to T cells (15, 16). With this in mind, experiments were performed to determine whether cell contact between ocular-like APCs and spleen cells was needed for inducing Tregs. Tregs were generated in vitro by culturing naive spleen cells with OVA-pulsed ocular-like APCs. However, in these experiments, a Transwell culture system was used in which ocular-like APCs and F4/80+ APC-depleted spleen cells were separated by a semipermeable membrane that prevented direct contact between the two cell populations. After 5–7 days in culture, the spleen cells were tested in a LAT assay to determine their capacity to directly suppress DTH responses. The LAT assay is based on the principle that OVA-specific CD8+ ACAID Tregs will directly suppress the expression of DTH responses produced by OVA-immunized T cells that normally induce an ear-swelling response when mixed with OVA and injected into the ears of naive mice. The results of this LAT assay showed that Tregs were generated, even when direct contact between the ocular-like APCs and spleen cells was inhibited (Fig. 1A). The generation of Tregs required ocular-like APCs and was not simply an effect of the TGF-beta that was added to the cell cultures; that is, culturing spleen cells in the presence of TGF-beta alone did not lead to the development of Tregs. The Transwell culture experiments were repeated and expanded to confirm that splenic B cells, rather than splenic APCs, were responsible for the induction of Ag-specific tolerance. In these experiments, ocular-like APCs were pulsed with either OVA or HEL and placed in the upper chambers of Transwell cultures. B cell suspensions were placed into the lower chambers. Two days later, the B cells were adoptively transferred to naive C57BL/6 mice. Seven days later, the B cell recipients were immunized s.c. with OVA emulsified in CFA. Ear-swelling responses to OVA and HEL (irrelevant Ag) were assessed 7 days after the s.c. immunization. The results demonstrate that B cells did not have to be in direct contact with the ocular-like APCs in order for them to induce ACAID in naive hosts (Fig. 1B). Moreover, B cells cocultured with HEL ocular-like APCs or untreated ocular-like APCs did not suppress OVA-specific DTH response. APCs not exposed to TGF-beta (regular APCs) but pulsed with OVA did not elicit the generation of ACAID-inducing B cells. Thus, ocular-like APCs deliver an Ag-specific signal to B cells that renders them tolerogenic and capable of inducing Ag-specific tolerance in third-party hosts. The transmission of the APC signal does not require direct cell-to-cell contact.


Figure 1
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  		FIGURE 1. Direct contact between ocular-like APCs and spleen cells is not needed for the generation of peripheral tolerance. A, OVA-pulsed ocular-like APCs were cultured either in the upper chamber or the lower chamber of Transwell culture plates. Spleen cell suspensions depleted of F4/80+ cells were placed in the lower chamber of Transwell culture plates. After 5–7 days, the spleen cell suspensions were tested in a LAT assay for their capacity to suppress DTH responses to OVA. B, Ocular-like APCs or nonocular APCs were placed in the upper chamber of Transwell culture plates, and B cell suspensions were placed in the lower chambers. Ocular-like APCs were pulsed with OVA, HEL, or no Ag. Nonocular regular (Reg.) APCs were pulsed with OVA. ACAID-inducing B cells (ACAID control) were generated by coculturing OVA-pulsed ocular-like APCs with B cells. Two days later, B cells from the bottom chambers and the ACAID control B cells were injected i.v. into panels of naive C57BL/6 mice. Seven days later, all mice were immunized SC with OVA plus CFA. DTH responses to OVA were assessed in a conventional ear-swelling assay 7 days after the s.c. immunization with OVA plus CFA. Positive controls consisted of C57BL/6 mice immunized s.c. with OVA plus CFA. Naive C57BL/6 mice served as negative controls. *, p < 0.01.

 
Additional experiments were performed to confirm that OVA was processed by ocular-like APCs and released to B cells for further processing and presentation to T cells. OVA-pulsed ocular-like APCs and ocular-like APCs not pulsed with OVA were thoroughly washed in HBSS and incubated for 48 h with splenic B cells. As a control, other splenic B cell suspensions were cocultured for 48 h with free OVA in the absence of ocular-like APCs. Both populations of B cells were washed thoroughly with HBSS and injected i.v. into naive C57BL/6 mice. After 7 days, the mice were immunized with OVA plus CFA. DTH to OVA was measured 7 days later using a conventional ear-swelling assay. The results of a typical experiment are shown in Fig. 2 and demonstrate that B cells incubated with ocular-like APCs that had been pulsed with OVA induced the generation of Tregs in naive mice, as shown by the suppressed DTH responses to OVA. By contrast, B cells incubated with OVA in the absence of ocular-like APCs did not induce suppression of DTH when adoptively transferred to naive mice. Likewise, ocular-like APCs not pulsed with OVA did not induce the generation of ACAID-inducing B cells. Thus, processing of OVA by ocular-like APCs is necessary for the induction of tolerogenic B cells.


Figure 2
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  		FIGURE 2. Ocular-like APCs process OVA and render B cells tolerogenic. Ocular-like APCs were pulsed either in the absence ("No Ag Ocular APC") or presence of OVA ("OVA Ocular APC") for 24–48 h in the presence of TGF-beta, washed thoroughly, and cocultured for 48 h with B cells. Other B cell suspensions were cultured with OVA alone for 48 h ("OVA No APC"). B cell suspensions were treated with anti-F4/80 Ab plus complement and injected i.v. into normal C57BL/6 mice that were then immunized s.c. with OVA plus CFA 7 days later. DTH responses to OVA were assessed 7 days after s.c. immunization using an ear-swelling assay. *, p < 0.01.

 
Expansion of splenic Ag-specific B cells following AC injection of Ag

Previous studies have demonstrated that the induction of ACAID requires that the host’s B cells express the BCR that recognizes the Ag that is introduced into the AC. For example, mice bearing the BCR transgene for HEL will develop ACAID if HEL is injected into the AC but will not develop ACAID if other Ags are used (16). Considering the small number of B cells expressing the BCR for OVA, we tested the hypothesis that AC priming with Ag stimulates the expansion of splenic B cells. HEL, OVA, or PBS was injected into the AC of HEL BCR-Tg mice. Seven days later, splenic B cell suspensions from each group of mice were examined by flow cytometry for the relative number of B cells that bound HEL. The results indicate that the number of B cells that bound HEL was dramatically increased in the HEL BCR mice that had been injected with HEL (Fig. 3). However, AC injection of either OVA or PBS did not result in an expansion in the number of HEL-binding B cells. It was not possible to determine whether the increased numbers of HEL-specific B cells was due to clonal expansion of splenic B cells or recruitment of HEL-specific B cells from other lymphoid tissues and the peripheral blood.


Figure 3
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  		FIGURE 3. Expansion of HEL-binding B cells in mice primed in the AC with HEL. HEL BCR-Tg mice were injected in the AC with OVA, PBS, or HEL. Seven days later, splenic B cells were isolated, incubated with biotinylated HEL, and the number of HEL-binding B cells was determined by flow cytometry using FITC-labeled avidin.

 
Role of MHC-II-bearing B cells in the induction of peripheral tolerance

Results shown in this study and elsewhere indicate that ACAID B cells can induce the development of Ag-specific Tregs when adoptively transferred to naive recipients (15, 16). ACAID is a form of peripheral tolerance that requires the participation of two independent populations of Tregs; one is CD4+ and promotes the development of the second population, which is CD8+ and suppresses the expression of DTH. Because B cells are needed for the generation of both populations of Tregs (16), we examined the role of MHC-I and II expression on the induction of ACAID by B cells.

ACAID B cells were generated in vitro as described earlier. However, B cells were obtained from either normal mice or MHC-II KO mice. After in vitro culture with OVA-pulsed ocular-like APCs, B cells were injected i.v. into normal mice. After 7 days, mice were immunized with OVA plus CFA, and DTH to OVA was measured 7 days later. As before, adoptive transfer of normal B cells cocultured with OVA-pulsed ocular-like APCs induced suppression of DTH responses to OVA (Fig. 4A). By contrast, B cells from MHC-II KO mice that were cocultured with OVA-pulsed ocular-like APCs were unable to induce suppression of DTH when adoptively transferred to normal mice. To further support this observation, B cell KO mice were reconstituted with B cells from either normal mice or MHC-II KO mice. B cell-reconstituted mice were then primed in the AC with OVA, and 7 days later, the mice were immunized s.c. with OVA plus CFA. Assessment of DTH responses to OVA 7 days after the s.c. immunization revealed that, as expected, reconstitution of B cell KO mice with B cells from normal donors restored the development of ACAID (Fig. 4B). By contrast, B cells from MHC-II KO mice failed to restore ACAID in the B cell-deficient hosts. In vitro spleen cell cultures were used to confirm that expression of MHC-II is required for the induction of ACAID Tregs. OVA-pulsed ocular-like APCs prepared from normal donors were cocultured with B cells from either normal or MHC-II KO mice and with spleen cells depleted of B cells and F4/80+ APCs. Seven days later, the spleen cells were tested for their capacity to suppress OVA DTH responses in a LAT assay. As in previous studies, spleen cells from normal mice cocultured with OVA-pulsed ocular-like APCs in the presence of B cells from normal mice inhibited the expression of DTH by spleen cells collected from normal mice that were immunized with OVA plus CFA (Fig. 4C). By contrast, B cells from MHC-II KO mice did not promote the generation of Tregs.


Figure 4
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  		FIGURE 4. B cells must express MHC-II molecules to induce peripheral tolerance. A, B cells from either normal or MHC-II KO mice were cocultured for 48 h with OVA-pulsed ocular-like APC prepared from normal mice. B cell suspensions were treated with anti-F4/80 Ab plus complement and injected i.v. into normal C57BL/6 mice that were immunized s.c. with OVA plus CFA 7 days later. DTH responses to OVA were assessed 7 days after s.c. immunization using an ear-swelling assay. B, B cell KO mice were reconstituted with B cells from either normal mice or MHC-II KO mice. B cell-reconstituted mice were injected in the AC with OVA 7 days prior to being immunized SC with OVA plus CFA. DTH responses to OVA were assessed 7 days after the SC immunization using an ear-swelling assay. ACAID Tregs were generated using in vitro spleen cell cultures. The three spleen cell culture groups contained OVA-pulsed ocular-like APCs and spleen cell suspensions that were depleted of B cells and F4/80+ APC. Spleen cell cultures were supplemented with B cells from normal mice or MHC-II-deficient mice. As a control, one spleen cell culture was not supplemented with B cells. All three spleen cell cultures were incubated for 5–7 days. Each spleen cell suspension was tested in a LAT assay for Tregs that suppressed OVA-specific DTH.

 
It has been reported that CD4+ Tregs are required for the development of CD8+ end-stage efferent suppressor cells in ACAID (14). Using the in vitro spleen cell culture model, we showed previously that the CD4+ Tregs in ACAID must produce IL-10 to promote the development of CD8+ Tregs (13). Moreover, CD4+ T cells from IL-10 KO mice are unable to support the development of ACAID, either in vitro or in vivo (13). However, exogenous IL-10 is able to replace CD4+ T cells, but not CD8+ T cells, in the induction of ACAID in vitro (13). Therefore, we suspected that MHC-II expression on ACAID B cells was required for the generation of CD4+ Tregs, whose primary function was to produce IL-10. To test this, B cell-depleted, F4/80+ APC-depleted spleen cell suspensions from normal mice were reconstituted with B cells from MHC-II KO donors and were cocultured with OVA-pulsed APCs from normal mice in the presence or absence of exogenous IL-10. Seven days later, the spleen cell cultures were tested for the presence of Tregs in a LAT assay. As before, spleen cell cultures containing B cells from MHC-II KO mice failed to support the development of Tregs that suppressed OVA DTH responses (Fig. 5). However, the addition of exogenous IL-10 to MHC-II KO B cell cultures restored the generation of Tregs that suppressed OVA DTH responses. Importantly, IL-10 alone did not induce the development of Tregs.


Figure 5
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  		FIGURE 5. IL-10 can substitute for MHC-II-bearing B cells in the induction of peripheral tolerance. B cell-depleted spleen cell suspensions that were treated with anti-F4/80 plus complement, were reconstituted with B cells from MHC-II KO mice or B cells from normal mice and cocultured for 5 days with OVA-pulsed ocular-like APCs in the presence or absence of exogenous IL-10 (10 ng/ml). Spleen cells were then tested for their capacity to suppress OVA-specific DTH in a LAT assay.

 
Expression of MHC-I on B cells is required for the induction of peripheral tolerance

Peripheral tolerance induced by either AC injection of Ag or by in vitro ACAID spleen cultures is mediated by CD8+ end-stage Tregs that are Ag specific. This implies that Ag is presented to the CD8+ Tregs by MHC-I molecules that are expressed on ACAID B cells. To test this, Tregs were generated in vitro by coculturing OVA-pulsed ocular-like APCs with B cells from either normal mice or beta2m KO mice. Two days later, B cells were isolated from the cultures and adoptively transferred to naive normal recipients that were s.c. immunized with OVA plus CFA 7 days later. DTH to OVA was assessed 7 days after the s.c. immunization. As before, B cells from normal mice were able to induce ACAID. By contrast, B cells from beta2m KO mice were unable to induce ACAID Tregs (Fig. 6A). In other experiments, B cell KO mice were reconstituted with B cells from either normal or beta2m KO mice. The reconstituted mice were primed in the AC with OVA and 7 days later were immunized SC with OVA plus CFA. DTH responses to OVA were assessed 7 days later and revealed that B cells from normal mice were able to reconstitute B cell KO mice and restore the development of ACAID in these hosts. By contrast, B cell KO mice reconstituted with B cells from beta2m KO mice failed to develop suppression and instead demonstrated positive DTH to OVA (Fig. 6B). In vitro spleen cell cultures were used to confirm that expression of MHC-I was required for the induction of ACAID Tregs. OVA-pulsed ocular-like APCs prepared from normal donors were cocultured with spleen cell suspensions depleted of F4/80+ APCs and B cells. The spleen cell cultures were reconstituted with B cells prepared from either normal or beta2m KO mice. Seven days later, the spleen cells were tested for their capacity to suppress OVA DTH responses in a LAT assay. As in previous studies, spleen cells from normal mice cocultured with OVA-pulsed ocular-like APCs in the presence of B cells from normal mice were able to inhibit the expression of DTH by spleen cells collected from normal mice that had been immunized with OVA plus CFA (Fig. 6C). By contrast, B cells from beta2m KO mice did not promote the generation of Tregs.


Figure 6
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  		FIGURE 6. Expression of MHC-I molecules on B cells is necessary for the induction of peripheral tolerance. A, B cells from either normal or beta2m KO mice were cocultured for 48 h with OVA-pulsed ocular-like APCs prepared from normal mice. B cell suspensions were treated with anti-F4/80 Ab plus complement and injected i.v. into normal C57BL/6 mice that were immunized s.c. with OVA plus CFA 7 days later. DTH responses to OVA were assessed 7 days after the s.c. immunization using an ear-swelling assay. B, B cell KO mice were reconstituted with B cells from either normal mice or beta2m KO mice. B cell-reconstituted mice were injected in the AC with OVA 7 days prior to being immunized s.c. with OVA plus CFA. DTH responses to OVA were assessed 7 days after the SC immunization using an ear-swelling assay. C, ACAID Tregs were generated using in vitro spleen cell cultures. The three spleen cell culture groups contained OVA-pulsed ocular-like APCs and spleen cell suspensions that were depleted of B cells and F4/80+ APC. Spleen cell cultures were either not reconstituted with B cells or were supplemented with B cells from either normal mice or beta2m KO mice. All three spleen cell cultures were incubated for 5–7 days. Each spleen cell suspension was tested in a LAT assay for its capacity to suppress OVA-specific DTH.

 
Reconstitution of B cell KO mice with B cells from MHC-II KO donors and beta2m KO donors restores the capacity to induce peripheral tolerance via the AC of the eye

If MHC-I- and MHC-II-expressing B cells are both needed for the induction of ACAID, it should be possible to restore peripheral tolerance by reconstituting B cell KO mice with B cells from MHC-II KO donors, which express normal MHC-I, and B cells from beta2m KO donors, which express normal MHC-II. This was tested by reconstituting B cell KO mice with equal numbers of B cells from MHC-II KO donors and beta2m KO donors. Reconstituted B cell KO mice were primed in the AC with OVA and 7 days later were s.c. immunized with OVA plus CFA. DTH responses to OVA were assessed 7 days after the s.c. immunization with OVA. The results demonstrate that reconstitution with a mixture of B cells from MHC-II KO donors and beta2m KO donors restored ACAID in B cell KO mice (Fig. 7).


Figure 7
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  		FIGURE 7. B cells from MHC-II KO mice and beta2m KO mice reconstitute peripheral tolerance in B cell KO mice. B cell KO mice were reconstituted with a splenic equivalent of a B cell suspension containing equal numbers of B cells from MHC-II KO mice and beta2m KO mice. OVA was injected into the AC 7 days after B cell reconstitution. Mice were immunized s.c. with OVA plus CFA 7 days after the AC injections with OVA. DTH responses to OVA were assessed 7 days after the s.c. immunization using an ear-swelling assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The unique immunological features of the eye were recognized >130 years ago by the Dutch ophthalmologist van Dooremaal (27), who noted the prolonged survival of mouse skin grafts placed in the AC of the dog eye. It was another 75 years until Medawar (28) recognized the full significance of the prolonged survival of skin allografts placed into the AC of rabbit eyes and termed the phenomenon "immune privilege." In the past 30 years, enormous insights have emerged in our understanding of immune privilege, and it has become clear that multiple anatomical, physiological, and immunoregulatory mechanisms contribute to immune privilege in the eye. Immune privilege is maintained, in large part, by the induction of peripheral tolerance, which is mediated by Tregs that are induced when Ag is introduced into the AC (5, 6, 7, 29).

The peripheral tolerance induced via the AC (i.e., ACAID) is generated through a complex series of cellular interactions that involve the eye, thymus, spleen, and elements of the sympathetic nervous system (30, 31, 32, 33). Ags introduced into the AC are processed by F4/80+ APCs in the eye, which emigrate to the thymus and the spleen (8, 32, 34). After arriving in the spleen, the F4/80+ ocular APCs set into motion a complex series of cellular interactions involving B cells (15, 16, 18), NKT cells (9, 35), {gamma}{delta} T cells (36, 37, 38), CD4+ Tregs (8, 13, 14), and CD8+ Tregs (14). In vitro and in vivo studies suggest that F4/80+ ocular APCs release peptide fragments that are captured by splenic B cells, which act as ancillary APCs and induce the generation of CD4+ Tregs and CD8+ Tregs (16). This conclusion is based on findings indicating that B cells collected from mice primed in the AC with OVA will induce OVA-specific ACAID when adoptively transferred to naive mice (15, 16).

Moreover, ACAID-inducing B cells can be generated in vitro by coculturing splenic B cells with OVA-pulsed ocular-like APCs (15, 16). ACAID B cells must express the BCR that recognizes the Ags that are processed by the ocular APCs. Additionally, acidification of the endosome is required for B cells to acquire the capacity to transfer ACAID (16). It is important to note that ACAID B cells, whether generated in vivo or in vitro, do not function as suppressor cells but instead induce the development of CD4+ Tregs and CD8+ Tregs (15, 16). Thus, the weight of evidence suggests that B cells act as ancillary APCs for the induction of ACAID.

The present results extend these findings and indicate that ocular APCs deliver a signal to B cells that renders them tolerogenic. The data also confirm that Ag processing by ocular APCs must occur in order for B cells to acquire the capacity to induce ACAID, as exposure to OVA alone, OVA-pulsed normal APCs, or untreated ocular-like APCs does not render B cells tolerogenic. However, cell contact between ocular-like APCs and B cells is not required; that is, tolerogenic B cells are generated even if the two cell populations are separated by a semipermeable membrane. AC injection of Ag results in a marked expansion in the numbers of B cells, which presumably increases the number of Ag-specific B cells that are available to present Ag to T cells for the induction of ACAID. Flow cytometric analysis of HEL BCR-Tg mice primed in the AC with HEL, OVA, or PBS indicates that splenic B cell proliferation is restricted to B cells that express the BCR that recognizes the Ag injected into the AC. ACAID B cells treated with either gamma irradiation or mitomycin C are not able to adoptively transfer ACAID, suggesting that clonal expansion of B cells is necessary for the induction of ACAID (data not shown). However, gamma irradiation or mitomycin C can alter protein synthesis and compromise cell viability. Thus, it is not possible to ascertain if the increased numbers of splenic B cells are the result of clonal expansion or recruitment of HEL-specific B cells from other lymphoid tissues and the peripheral blood.

ACAID can be induced by either AC injection of Ag or by the adoptive transfer of splenic B cells from mice primed in the AC with Ag to naive recipients (15, 16). In both cases, Ag-specific CD8+ Tregs and CD4+ Tregs are induced, which leads to the question as to how B cells can simultaneously induce two populations of Tregs that are MHC-I and MHC-II restricted, respectively. The present results indicate that a defect in either the assembly or expression of either MHC-I or MHC-II molecules on B cells prevents B cells from inducing ACAID. However, if both B cell populations are combined, they are capable of restoring ACAID in B cell KO mice, suggesting that B cells present Ag on both MHC-I and MHC-II molecules.

ACAID shares many of the properties with orally induced immune tolerance to nickel, including the participation of B cells and NKT cells (15, 18, 25, 39, 40, 41, 42). Orally induced tolerance to nickel can be adoptively transferred with B cells; however, the B cells induce a form of infectious tolerance that can be transferred to third-generation hosts with either T cells or APCs. It will be important to determine whether a similar form of infectious tolerance occurs in ACAID.

The peripheral tolerance induced by AC injection of Ag is more than a curious artifact and has important clinical implications. ACAID not only mitigates ocular autoimmune diseases (43), but also promotes corneal allograft survival (11, 44, 45, 46). Orthotopic corneal grafts are in direct contact with the AC of the eye, and thus, can induce ACAID, which is intimately associated with corneal allograft survival (11, 46). Moreover, induction of ACAID by AC injection of donor-derived cells before corneal transplantation enhances corneal allograft survival, while maneuvers that prevent the corneal allograft’s capacity to induce ACAID result in increased graft rejection (44, 45).

DTH responses are notorious for producing ischemic necrosis and damaging innocent bystander cells. Thus, the capacity of ACAID to inhibit DTH to Ags that enter the eye or to tissue-specific Ags expressed in the eye has obvious benefits for the survival of corneal endothelial and retinal cells, which are terminally differentiated and cannot proliferate.

Thus, the peripheral tolerance that is induced in immune privileged sites, such as the eye, not only prevents immune-mediated responses directed at self Ags, but also regulates immune-mediated responses that indirectly injure innocent bystander cells that cannot regenerate and are crucial for the normal function of an organ such as the eye.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants EY005631 and EY016664 and an unrestricted grant from Research to Prevent Blindness, New York, NY. Back

2 Address correspondence and reprint requests to Dr. Jerry Y. Niederkorn, Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390. E-mail address: jerry.niederkorn{at}utsouthwestern.edu Back

3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; AC, anterior chamber; ACAID, AC-associated immune deviation; Tg, transgenic; HEL, hen egg lysozyme; Treg, regulatory T cell; beta2m, beta2 microglobulin; KO, knockout; LAT, local adoptive transfer; MHC-II, MHC class II. Back

Received for publication December 15, 2005. Accepted for publication February 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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