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Purpose: Severing corneal nerves during corneal transplantation does not affect first corneal transplants, but abolishes immune privilege of subsequent corneal allografts. This abrogation of immune privilege is attributable to the disabling of T regulatory cells (T regs) induced by corneal transplantation. The goal of this study was to determine if severing corneal nerves induces the development of contrasuppressor (CS) cells, which disable T regs that impair other forms of immune tolerance., Methods: Effect of corneal nerve ablation on immune tolerance was assessed in four forms of immune tolerance: anterior chamber-associated immune deviation (ACAID); oral tolerance; corneal transplantation, and intravenously (IV) induced immune tolerance. T regulatory cell activity was assessed by adoptive transfer and by local adoptive transfer (LAT) of suppression assays., Results: Corneal nerve ablation prevented ACAID and oral tolerance, but did not affect IV-induced immune tolerance. Contrasuppressor cells blocked the action of T regs that were generated by anterior chamber injection, oral tolerance, or orthotopic corneal transplantation. The neuropeptide substance P (SP) was crucial for contrasuppressor activity as CS cells could not be induced in SP-/- mice and the SP receptor inhibitor, Spantide II, prevented the expression of CS cell activity in vivo. Contrasuppressor cells expressed CD11c surface marker that identifies dendritic cells (DC)., Conclusions: The loss of immune privilege produced by corneal nerve ablation following corneal transplantation extends beyond the eye and also affects immune tolerance induced through mucosal surfaces and appears to be mediated by a novel cell population of CD11c+ CS cells that disables T regs.

Purpose: We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance., Method: The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice., Results: The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%)., Conclusions: The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.

Immune tolerance can be induced by numerous methods. This review article aims to draw lines of similarity and contrast between two unique models of immune tolerance, namely Anterior Chamber Associated Immune Deviation (ACAID) and Nickel-induced oral tolerance. ACAID is an immune tolerance model that leads to the generation of CD4(+) T regulatory cells and CD8(+) T regulatory cells in the periphery after the injection of an antigen into the anterior chamber of the eye. Nickel-induced oral tolerance is another immune tolerance model that is induced by the contact allergen Nickel and leads to the generation of Nickel-specific CD4(+) CD25(+) T regulatory cells after oral exposure. The goal of comparing different models of immune tolerance is to identify which mechanisms are universal and which mechanisms are model-specific. The knowledge of such mechanisms would allow scientists and clinicians to better intervene in different immune deregulation scenarios.

Type II collagen (CII) is a cartilage structural protein that plays important roles in joint function, arthritis and ageing. In studying the ability of CII to induce eye-mediated specific immune tolerance, we have recently proven that CII is capable of inducing anterior chamber-associated immune deviation (ACAID) in Balb/c mice. Here, we study the ability of CII to induce eye-mediated immune tolerance in strains of mice that are prone to the induction of rheumatoid arthritis. Thus, we hypothesized that CII induces ACAID in DBA/1 mice and in C57BL/6 mice through the AC route (direct injection) or the intravenous route (adoptive transfer of in vitro-generated CII-specific ACAID macrophages or of CII-specific in vitro-generated T regulatory cells). Specific immune tolerance induction was assessed using both delayed-type hypersensitivity (DTH) and local adoptive transfer (LAT) assays. Results indicated the ability of CII to generate CII-specific ACAID-mediated immune tolerance in vivo and in vitro in both DBA/1 mice and C57BL/6 mice. These findings could be beneficial in studies of immune tolerance induction using CII., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Introduction of antigens into the anterior chamber (AC) of the eye generates a specific systemic form of tolerance that is termed AC-associated immune deviation (ACAID). Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human CNS demyelinating diseases, including multiple sclerosis (MS) and acute disseminated encephalomyelitis. We investigated whether the encephalitogenic antigens myelin oligodendrocyte glycoprotein (MOG35-55) or myelin basic protein (MBP) induce ACAID in the EAE-prone C57BL/6 mice. We hypothesized that injection of MOG35-55/MBP induces antigen-specific tolerance whether via the AC route, the adoptive transfer of in vitro-generated MOG35-55-specific/MBP-specific ACAID antigen presenting cells (APCs), or the adoptive transfer of MOG35-55-specific/MBP-specific ACAID T regulatory cells (Tregs). ACAID is characterized by the specific impairment of delayed-type hypersensitivity (DTH) responses. Thus, DTH assays were used to test for ACAID following the AC injection of MOG35-55/MBP, or the intravenous injection of MOG35-55-specific/MBP-specific ACAID APCs. The functional local adoptive transfer (LAT) assays were used to examine the putative regulatory functions of in vitro generated MOG35-55-specific/MBP-specific Tregs. This report is the first to demonstrate the in vivo and in vitro induction of MOG35-55-specific/MBP-specific ACAID-mediated tolerance in C57BL/6 mice. These findings highlight the need for novel immunotherapeutic strategies for MS and optic neuritis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aims: Administration of antigens into the anterior chamber (AC) of the eye induces a form of antigen-specific immune tolerance termed anterior chamber-associated immune deviation (ACAID). This immune tolerance effectively impairs host delayed-type hypersensitivity (DTH) responses. We hypothesized that ACAID could be generated in BALB/c mice following AC inoculation of the encephalitogenic antigens myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP)., Methods: We used DTH assays and local adoptive transfer (LAT) assays to test whether MOG/MBP-induced ACAID following their administration into the AC, whether they elicited this immune tolerance via CD8(+) T cells, and whether their AC coadministration (MOG/MBP) induced specific immune tolerance to one or both antigens., Results: We showed that MOG/MBP-induced AC-mediated specific immune tolerance, as evident from impaired DTH responses. This antigen-driven DTH suppression was solely mediated via splenic CD8(+) T cells as confirmed by LAT assays. Finally, a single AC injection with both antigens was sufficient to induce specific immune tolerance to these antigens, as evident from DTH and LAT assays., Conclusion: ACAID T-cell regulation could be used as a therapeutic tool in the treatment of complicated autoimmune diseases that involve multiple antigens such as multiple sclerosis., (© 2013 John Wiley & Sons Ltd.)

Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required Fas ligand-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by CD8(+) regulatory T cells. This study examined the mechanism by which these CD8(+) regulatory T cells mediate tolerance after AC injection of Ag. AC injection of Ag did not prime CD4(+) T cells and led to increased TRAIL expression by splenic CD8(+) T cells. Unlike wild-type mice, Trail(-/-) or Dr5(-/-) mice did not develop tolerance to Ag injected into the eye, even though responding lymphocytes underwent apoptosis in the AC of the eyes of these mice. CD8(+) T cells from Trail(-/-) mice that were first injected via the AC with Ag were unable to transfer tolerance to naive recipient wild-type mice, but CD8(+) T cells from AC-injected wild-type or Dr5(-/-) mice could transfer tolerance. Importantly, the transferred wild-type (Trail(+/+)) CD8(+) T cells were also able to decrease the number of infiltrating inflammatory cells into the eye; however, Trail(-/-) CD8(+) T cells were unable to limit the inflammatory cell ingress. Together, our data suggest that "helpless" CD8(+) regulatory T cells generated after AC injection of Ag enforce systemic tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.

The phrase "immune privilege" was coined by Peter Medawar to describe the absence of an immune response to allografts placed into the anterior chamber of the eye or brain. We now understand that immune privilege is more than a passive microenvironment with a distinctive anatomical structure that holds back immunity. The ocular microenvironment actively engages the immune system with immunosuppressive biochemical mechanisms. The unique characteristics of ocular immune privilege appear designed to protect the eye from damage while preserving foveal vision, thus providing the host with a definite survival advantage. However, the protection is not always sufficient and the eye becomes susceptible to uveitis. Uveitis is an intraocular inflammatory disorder that encompasses a wide range of underlying etiologies. It may be idiopathic or associated with systemic disease or infection. Understanding the biochemistry of immune privilege has the potential to identify its weaknesses that allow for immunity to break through.

The original evidence for the existence of immunologically privileged sites in the body was based on the prolonged survival of genetically disparate transplanted tissue in the anterior chamber of the eye. The failure of the immune system to elicit an immune response in this and other such sites constitutes the hallmark of the immune privilege status. The remarkably successful field of corneal transplantation in clinical practice is undoubtedly associated with corneal immune privilege. Several investigations have addressed the regulatory mechanisms governing this phenomenon, which involves a complex interplay between multiple molecular and cellular pathways. Furthermore, the use of various transgenic mouse models has facilitated the identification of critical pathways, which upon disruption can modify the immune privileged status of the eye. Understanding these pathways not only reveals the mechanisms underlying various ocular inflammatory disease conditions, but also has clinical implications for the transplantation field and for the treatment of autoimmunity.

It has been over 60 years since the phrase immune privilege was used by Sir Peter Medawar to describe the lack of an immune response against allografts placed into the ocular microenvironment. Since then, we have come to understand that the mechanisms of ocular immune privilege include unique anatomical features of a blood barrier and a lack of direct lymphatic drainage. Also, we know that the ocular microenvironment is rich with immunosuppressive molecules that influence the activity of immune cells. Moreover, the placement of foreign antigen into the ocular microenvironment can induce a systemic form of tolerance to the foreign antigen called anterior chamber-associated immune deviation (ACAID). Many soluble immunomodulators are found in aqueous humour, and are a mixture of growth factors, cytokines, neuropeptides, and soluble receptors. This is a continuously growing list. The mechanisms of ocular immune privilege induce apoptosis, promote the production of anti-inflammatory cytokines, and mediate the activation of antigen-specific regulatory immunity. These mechanisms of immune privilege also attempt to impose themselves upon immunity within the uveitic eye. The adaptation of several anatomical and biochemical mechanisms to establish an immune privileged microenvironment within the eye makes the eye immunologically unique. It is a tissue site where we may learn how immunity is regulated in inflammation and at rest. Success in translating the lessons of ocular immune privilege to other tissues has the potential to drastically change the therapy and clinical outcomes of autoimmune diseases and allograft survival.

Background CD8+ regulatory T cells (Treg) have been considered to be involved in a model of ocular-induced tolerance, known as anterior chamber-associated immune deviation (ACAID). The mechanisms of suppression by CD8+ T cells in ACAID remain only poorly understood. TGF-beta1 is considered as an inhibitory cytokine for immunosuppression in some models. The production of TGF-beta1 by CD8+ T cells in ACAID, and whether CD8+ T cells exert suppression through TGF-beta1, is unknown. Methods The suppressive effect of CD8+ T cells in ACAID mice was determined by a local adoptive transfer (LAT) assay. The production of TGF-beta1 by CD8+ T cells was measured by enzyme-linked immunosorbent assay (ELISA). Anti-TGF-beta1 antibodies were used in the LAT assay to test if they could block the inhibitory effect of CD8+ T cells. Results CD8+ T cells from ACAID mice were shown to block the delayed-type hypersensitivity (DTH) response in an antigen-specific manner in a LAT assay. These CD8+ T cells secreted TGF-beta1, and their suppression could partially be blocked by anti-TGF-beta1 antibodies. Conclusions Our study confirms that CD8+ T cells from ACAID mice possess inhibitory properties. This population exerts part of its suppressive function via the production of TGF-beta1.

Background: CD8+ regulatory T cells (Treg) have been considered to be involved in a model of ocular-induced tolerance, known as anterior chamber-associated immune deviation (ACAID). The phenotype and characteristics of CD8+Treg in ACAID remain only poorly understood. Recent studies have reported that the CD94-Qa-1 system is implicated in the induction of ACAID CD8+Treg, but the functions and characteristics of CD8+CD94+T cells remain unclear., Results: Both mRNA and protein of CD94 and NKG2A were markedly up-regulated on splenic CD8+T cells of ACAID mice compared with controls. Flow cytometric analysis showed that very few CD8+CD94+T cells express granzyme B, perforin and Foxp3. CD8+CD94+T cells, but not CD8+CD94-T cells, magnetically isolated from the spleens of ACAID mice, produced large amounts of TGF-beta1 and exhibited suppressive activity in vitro. Neutralization of TGF-beta1 caused reversal of suppression mediated by CD8+CD94+T cells., Conclusion: CD8+CD94+T cells from ACAID mice exhibited suppressive activity in association with enhanced expression of TGF-beta1, suggesting that CD8+Treg are mainly distributed in CD94+T cell subpopulations.

Purpose: Anterior chamber-associated immune deviation (ACAID) is a form of peripheral tolerance achieved via intracameral antigen inoculation. It is well known that ACAID effectively down-regulates potentially destructive immunities such as delayed-type hypersensitivity (DTH) at extraorbital sites. However, what has not been specifically addressed is whether local intraocular tissues are negatively affected from intracamerally placed antigen. Thus, the current study was undertaken to detect and characterize potential pathological effects on intraocular tissues following intracameral inoculation with alloantigen., Methods: ACAID induced in C57BL/6 hosts via intracameral inoculation with allogeneic (BALB/c) splenocytes was confirmed by the absence of DTH reactivity in the periphery. Injuries to the anterior segment and neuroretina following intracameral inoculation were evaluated pathologically via histological evaluation, molecularly via upregulation of glial fibrillary acidic protein (GFAP), and functionally via assessment of photoreceptor degeneration and electroretinogram (ERG) out to 24 days. In all experiments, intracamerally inoculated mice were compared to sham-operated, and controlled lens-punctured mice--a procedure that elicits intracameral inflammation for positive identification of immunopathological changes., Results: Inflammation of anterior segment tissues persisted out to eight days, despite evidence that significant clearance of allogeneic cells took place within 6 h. In the neuroretina, a transient loss in ERG B-wave amplitudes was detected, but photoreceptor degeneration and GFAP upregulation were not., Conclusions: Intracameral inoculation with alloantigen leads to anterior segment inflammation and ERG dysfunction; however, this was markedly reduced and transient when compared to strong anterior segment inflammation induced by a more serious lens-puncture wound.

The eye possesses a critical method of self-preservation in response to intraocular antigen presentation. Instead of conventional immunity by means of delayed-type hypersensitivity (DTH), the eye participates in a systemic immune response involving the thymus and spleen, ultimately leading to suppression of cell-mediated (T helper 1) immunity. The immune response begins with intraocular capture of antigen by specialized ocular antigen-presenting cells (APCs). These activated APCs then migrate preferentially to the marginal zone of the spleen, where they become part of an intricate and highly specific cluster of immune cells. The end result is the emergence of a population of antigen-specific T-regulatory lymphocytes that return to the eye and suppress DTH.

The present study determined whether topical latanoprost, a prostaglandin (PG) F(2alpha) analog, influences the induction of anterior chamber-associated immune deviation (ACAID), corneal neovascularization (NV) or survival of corneal allografts in mice. BALB/c mice received topical latanoprost or PGE(2) once or multiple times daily starting 4 weeks prior to or the day of anterior chamber injection of C57BL/6 splenocytes. Induction of allo-specific ACAID was assessed by ear challenge with C57BL/6 splenocytes 1 week after subcutaneous immunization. In a separate experiment, orthotopic corneal transplantation was performed using C57BL/6 mice as donors and BALB/c mice as recipients. Recipients were randomized in a masked fashion to receive topical latanoprost or PGE(2). Graft fate was assessed clinically under surgical microscopy. Presence of MHC class II(+) CD11c(+) or CD11b(+) cells in normal BALB/c mouse eyes following latanoprost or PGE(2) administration was assessed immunohistochemically. Control mice received topical 20% dimethyl sulfoxide or no treatment. Allo-specific ACAID was induced after 2 or 6 weeks of once daily treatment with latanoprost, and was induced even after 6 weeks of multiple treatments with latanoprost. Conversely, mice receiving PGE(2) failed to develop ACAID. Opacity and corneal NV scores for allografts treated with latanoprost were statistically indistinguishable from those for control allografts (p>0.05), whereas all allografts treated with PGE(2) were rejected. Opacity and NV scores were significantly higher in these allografts than in controls (p<0.05). A number of MHC class II(+) CD11c(+) cells were present in the central cornea after PGE(2) treatment. Topical application of latanoprost does not influence induction of ACAID or graft outcomes including opacity and NV, whereas PGE(2) does. Immune privilege of corneal allograft is maintained after topical latanoprost application in mice.

Purpose: Anterior chamber associated immune deviation (ACAID) is an antigen-specific form of peripheral immune tolerance that is induced to exogenous antigens placed in the ocular anterior chamber, which leads to a suppression in delayed-type hypersensitivity (DTH). Considerable work has been done on ACAID induction to major histocompatibility (MHC) alloantigens. However, its role on cytotoxic T lymphocyte (CTL) activity is currently unknown., Methods: C57BL/6 (H-2(b)) mice received an intracameral (IC) inoculation with BALB/c (H-2(d)) splenocytes. Splenic CD4(+) and CD8(+) T cell populations were characterized by flow cytometry and proliferation assays during induction and expression phases of ACAID. Percentages of CD4(+)CD25(+)FoxP3(+) T regulatory cells (Treg) were also followed. Lastly, CTL function was measured at various time points during ACAID expression, and Treg were added to identify potential alterations in CTL function., Results: CD4(+) and CD8(+) T cell percentages and proliferation increased in the spleen during ACAID induction but then sharply decreased in response to an allospecific immunization. Expression of ACAID also exhibited a significant drop in CTL function. However, while Treg expansion was observed, these cells did not directly mediate the CTL inhibition., Conclusions: ACAID mediates an inhibition of CTL function against MHC alloantigens. Furthermore, we found that ACAID induction leads to the expansion and proliferation of CD4(+) and CD8(+) T cells while ACAID expression is associated with a diminishment in T cell percentages due to proliferation impairment. Lastly, Treg also expand during ACAID induction. However, our data suggest that Treg do not directly inhibit CTL activity.

In a model of peripheral tolerance called anterior chamber-associated immune deviation (ACAID), the differentiation of the T regulatory cells depends on NKT cells and occurs in the spleen. In this study, we show that NKT cells that express the invariant (i) TCR and are the CD1d-reactive NKT cells (required for development of peripheral tolerance) actually produced urokinase-type plasminogen activator (uPA) during tolerance induction. The RT-PCR and in vitro plasmin assay showed that splenic iNKT cells derived uPA-converted plasminogen to plasmin. Moreover, uPA was required for tolerance induction because uPA knockout (KO) mice did not develop peripheral tolerance or develop CD8(+) T regulatory cells after Ag inoculation into the anterior chamber. In contrast, other aspects of ACAID-induced tolerance, including recruitment of iNKT cells to the spleen and production of IL-10 by iNKT cells, were unchanged in uPA-deficient mice. The adoptive transfer of splenic NKT cells from wild-type mice restored ACAID in Jalpha18 KO mice (iNKT cell deficient), but NKT cells from uPA KO mice did not. We postulate that the mechanism of action of uPA is through its binding to the uPAR receptor, and enzymatic cleavage of plasminogen to plasmin, which in turn activates latent TGFbeta. In conclusion, uPA derived from iNKT cells is required to induce peripheral tolerance via the eye.

Purpose: To examine the expression of Foxp3 on CD8+ T cells in the spleen during anterior chamber-associated immune deviation (ACAID)., Methods: Ovalbumin (OVA) was injected into the anterior chamber (AC) of C57BL/6 mice, and the delayed-type hypersensitivity (DTH) response was measured to evaluate the development of ACAID. The suppressive effect of CD8+ T cells in ACAID mice was determined by a local adoptive transfer (LAT) assay. Flow cytometry was used to assay the frequency of CD8+ Foxp3+ T cells from normal mice, ACAID mice, and control mice receiving an AC injection of PBS (PBS-AC-injected mice). These frequencies were also tested after polyclonal or specific antigen stimulation. The mRNA level of Foxp3 in CD8+ splenocytes from different groups with or without stimulation were determined by reverse transcription-polymerase chain reaction., Results: OVA injection into the AC induced ACAID, and CD8+ T cells from ACAID mice inhibited the ear-swelling response by OVA-primed responder cells in LAT assay. Flow cytometry analysis showed that the frequency of CD8+ Foxp3+ cells in splenocytes was upregulated in ACAID mice following polyclonal or specific antigen stimulation. Foxp3 mRNA was only detected in CD8+ T cells from ACAID mice after polyclonal stimulation., Conclusions: An upregulated Foxp3 expression in CD8+ T cells is associated with the development of ACAID, suggesting an involvement of CD8+ Foxp3+ T cells in this model of immune tolerance.

CD8(+) regulatory (suppressor) T cells are induced by complex cellular pathways in the spleens of mice that have received an injection of antigen into the anterior chamber (AC) of an eye, an immune-privileged site. Although these CD8(+) regulatory T cells perform an antigen-specific regulatory function for an immune response to self and non-self antigens, the mechanisms of the activation or function of these regulatory cells are not clear. Here, we describe a novel mechanism for the activation of splenic CD8(+) regulatory T cells induced by injection of antigen into the AC. Immunization of mice with trinitrophenyl and bovine serum albumin (TNP-BSA) amplified AC-induced splenic CD8(+) regulatory T cells that suppressed the initiation of contact sensitivity when transferred to immunized, challenged mice. These CD8(+) regulatory T cells were produced independently of perforin, indicating that they are not canonical cytotoxic T cells. Fas ligand (FasL)-deficient CD8(+) regulatory T-cell function was rescued by inclusion of exogenous interferon-gamma (IFN-gamma), demonstrating that the expression of FasL by CD8(+) regulatory T cells was dispensable, but IFN-gamma was not. Ultimately, we demonstrated that the generation of these CD8(+) regulatory T cells occurred independently of IFN-gamma, but their suppressor function required IFN-gamma receptor stimulation.

The immune-privileged status of the anterior chamber of the eye is altered in experimentally induced intraocular inflammation and in the pigment dispersion syndrome of DBA/2J mice. However, the eye has developed multiple mechanisms to maintain ocular immune privilege even in the presence of intraocular inflammation.

Evidence of ocular immune privilege was noted almost 130 years ago. The past 30 years have witnessed an explosion in research on ocular immune privilege. One of the primary mechanisms that contribute to ocular immune privilege is the unique form of immune deviation that is invoked when antigens are introduced into the anterior chamber (AC) of the eye - a phenomenon termed AC-associated immune deviation (ACAID). ACAID embodies a constellation of cellular interactions and at least four different organ systems: eye, thymus, spleen, and sympathetic nervous system. At least four different cell populations interact to generate CD8+ T regulatory cells that suppress both Th1- and Th2-mediated inflammation. The interactions that occur between F4/80+ antigen-presenting cells, CD4+ T regulatory cells, NK1.1+ T cells, gammadelta T cells, B cells, and CD8+ T cells remain to be fully elucidated. Ocular immune privilege was originally perceived as a simple anatomic anomaly that has evolved to be one of the most sophisticated and intriguing forms of immune regulation.

Anterior chamber (AC)-associated immune deviation (ACAID) is a form of ocularderived peripheral tolerance that helps to maintain the immune privilege of the eye by suppressing both the priming and elicitation of adaptive immune responses. ACAID is known to facilitate the survival of corneal grafts and suppression autoimmune uveitis in the eye. Intravenous inoculation of in vitro generated ACAID tolerance-inducing antigen presenting cells (APCs) treated with transforming growth factor-Beta2 (tolerogenic APCs) generates the kind of T regulatory cells found in in vivo ACAID when antigen is inoculated into the AC of the eye. Here, we review the application of peripheral tolerance induction by ACAID with either AC inoculation or in vitro generated tolerogenic ACAID-APCs in suppressing ongoing Th1- and Th2-mediated immune pathogenesis in naive and presensitized hosts. Transfer of tolerogenic APCs has suppressed antigen-specific immune inflammation in animal models of experimental autoimmune encephalomyelitis, hapten immune pulmonary interstitial fibrosis, and ovalbumin-induced allergic pulmonary inflammation. The possibility of immune therapy by in vitro generated ACAID-like tolerogenic APCs in humans is discussed.

The visual axis of the eye focuses light images precisely on the retina. The retina is intolerant of distortion that might be induced by innate or immune inflammation. In addition, the corneal endothelium and the neurosensory retina are unable to regenerate if injured by trauma or inflammation. Within the environment of this visual organ a phenomenon called ocular immune privilege provides the eye with the necessary immune protection against infectious agents by allowing the expression of the least deleterious immune effector mechanisms. Moreover, the mechanisms of immune privilege are multiple, overlapping, and include both active and passive suppression of innate and immune inflammation. At the very basis of an effective immune response are cellular interactions and their cross talk. Central to the ability of cells to communicate are the intercellular channels that are established to isolate signals and movement of proteins between cells. Within this secure nano-environment, cells signal each other and even exchange proteins. Studies reviewed here are centered on knowledge and exploration of the tolerogenic synapse rather than the immunogenic synapse. The unique cells (invariant natural killer T cells, F4/80+ antigen-presenting cells, and T and B lymphocytes) that cluster within the marginal zone following injection of antigen in the anterior chamber (AC) express a phenotype of cell surface molecules that that seem to be uniquely critical for the development of AC-associated immune deviation. How these cell surface molecules behave during the cellular interactions that result in the development of regulatory T cells and peripheral tolerance induced through the eye is discussed.

Antigens injected into the anterior chamber (AC) of the eye induce a form of peripheral immune tolerance termed anterior chamber-associated immune deviation (ACAID). ACAID is initiated by F4/80+ ocular antigen-presenting cells (APC) which capture ocular antigens and migrate to the spleen where they transfer antigenic peptides to B cells, which act as ancillary APC for the induction of T-regulatory cells (T(reg)) that inhibit delayed-type hypersensitivity (DTH) responses. Here we show that ocular-like APC induce the expansion of tolerogenic splenic B cells. Furthermore, we show that inhibiting B-cell proliferation with either mitomycin-c or gamma-irradiation abolishes the ability of B cells to induce T(reg). To our knowledge, this is the first study to report that B-cell proliferation is needed for B-cell-induced T-cell tolerance., (Copyright 2007 S. Karger AG, Basel.)

Anterior chamber-associated immune deviation (ACAID) is a form of peripheral tolerance that is induced by introducing Ags into the anterior chamber (AC) of the eye, and is maintained by Ag-specific regulatory T cells (Tregs). ACAID regulates harmful immune responses that can lead to irreparable injury to innocent bystander cells that are incapable of regeneration. This form of immune privilege in the eye is mediated through Tregs and is a product of complex cellular interactions. These involve F4/80+ ocular APCs, B cells, NKT cells, CD4+CD25+ Tregs, and CD8+ Tregs. gammadelta T cells are crucial for the generation of ACAID and for corneal allograft survival. However, the functions of gammadelta T cells in ACAID are unknown. Several hypotheses were proposed for determining the functions of gammadelta T cells in ACAID. The results indicate that gammadelta T cells do not cause direct suppression of delayed-type hypersensitivity nor do they act as tolerogenic APCs. In contrast, gammadelta T cells were shown to secrete IL-10 and facilitate the generation of ACAID Tregs. Moreover, the contribution of gammadelta T cells ACAID generation could be replaced by adding exogenous recombinant mouse IL-10 to ACAID spleen cell cultures lacking gammadelta T cells.

Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the tryptophan catabolism, has been shown to play an important role in various forms of immune tolerance. Since anterior chamber associated immune deviation (ACAID) is a systemic immune tolerance elicited by introducing exogenous antigens into the anterior chamber of the eye, we investigated the expression and function of IDO in the development of this ocular tolerance. ACAID was induced in BALB/c mice by an intracameral injection of 50mug ovalbumin (OVA). The IDO expression in the splenocytes during ACAID was determined by fluorescent quantitative real-time PCR, Western blot analysis and immunohistochemistry. The development of ACAID was evaluated by the delayed-type hypersensitivity (DTH) response after intraperitoneal injection of an IDO inhibitor 1-methyl-dl-tryptophan (1-MT). Secretion of IFN-gamma and IL-4 by splenocytes and lymph node cells from the mice treated with or without 1-MT were also evaluated using intracellular cytokine staining. Our results showed that the IDO expression was significantly increased at both mRNA and protein levels following OVA intracameral injection. Inhibition of IDO with 1-MT prevented the development of ACAID, which was indicated by the re-appearance of the OVA-specific DTH response. IL-4 was significantly reduced and IFN-gamma was partially recovered after the treatment of 1-MT. Our study reveal that IDO is up-regulated during ACAID and IDO inhibitor prevents ACAID generation, suggesting that IDO is involved in the development of this immune tolerance.

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.

The introduction of antigen into the anterior chamber of an eye induces the antigen-specific suppression of cell-mediated immunity and the antigen-induced production of immunoglobulin G2 antibodies. To define further the role of iris monocytic cells in the systemic suppression of cell-mediated immunity that follows the entry of foreign antigen into the anterior chamber, murine iris wholemounts or cell suspensions of iris cells were stained with fluorescent anti-F4/80 and/or anti-CD11c, anti-CD11b antibodies and examined by confocal microscopy or flow cytometry, respectively. Monocytic cells in iris cell suspensions were recovered from mice receiving an injection of trinitrophenylated bovine serum albumin (TNP-BSA) into an anterior chamber and Percoll-enriched iris cells separated into cells expressing F4/80 or CD11c were injected intravenously into TNP-BSA-immunized or naive recipients. The recipients were challenged to induce delayed-type hypersensitivity (DTH) or were provided with splenocytes or thymocytes that transfer the suppression of DTH. The homing of monocytic bone marrow cells to the iris was determined by the intravenous injection of bone marrow cells from green fluorescent protein (GFP)-transgenic donors into C57 mice, and the staining of recipient iris wholemounts with anti-F4/80 antibodies. Iris cells with a dendritic morphology expressing both F4/80 and/or CD11c and CD11b, some cells expressing only F4/80 or CD11c, were detected. The irides of irradiated GFP- mice that received intravenous GFP+ bone marrow cells contained GFP+ F4/80+ cells. F4/80+ and CD11c+ cells from the irides of donors that received intracameral TNP-BSA transferred the suppression of DTH when injected intravenously into TNP-BSA-immunized recipients, activated immunoregulatory thymocytes and activated antigen-specific splenic regulatory effector cells. These results support the hypothesis that iris monocytic cells may participate in the systemic induction of regulatory T cells.

Purpose: Regulatory CD4+ T cells (T regs) arise in the spleens of mice with anterior chamber-associated immune deviation (ACAID), an eye-derived tolerance evoked by injection of antigen into the ocular anterior chamber (AC). The current study was conducted to investigate the possibility that these T regs express CD25 and are derived from natural CD4+CD25+ T cells., Methods: Naïve T cells from DO11.10 mice were activated in vitro by ovalbumin (OVA)-pulsed, TGFbeta-treated antigen-presenting cells (APCs), and the expression of CD25 assayed by flow cytometry. OVA-specific ACAID T regs were obtained from the spleens of DO11.10 mice with ACAID to OVA. Immunomagnetic enrichment was used to sort out CD4+CD25+, and CD4+CD25- ACAID T cells before they were injected into OVA-immunized mice or examined for mRNA expression of the regulatory T-cell transcription factor Foxp3. In addition, before AC injection of OVA, systemic depletion of CD25+ T cells was performed with injections of anti-IL-2 receptor antibody into the mice., Results: OVA-specific T cells from DO11.10 mice expressed CD25 when exposed to OVA-pulsed, TGFbeta-treated APCs, even when the DO11.10 T cells were depleted of CD25+ cells before their in vitro stimulation. In addition, DH was suppressed in naïve mice that were injected with CD4+CD25+ or CD4+CD25- ACAID T cells. The CD4+CD25+, but not the CD4+CD25-, ACAID T regs expressed Foxp3. Finally, OVA induced ACAID in mice depleted of CD25+ cells., Conclusions: Some of the CD4+ T regs of ACAID arise from CD25- precursors, and the induction of ACAID is not dependent on the presence of natural CD4+CD25+ T regs.

Purpose: To characterize anterior chamber immune deviation (ACAID) in 129-strain and mixed 129-strain mice., Methods: ACAID was assayed using standard protocols with herpes simplex-1 (HSV-1) and trinitrophenol-hapten-spleen cells (TNP-spleen) in C57B1/6, 129P2, 129X1, and intercrossed strains. Systemic tolerance induction was assayed using an ultraviolet light skin tolerance protocol to 2,-4,6-trinitro-l-chlorobenzene (TNCB)., Results: 129X1 and C57Bl/6xl29Xl Fl mice did not show ACAID to HSV-1. C57Bl/6xl29P2 mice did not show ACAID to TNP-spleen. C57Bl/6xl29P2 mice did show normal peripheral immune deviation to TNCB. (C57Bl/6xl29Xl) x C57B1/6 N2 backcrossed mice showed a bimodal ACAID response to HSV-1 suggesting a single dominant allele in the 129X1 background responsible for suppressing ACAID., Conclusion: ACAID to multiple antigens is significantly reduced in 129-strain mice and their outcrossed progeny. Since 129-strain embryonic stem cells are widely used to generate knockout and transgenic mice, care must be taken to extensively backcross resultant strains in order to assess the effect of particular genes on ACAID.

Ags expressed at immune privileged sites and other peripheral tissues are able to induce T cell tolerance. In this study, we analyzed whether tolerance toward an intraocular tumor expressing a highly immunogenic CTL epitope is maintained, broken, or reverted into immunity in the event the anatomical integrity of the eye is lost. Inoculation of tumor cells into the anterior chamber of the eye of naive B6 mice leads to progressive intraocular tumor growth, an abortive form of CTL activation in the tumor-draining submandibular lymph node, and systemic tolerance as evidenced by the inability of these mice to reject an otherwise benign tumor cell inoculum. Loss of anatomical integrity of the eye as a consequence of phthisis resulted in loss of systemic tolerance and the emergence of effective antitumor immunity against an otherwise lethal tumor challenge. Phthisis was accompanied by dendritic cell maturation and preceded the induction of systemic tumor-specific CTL immunity. Our data show that normal tissue homeostasis and anatomical integrity is required for the maintenance of ocular tolerance and prevention of CTL-mediated immunity. These data also indicate that tissue injury in the absence of viral or microbial infection can act as a switch for the induction of CTL immunity.

The peripheral tolerance that is elicited by the anterior chamber-associated immune deviation (ACAID) protocol is characterized by impairment of Th1 responses such as delayed-type hypersensitivity. It has been proposed that suppression of Th1 responses is mediated by a deviation toward Th2 responses. Because NKT cells have a prominent role in ACAID and NKT cell-derived IL-13 is required in a tumor model of tolerance, we postulated that NKT cell-derived Th2 cytokines might have a role in ACAID. However, contrary to the tumor model, in this study we show that NKT cells from IL-13-deficient mice or IL-4/IL-13 double deficient mice were able to reconstitute the capability of J alpha18-deficient mice (lacking invariant NKT) to develop peripheral tolerance postintracameral inoculation of Ag. Also, we were able to induce peripheral tolerance directly in IL-13-deficient, IL-4/IL-13-double deficient, and STAT6-deficient mice by inoculation of Ag into their eye. We conclude that neither IL-4 nor IL-13 cytokines are required for the generation of efferent CD8+ T regulatory cells during eye-induced peripheral tolerance. We propose that Ags inoculated into the anterior chamber of the eye induce the immunoresponse to deviate from producing immune T effector cells to producing efferent T regulatory cells, rather than deviating from Th1- to Th2-type effector cells.

The eye is an immune-privileged site that uses specialized mechanisms to protect itself from damage and to preserve its visual acuity. Among the mechanisms that contribute to the eye's privileged existence is its ability to induce both local and peripheral tolerance to antigens that may transgress its chambers. Experimentally, antigens that are inoculated into the anterior chamber induce an associated immune deviation of immune lymphocytes from their potential T-helper inflammatory responses to T regulatory cells. The prominent role of a somewhat rare cell called invariant (i)NKT cell in the process of tolerance induced through the anterior chamber-associated immune deviation model, ACAID, has revealed novel biological characteristics for the iNKT cell, as well as novel mechanisms for the induction of tolerance that have not been previously known or considered. The role of the iNKT cell in ACAID and its novel story is discussed in this mini-review.

Purpose: We tested whether antigen administration via the anterior chamber (a.c.) was equivalent to intravenous (i.v.) or mucosal administration antigen., Methods: Ovalbumin (OVA)-specific CD8(+) T cells (OT-I) were enumerated in lymphoid tissues of C57Bl/6 (B6) mice via adoptive transfer after the same amount of antigen was administered via a.c., i.v., or mucosal routes. Lytic activity was measured in B6 and gammadeltaT cell-deficient B6 mice given OVA via a.c., i.v, or mucosal routes after injection with OVA in adjuvant., Results: OVA a.c. induced a pattern of T-cell proliferation distinct from i.v. or mucosal administration. A.c. and i.v., but not mucosal, OVA induced cytolytic T lymphocyte (CTL) tolerance. The inhibition of CTL responses was significantly greater in mice given OVA a.c. rather than i.v. gammadeltaT cells contributed to a.c.-, but not i.v.-, induced CTL tolerance., Conclusions: A.c. administration of antigen not de-facto i.v. or mucosal administration of antigen.

The introduction of antigens into the anterior chamber (AC) of the eye, an immune-privileged site, induces immune responses that effectively eliminate ocular pathogens while minimizing tissue damage that can cause blindness. This specialized immune response, termed AC associated immune deviation (ACAID) is thought to be an evolutionary compromise to preserve the delicate microanatomy of the eye while maintaining ocular immune responses. The injection of soluble antigen in the AC of mice results in systemic tolerance characterized by reduced priming for antigen-specific delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses. Similarly, the injection of histo incompatible tumors into the AC of mice reduces priming for DTH responses specific to minor antigens. However, robust tumor-specific CTL responses are induced systemically following this treatment that are capable of eliminating a subsequent injection of the same tumors in the skin or the opposite eye. Interestingly, CTL responses induced by administration of tumors in the AC fail to eliminate the primary ocular tumor. In this review, we compare and contrast CTL responses generated by the injection of soluble or tumor-associated antigens in the AC and discuss mechanisms employed to induce ocular CTL tolerance.

Pigment dispersion syndrome causes iris pigment release and often progresses to elevated intraocular pressure and pigmentary glaucoma (PG). Because melanin pigment can have adjuvant like properties and because the Gpnmb gene, which contributes to pigment dispersion in DBA/2J (D2) mice, is expressed in dendritic cells, we tested the hypothesis that ocular immune abnormalities participate in PG phenotypes. Strikingly, we show that D2 eyes exhibit defects of the normally immunosuppressive ocular microenvironment including inability of aqueous humor to inhibit T cell activation, failure to support anterior chamber (AC)-associated immune deviation, and loss of ocular immune privilege. Histologic analysis demonstrates infiltration of inflammatory leukocytes into the AC and their accumulation within the iris, whereas clinical indications of inflammation are typically very mild to undetectable. Importantly, some of these abnormalities precede clinical indications of pigment dispersal, suggesting an early role in disease etiology. Using bone marrow chimeras, we show that lymphohematopoietic cell lineages largely dictate the progression of pigment dispersion, the ability of the eye to support induction of AC-associated immune deviation, and the integrity of the blood/ocular barrier. These results suggest previously unsuspected roles for bone marrow-derived cells and ocular immune privilege in the pathogenesis of PG.

Unlabelled: Corneal graft rejection can be prevented by local macrophage depletion, via subconjunctival injections with clodronate liposomes. To unravel the underlying immunological mechanism responsible for prolonged graft survival in this circumstance, the effect of this regimen on induction of donor-specific delayed type hypersensitivity (DTH) and anterior chamber associated immune deviation (ACAID) was determined. The study showed that although subconjunctival clodronate liposome-treatment failed to alter systemically induced DTH and ACAID, both types of immune response were absent in clodronate liposome-treated rats after corneal transplantation. Thus, elimination of macrophages from the corneal transplant site renders corneal allografts immunologically "invisible" to the recipient., Purpose: To determine whether local macrophage depletion, via administration of clodronate liposomes, alters delayed type hypersensitivity (DTH) responses and induction of anterior chamber associated immune deviation (ACAID) after corneal allotransplantation., Methods: Clodronate liposome-treated and untreated rats received orthotopic corneal allografts and were tested for DTH responses and induction of ACAID towards donor antigens. Also in subconjunctivally treated and untreated rats, DTH responses were measured after subcutaneous immunization or induction of ACAID with allogeneic spleen cells., Results: Subconjunctival injected clodronate liposomes prevented grafted rats from developing donor-specific DTH as well as ACAID. By contrast, subconjunctivally injected clodronate liposomes had no effect on donor-specific DTH responses after systemic immunization or on the induction of ACAID with allogeneic cells., Conclusions: Depletion of macrophages at the time of corneal allografting seems to render the grafts immunologically invisible to the recipients. This could explain why these grafts survive "indefinitely" without any other form of therapy.

Purpose: Delivery of antigen to the anterior chamber (AC) of the eye induces a systemic form of tolerance, referred to as anterior chamber-associated immune deviation (ACAID). ACAID is characterized by decreases in delayed-type hypersensitivity responses and complement-fixing antibodies on subsequent challenge with an immunogenic form of the antigen. The current study was designed to test whether priming of antigen-specific CD8(+) cytotoxic T-lymphocytes (CTLs) are inhibited by injection of soluble antigen into the AC and whether gammadelta T cells play a role in the inhibition of such responses., Methods: Antigen was administered through the AC to normal gammadelta T-cell-deficient or reconstituted gammadelta T-cell-deficient mice. Seven days after the AC injection, the mice were primed with antigen in adjuvant and 10 days later, their spleen cells were cultured for 5 to 7 days and the CTL responses measured., Results: CTL responses were inhibited by antigen delivered through the AC in normal but not gammadelta T-cell-deficient mice. Tolerance was reconstituted in delta-chain knockout mice by the adoptive transfer of gammadelta T cells from normal mice. Moreover, spleen cells and splenic gammadelta(+) T cells, but not gammadelta(-) T cells, from mice injected with antigen through the AC inhibited development of CTL responses when cultured together with primed effector T cells., Conclusions: These data show, for the first time, that administration of soluble antigen in the AC inhibits development of CD8(+) cytotoxic T-cell responses and that gammadelta T cells play a critical role in inhibition of CTL responses.

Anterior chamber-associated immune deviation (ACAID), a manifestation of ocular immune privilege, prevents Th1-dependent delayed hypersensitivity from developing in response to eye-derived Ags, thereby preserving vision. Since Th2-type cells have recently been shown to mediate destructive inflammation of the cornea, we wondered whether pre-emptive induction of ACAID could inhibit Th2 responses. Using a murine model of OVA -specific, Th2-dependent pulmonary inflammation, we pretreated susceptible mice by injecting OVA alone into the anterior chamber, or by injecting OVA-pulsed, TGF-beta2-treated peritoneal exudate cells i.v. These mice were then immunized with OVA plus alum strategy that generates Th2-mediated OVA-specific pulmonary pathology. When pretreated mice were challenged intratracheally with OVA, their bronchoalveolar lavage fluids contained far fewer eosinophils and significantly less IL-4, IL-5, and IL-13 compared with that of positive, nonpretreated controls. Similarly, lung-draining lymph node cells of pretreated mice secreted significantly less IL-4, IL-5, and IL-13 when challenged in vitro with OVA. Moreover, sera from pretreated mice contained much lower titers of OVA-specific IgE Abs. We conclude that Ags injected into the anterior chamber of the eye impair both Th1 and Th2 responses. These results reduce the likelihood that ACAID regulates Th1 responses via a Th2-like mechanism. Thus, immune privilege of the eye regulates inflammation secondary to both Th1- and Th2-type immune responses.

The induction of peripheral tolerance via immune privileged sites such as the eye requires splenic colocalization of NKT cells and CD1d(+) tolerogenic F4/80(+) APCs, both of which are needed for the generation of CD8(+)-regulatory T (Tr) cells. Whereas tolerogenic APCs secrete the chemokine macrophage-inflammatory protein-2 for the purpose of recruiting NKT cells, the signals responsible for recruiting potential Tr cells and additional APCs to the spleen are not known. Here we examined the ability of CD1d-stimulated NKT cells to produce chemokines that can recruit other cells needed for tolerance. Our results show that NKT cells stimulated by either CD1d-transfected fibroblasts in vitro or CD1d(+) tolerogenic APCs both in vivo and ex vivo produced RANTES in a CD1d-dependent manner. The requirement for RANTES in tolerance was demonstrated by studies in which RANTES blockade in vivo prevented not only APC accumulation in the spleen but also the generation of CD8(+) Tr cells that suppress Th1 immunity. Thus, CD1d-restricted NKT cells provide critical signals for orchestrating the accumulation of cells needed for tolerance induction. These data expand our current knowledge of RANTES beyond its role in Th1 immune responses to show its importance in tolerance induction and add a novel aspect to our understanding of the role of NKT cells in tolerance. Understanding the precise mechanisms involved in tolerance induction may lead to more effective therapeutic strategies for autoimmunity and graft rejection.

A form of systemic tolerance is created when antigenic material is placed in the anterior chamber of the eye, an immune privileged site. Termed anterior chamber associated immune deviation (ACAID), this form of tolerance insures that the systemic immune response to eye-derived antigens is devoid of T cells that mediate delayed hypersensitivity and antibodies that fix complement. As a consequence, the eye is spared the disruptive blinding influence of immunogenic inflammation. ACAID arises when antigen is captured by intraocular antigen presenting cells, then carried to the marginal zone of the spleen where, in the presence of natural killer T cells and marginal zone B cells, a microenvironment is created that activates antigen-specific T cells to differentiate into regulatory cells that interfere with the induction of delayed hypersensitivity as well as its expression. Transforming growth factor beta-2 (TGFbeta-2), constitutively present in the aqueous humor of the eye, imposes distinctive properties on antigen presenting cells. Thrombospondin is an early activated gene following TGFbeta-2 treatment and as a consequence the eye-derived antigen presenting cells fail to secrete IL-12 or express CD40. The lack of these potent stimulators of Th1 cell differentiation is central to the induction of ACAID.

Immune privilege was first explored in the late 1800s by van Dooremaal, and was then extended by Medawar in the mid 1900s to fit in with emerging concepts of transplantation immunology. Modern concepts and understanding of immune privilege come from subsequent studies produced by Medawar, Billingham, and Streilein. The exploitation of the model of anterior chamber immune deviation (ACAID) in mice has allowed us to look at both cellular and molecular mechanisms involved in the prevention of potentially damaging immune responses in such privileged sites. This review gives a historical perspective of the immune privilege research and provides up-to-date information of molecules, cells, and concepts newly recognized as contributing to tolerance induction induced in such specialized areas of the body. Evidence is given to support the idea that application of such information may lead to potential for therapeutic applications of ACAID mechanisms in prevention of progression of immune-inflammatory diseases in humans.

Since the 19th century, it has been recognized that the anterior chamber of the eye permits the prolonged, and sometimes permanent, survival of foreign tissue and tumor grafts. It was initially believed that the absence of patent lymphatics draining the interior of the eye prevented antigens from reaching regional lymphoid tissues. However, sequestration of intraocular antigens alone cannot account for ocular immune privilege, and a clear picture is now emerging that a constellation of anatomical, physiological, and dynamic immunoregulatory factors contribute to ocular immune privilege. Ocular fluids contain a potpourri of immunosuppressive and immunoregulatory factors that suppress T-cell proliferation and secretion of proinflammatory cytokines. The interior of the eye is decorated with Fas ligand (CD95L), which induces apoptosis of infiltrating inflammatory cells. Antigens introduced into the eye induce a unique immune deviation in which TH1 responses, namely delayed-type hypersensitivity (DTH), are actively suppressed. Thus, ocular immune privilege is sustained by factors that suppress immune cell proliferation and purge immune cells that enter the eye and by a dynamic immunoregulatory process that suppresses antigen-specific DTH. Suppression of certain inflammatory responses is an important adaptation for preventing immune-mediated injury to ocular tissues that have little or no capacity to regenerate. Thus, immune privilege is a crucial adaptation for preserving vision.

Anterior chamber-associated immune deviation (ACAID) is a systemic form of tolerance that is elicited by introducing antigens into the anterior chamber of the eye. ACAID is characterized by deficiencies in delayed-type hypersensitivity and complement-fixing antibodies upon subsequent challenge with antigen. The mechanisms responsible for the generation of this form of tolerance are not yet completely clear. Here we asked whether gammadelta T cells, which are critical in the induction of oral tolerance and nasal tolerance, play a role in ACAID. The percentage of splenic gammadelta T cells was higher in mice that received antigen via the anterior chamber compared to untreated mice. In addition, CD44 was up-regulated on some splenic gammadelta and alphabeta T cells after the intraocular injection of antigen. Moreover, administration of antigen into the anterior chamber did not induce ACAID in the C57BL/6 mice pretreated with anti-mouse delta-chain monoclonal antibody or in the gammadelta T-cell-receptor-deficient (delta-/-) mice. gammadelta T cells from wild-type mice reconstituted ACAID when transferred into the delta-/- mice before injection of antigen, verifying that the deficiency in delta-/- mice results from the lack of gammadelta T cells rather than from an inadvertent change caused by deletion of the delta-chain. These findings indicate that gammadelta T cells play a very important role in ocular tolerance.

In a model of systemic tolerance called Anterior Chamber-Associated Immune Deviation (ACAID), the differentiation of the T regulatory (Tr) cells depends on NK T cells and occurs in the spleen. We now show that the CD1d-reactive NK T cell subpopulation, required for development of systemic tolerance, expresses the invariant V alpha 14J alpha 281 TCR because J alpha 281 knockout (KO) mice were unable to generate Ag-specific Tr cells and ACAID. The mechanism for NK T cell-dependent differentiation of Ag-specific Tr cells mediating systemic tolerance was studied by defining the cytokine profiles in heterogeneous and enriched NK T spleen cells. In contrast to there being no differences in most regulatory cytokine mRNAs, both mRNA and protein for IL-10 were increased in splenic NK T cells of anterior chamber (a.c.)-inoculated mice. However, IL-10 mRNA was not increased in spleens after i.v. inoculation. Finally, NK T cells from wild-type (WT) mice, but not from IL-10 KO mice, reconstituted the ACAID inducing ability in J alpha 281 KO mice. Thus, NK T cell-derived IL-10 is critical for the generation of the Ag-specific Tr cells and systemic tolerance induced to eye-inoculated Ags.

Ocular immune privilege is the result of several unique features of the eye, including the systemic down-regulation of Th1 immune responses to Ags encountered in the anterior chamber of the eye-a phenomenon termed anterior chamber-associated immune deviation (ACAID). The induction of ACAID requires the participation of three cell populations: the ocular ACAID APC, the splenic B cell, and the splenic T cell. Because B cells have been implicated in tolerogenic Ag presentation in other systems, we hypothesized that B cells were responsible for the induction of regulatory T cells in ACAID. The central hypothesis for this study is that APC from the eye migrate to the spleen where they release antigenic peptides (OVA) that are captured and presented to T cells by splenic B cells. A combination of in vitro and in vivo studies demonstrated that splenic B cells, incubated with ACAID APC in vitro, were capable of inducing ACAID when transferred to naive mice. The induction of ACAID required the normal expression of ss(2)-microglobulin on both the B cell and ACAID APC, but not on the T suppressor cells. Moreover, the induction of ACAID regulatory cells required histocompatibility between the B cells and regulatory T cells at the TL/Qa region. The results indicate that: 1) B cells are necessary for the induction of ACAID; 2) ACAID B cells do not directly suppress the expression of delayed-type hypersensitivity; and 3) the induction of Ag-specific regulatory T cells by ACAID B cells requires histocompatibility at the TL/Qa region.

Peripheral tolerance occurs after intraocular administration of Ag and is dependent on an increase in splenic NKT cells. New data here show that macrophage inflammatory protein-2 (MIP-2) is selectively up-regulated in tolerance-conferring APCs and serves to recruit NKT cells to the splenic marginal zone, where they form clusters with APCs and T cells. In the absence of the high-affinity receptor for MIP-2 (as in CXCR2-deficient mice) or in the presence of a blocking Ab to MIP-2, peripheral tolerance is prevented, and Ag-specific T regulatory cells are not generated. Understanding the regulation of lymphocyte traffic during tolerance induction may lead to novel therapies for autoimmunity, graft acceptance, and tumor rejection.

Purpose: Topical treatment with interleukin 1 receptor antagonist (IL-1ra) can promote corneal allograft survival by suppressing induction of allodestructive immunity. The purpose of these experiments was to determine whether IL-1ra could also promote induction of allo-protective tolerogenic pathways, including anterior chamber-associated immune deviation (ACAID), which has been shown to participate in long-term survival of corneal transplants., Methods: Corneal buttons from BALB/c (syngeneic) or C57BL/6 (fully mismatched allogeneic) mice were orthotopically grafted onto BALB/c recipients. Topical IL-1ra or vehicle alone was applied to grafts three times daily. Donor-specific ACAID was measured in allogeneic grafted mice at 4 and 8 weeks after transplantation by ear-challenging grafted hosts with donor-derived splenocytes 1 week after SC immunization. In separate experiments, grafted mice were treated for 4 weeks before injecting ovalbumin (OVA) into their anterior chambers to determine their capacity to induce antigen-specific ACAID., Results: Treatment with IL-1ra did not promote, or inhibit, induction of donor-specific ACAID compared with vehicle-treated controls at either the early or late time points studied. However, IL-1ra treatment after transplantation led to significantly earlier restoration of the grafted eyes' capacity for inducing ACAID to soluble antigen (OVA)., Conclusions: Promotion of OVA-specific ACAID by IL-1ra suggests that suppression of IL-1-mediated mechanisms contributes to recovery of the anterior segment's immunosuppressive microenvironment at least 1 month earlier than would otherwise be seen after corneal transplantation. However, IL-1ra treatment does not alter induction of donor-specific ACAID after transplantation, suggesting that its anti-inflammatory activities do not lead to an ACAID-inducing signal per se. This suggests that IL-1ra promotes graft survival almost exclusively by virtue of suppressing inflammation and not by directly promoting tolerance or antigen-specific regulatory pathways.

Ocular immune privilege arises from interactions between the immune apparatus and the eye itself, thereby providing immune protection for the eye that is devoid of sight-threatening inflammation. On the one hand, antigens injected intraocularly elicit deviant systemic immune responses that are devoid of immunogenic inflammation (Anterior Chamber-Associated Immune Deviation, ACAID). On the other hand, the ocular microenvironment (aqueous humor, secreted by cells that surround this chamber) suppresses intraocular expression of immunogenic inflammation. Several lines of evidence indicate that ocular immune privilege is under neural control. First, aqueous humor contains neuropeptides (alpha-MSH, VIP, CGRP) that inhibit and alter the functional properties of T lymphocytes and macrophages. Second, when corneal nerves are severed, the tissues surrounding the anterior chamber cease secreting immunosuppressive factors and ACAID fails--until the nerves regrow. Third, light deprivation abolishes the capacity of the anterior chamber to support ACAID induction, a process that is sensitive to neuropeptides and melatonin. The photoreceptor(s) responsible for ACAID are connected to the nervous system and may reside in the anterior segment and/or the retina. Thus, neural elements from the central nervous system and within the eye help to shape both the induction and the expression of ocular immunity, thereby promoting immune privilege.

Purpose: The importance of the spleen to the induction of anterior chamber-associated immune deviation (ACAID) in mice has been known for some time. Removal of the spleen within the first 4 days of injecting antigen into anterior chamber can abolish immune deviation and result in immunity. The purpose of this study is to explore the role of spleen in ACAID in other different species of animals., Methods: After splenectomy and sham-splenectomy, bovine serum albumin (BSA) as a soluble antigen was inoculated into the anterior chamber (AC) of different animals (rat, rabbit and monkey) respectively. Recipient animals were immunized with BSA and complete Freund's adjuvant. Delayed-type hypersensitivity (DTH) was assessed by skin challenge. The maintenance time of deviant immune response was evaluated in the fixed interval., Results: All the animals with receiving intraocular antigen inoculation, both in splenectomized group and in sham-splenectomized group, did not display DTH reaction. In addition, the maintenance time of immune deviation in both groups after AC inoculation of antigen was not significantly different. In contrast, all the animals without receiving intraocular antigen inoculation, both in splenectomized group and in sham-sple-nectomized group, displayed positive DTH reaction., Conclusions: The spleen may not play an important role in the induction and maintenance of ACAID in rat, rabbit and monkey. These data suggest that as the evolution progresses, the function of spleen may be replaced by other lymphatic organs.