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Immune regulation and the eye
Review
Immune regulation and the eye Joan Stein-Streilein Schepens Eye Research Institute, 20 Staniford Street, Boston, MA02114, USA
The eye is an immune privileged site that is styled to maintain the visual pathway while at the same time provide defense against invading organisms. The eye does this by selecting immune responses that function in the absence of inflammation. Immune regulation by the eye takes the form of several active processes including a local immunosuppressive environment, the contribution of soluble factors, Fas-FasL-induced apoptosis and unique suppressive mechanisms used by pigment epithelial cells in the eye. These processes are so effective that antigens encountered in the eye result in specific systemic tolerization; a phenomenon akin to gut-induced oral tolerance. This review discusses the cellular and molecular basis of tolerance induction by the eye and notes the parallels to gut-induced peripheral tolerance. Regional specialization of immune responses The immune response seems to be customized to the organ in which it is initiated, as well as being specialized for the region in which it has to function. Thus, it is not surprising that there are differences in responses to antigen depending on the route of its administration. Immunologists working in the field know these facts and therefore select the route of immunization to bias the response to their desired outcome. For instance, injection of antigen into the eye or gut is used in animal models to induce tolerance and a subcutaneous route used to produce inflammation. The eye and the gut are immunization routes classically used for inducing peripheral tolerance. However, the eye is far more well-studied as a model of immune privilege. Immune privileged sites are known for their acceptance of foreign tissue grafts. Indeed, in humans, mismatched cadaver cornea grafts are accepted 60–70% of the time, whereas 50% are rejected in eyes with preexisting inflammation [1]. The mechanisms of immune privilege in the eye are multiple and overlapping, but what is not generally recognized is that other tissues share some of the mechanisms used to down-regulate inflammation and induce tolerance in their own environments. Thus, what is learned from ocular immunology studies might be generally applicable to other tissues. Every location within the eye is immune privileged (Figure 1). In other words, antigen injected into the anterior chamber [2], vitreous [3] or sub-retinal space [4] induces peripheral tolerance to that antigen. Furthermore, parts of the eye like the cornea are considered to be intrinsically immune privileged tissues because when transplanted to sites perfectly capable of rejecting foreign antigens, they enjoy acceptance. For instance, a cornea is Corresponding author: Stein-Streilein, J. ([email protected]).
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accepted when transplanted under the kidney capsule, a site that is not immune privileged [5,6]. Mechanisms of immune privilege in the eye To understand the eye’s immune response one has to consider the multiple overlapping mechanisms that contribute to the establishment and maintenance of privilege. First, the eye is filled with immunosuppressive factors including neuropeptides, aMSH (melanocyte stimulating hormone), somatostatin, vasoactive intestinal peptide, calcitonin gene related peptide [7,8], cytokines (e.g. TGFb-2), complement inhibitors [9], and an inhibitor of NK cell activity (macrophage inhibitory factor) [10]. Second, the low expression of MHC class II in the eye limits antigen presentation. Third, stromal cells from the iris, ciliary body and retina of the eye are able to convert immune T cells to regulatory (Treg) cells [11–15] (also see Figure 1). Furthermore retinal pigment epithelial (RPE) cells that line the borders of the eye are able to directly inhibit primed T cells [16]. Fourth, death inducing molecules like PDL-1 and FasL are expressed by stromal cells in the eye and induce apoptosis of immune cells that transgress ocular boundaries [17,18]. We will revisit these mechanisms in more detail below. Fas ligand (FasL) Fas-FasL interactions contribute to immune privilege mechanisms. Unlike cytolysis or necrotic death that destroys tissues, the Fas-FasL system leads to apoptosis or programmed cell death without inflammation [19]. The importance of FasL in immune privilege has been studied in the eye and other immune privilege sites [17]. FasL is expressed at strategic locations throughout the eye including the cornea, retina, iris, and ciliary body [20]. The locations are strategic because they guard the junctions and barriers of the eye that encounter the systemic immune system. The FasL system appears to be poised at these locations to induce apoptosis of transgressing inflammatory or tumor cells. Soluble FasL (sFasL) can inhibit FasL-mediated apoptosis through its competition with the Fas receptor and its presence in the serum and aqueous humor of uveitis patients is indicative of a breakdown in this protective system. It is known that apoptosis (by any mechanism), but not necrosis, contributes to tolerance pathways [21]. Besides eliminating Fas-expressing cells, apoptotic cells making IL-10 induce the phagocytic cells that engulf them to produce IL-10 and express tolerogenic characteristics. Anterior chamber associated immune deviation (ACAID) A well-studied model of immune privilege called ACAID was first developed in the mid-1970s [22]. Many aspects of
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Review
Figure 1. Anatomy of the human eye. The major elements of the eye are shown. All the internal components of the eye are immune privileged including the cornea, anterior chamber, ciliary body, retina, retinal pigmented epithelium and vitreous.
ACAID are important and active in other immune privileged sites and it furthermore shares characteristics with oral tolerance induction (also see the article by Tsuji and colleagues in this issue) [23]. In this model, the placement of antigen into the anterior chamber of the eye (i.e. the region between the cornea and the lens, see Figure 1) induces a characteristic immune response that includes the absence of complement fixing antibodies and Th1 and Th2 immune responses specifically to that antigen. However, the animal remains perfectly capable of responding to the antigen and protecting the eye by means of an immune response without inflammation. Much of our understanding of immune privileged sites and their specialized immune responses comes from studies using the ACAID model in the eye [2]. A popular posit is that during ACAID induction, the antigen that is injected into the eye is picked up by indigenous F4/80+ cells, carried through the trabecular meshwork into the bloodstream, and after a possible detour into the thymus [24–26] the APC associated antigen makes its way into the spleen. Within the spleen the F4/80+ APC interact with T cells, NKT cells and marginal zone B cells to induce antigen-specific Treg cells that limit both local and systemic Th1 and Th2 response (Figure 2). The actual F4/80+ cells that leave the eye have never been tracked, because only a few eye-derived F4/80+ cells seed the periphery. It is thought that eye-derived APC recruit and modulate F4/80+ cells from the periphery toward tolerance and thus increase their tolerance potential. Studies that quantify the numbers of F4/80+ cells in the blood or spleen after anterior chamber inoculation show at least a twofold increase in the normal numbers in these tissues, clearly more than could come from the eye [27]. This increase in
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F4/80+ cells is induced by signals from the eye-derived cells and other cells involved in inducing peripheral tolerance to the antigen [28]. It is difficult to characterize the ocular F4/80+ cell as either a macrophage or dendritic cell (DC) because although the F4/80 protein is generally used as a marker of tissue macrophages, it is also found on a small subset of DC. Phenotypically the F4/80+ APC that can induce ACAID express CD11b and CD11c; have low CD40, low MHC class II, low B7–1 and -2 [29], high CD1d; and produce IL-10, TGFb [30] and MIP-2 [27]. The F4/80+ cells do not express the chemokine receptors CCR7 or CCR6 but do express CXCR4 [29]. If the ACAID tolerogenic APC had to be fitted into the DC Procrustean bed, this F4/80+ APC might be called a semimature DC [31] because it lacks CCR6, a marker of immature DC, but also lacks CCR7, a marker for mature DC. During ACAID induction, many F4/80+ APC accumulate in the marginal zone (MZ) of the spleen with other bone marrow-derived cells (Figure 2). Each of the accumulating cell types is critical for the tolerance outcome of the inoculation. The F4/80+ APC interacts through its CD1d molecule with an invariant NKT (iNKT) cell. The iNKT cell is restricted by CD1d, thus this rare population is absent in CD1d knockout mice. Moreover, CD1d deficient or iNKT cell deficient mice are unable to develop ACAID, showing that iNKT cells are crucial for the development of ACAID [32]. Other cells that are also needed include CD1d+ MZ B cells [33] and, of course, the T cells that differentiate into the Tregs. Previous studies have suggested that B cells were additional APC required to actually present antigen to the T cells [34,35]. It appears that the iNKT cells interact with CD1d on the F4/80+ and MZ B cell APC. Why the CD1d restricted iNKT cell needs to interact with two types of CD1d+ APCs (i.e. MZ B cells and the F4/80+ cells) is not understood. Regional specialization of APC indigenous to the eye The eye is filled with immune-suppressing molecules [8] and the fact that indigenous F4/80+ APC are constantly exposed to this immunosuppressive environment might explain their innate tolerogenic ability. Early ACAID investigators showed that aqueous humor directed in vitro APC to become tolerogenic and a major mediator of this signal was TFGb [36–38]. Thus, TGFb has been used in vitro to generate surrogate tolerogenic ACAID APC [39]. The ACAID-like tolerogenic APC, whether ex vivo or induced in vitro with TGFb and antigen, express low class II, low CD40, nondetectable IL-2 and no CCR7 or CCR6. The ACAID-like APC also express increased levels of CD1d and produce IL-10 and TGFb. Because of the similarities with ACAID in vivo, the in vitro model of ACAID is a valuable tool for studying the cellular and molecular mechanisms of tolerance induction that occur in the marginal zone of the spleen. The ACAID F4/ 80+ APC that leave the eye are absolutely critical to the induction of ACAID and like their in vitro-generated counterparts, can induce ACAID when adoptively transferred intravenously to naı¨ve mice. The cell surface molecules that are involved in the interactions between the APC and the other cells needed for ACAID have been studied both in vitro and in vivo. 549
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Figure 2. The immune tolerance reflex arc. The afferent portion of the arc begins with the entrance of antigen to the eye. Central processing takes place in the marginal zone of the spleen. The efferent phase of the tolerance response occurs in the periphery, which includes both local and systemic regulation of the antigen-specific immune response. The cellular and molecular interactions between cells within the marginal zone aggregates. The F4/80+ APC interacts with iNKT cells, MZ B cells, and T cells in the spleen to generate Treg cells. All molecules shown on the surface of or produced by cells have a role in the efficient production of Treg cells after anterior chamber inoculation of antigen. Most studies were done using an assay for suppression of effector responses and therefore relate to the generation of Foxp3+ CD8+ Treg cells. It is thought that the same collection of cells in the MZ also leads to the generation of the CD4+ Treg after anterior chamber inoculation of antigen, but this hypothesis awaits testing.
Critical molecules expressed by tolerogenic ocular APC The F4/80 protein The F4/80 protein turns out to be not only a marker of tolerogenic APC in the ACAID model but also a critical protein that must be expressed by the APC that transports the antigen from the eye to the spleen [40]. ACAID cannot be induced in F4/80-deficient mice but when F4/80-null APC are treated with TGFb they are perfectly capable of inducing tolerance when transferred to F4/80-sufficient, but not F4/80-deficient, recipient hosts. Thus, TGFb receptors are functional on F4/80-null APC and the cells are able to produce the necessary cytokines and receptors for inducing tolerance in a wild-type mouse. Although the exact function of the F4/80 protein remains unknown, its structure might give a clue to its function (reviewed in Ref. [41]). F4/80 has a hybrid structure (Figure 3) consisting of two different protein superfamilies [42] and is a prototypic member of the epidermal growth factor-seven span transmembrane (EGF-TM7) receptor family [43]. EGF like molecules has been observed in a wide variety of extracellular proteins involved in adhesion, receptor-ligand interactions, and extracellular matrix structure. Because of the extracellular module, it is possible that the F4/80 protein is needed for stabilization of cells to each other or to the MZ stromal cells for the induction of tolerance [27]. However, the TM7/G proteincoupled receptor domain is known to mediate signal transduction of an extensive variety of exogenous stimuli [44]. Because dual action was shown for this molecule in other systems, it is possible that the binding of the outer portion of the F4/80 molecule will be followed by an important signal to the APC during induction of ACAID or low-dose oral tolerance [40]. 550
The CD1d MHC class I-like molecule Although it is textbook information that APC present antigen on MHC class II to CD4+ T cells and MHC class I to CD8+ T cells, it turns out that CD1d, a class I like molecule, is important for tolerogenic APC’s communication with NKT cells. CD1d is an MHC class I-like molecule that presents endogenous lipids to T cells [45]. As a
Figure 3. F4/80 Structure. The F4/80 molecule appears to be a crucial component of tolerogenic function of ocular APC. The F4/80 hybrid structure consists of two different superfamily motifs: epidermal growth factor (EGF) and seven span transmembrane (TM7) like families. The EGF-like domains are located at the extracellular NH2 terminus and are probably involved in adhesive interactions with cells and extracellular matrix, whereas the seven span TM7 portion is similar to class B G-protein-coupled receptors is located at the C-terminus is probably responsible for signal transduction. The molecular details of F4/80 involvement in tolerance induction remain unclear.
Review surrogate ligand many studies load a lipid isolated from a marine sponge (a-galactosylceramide, a-GalCer) onto the CD1d molecule to stimulate CD1d-restricted NKT cells. In our hands, the treatment of the tolerogenic ocular APC with a-GalCer is one of the few ways to abrogate their tolerogenic function. Furthermore, injection of this reagent appears to abrogate ACAID and causes increased cornea graft rejection (unpublished observations). F4/80+ APC and the MZ B cell are required to express CD1d in ACAID, possibly to allow interaction with the iNKT cell. Important molecules on the iNKT cell The iNKT cells involved in peripheral tolerance induction via the eye comprise an even smaller subpopulation of this already rare population of cells. About 85% of the NKT cells in the mouse are restricted by CD1d and express the invariant Va14 Ja18 TCR. The majority of iNKT cells are double negative (i.e. CD4- CD8 ) and only a minor population is CD4+ [46]. The iNKT cell required for ACAID expresses CD4 [47] and produces the immunosuppressive molecules IL-10 and TGFb [32,48], the chemokine RANTES [28] and the serine protease urokinase [49]. Because ACAID can be induced in MHC class II knockout mice and antibodies to CD4 protein removes the ability to induce ACAID in these mice [47], CD4+ iNKT cells but not conventional CD4+ T cell may play a role in this process. The actual function of the CD4 molecule on the iNKT cell in ACAID is unknown. Ly49 C/I NK inhibitory receptor Within this small CD4+ iNKT cell population required for ACAID is another minor subpopulation that express the NK inhibitory molecule, Ly49 C/I. Specifically it is this CD4+ Ly49 C/I+ iNKT cell subpopulation that is required for the development of peripheral tolerance via the eye [50]. Ly49 receptors were initially identified on NK cells where they serve to recognize abnormal or foreign cells that do not express conventional levels of self-MHC class I molecules; a concept put forth as the ‘missing-self’ hypothesis [51]. In addition to NK cells, NKT cells, gd T cells, memory CD8+ T cells, B-1B cells, and a subset of DC also express Ly49 molecules [52,53]. Although the family of Ly49 molecules contains both inhibitory and activating receptors, there are no reports of activating Ly49 receptors being expressed by NKT cells [54]. Ly49 receptor expression on NKT cells is governed by several interacting mechanisms including NKT cell maturity, MHC context, and the nature of the TCR [55]. Similar to their role on NK cells, Ly49 molecules appear to influence the type of responsiveness of NKT cells during innate and adaptive immune responses. Engagement of Ly49 receptors inhibits inflammatory cytokine production by NKT cells [56], NKT cell proliferation [57] and cytotoxic activity [54]. Recent studies have extended the understanding of the role of Ly49 molecules in immune regulation to include the idea that NK inhibitory receptors provide positive signals for the efficient production of the IL-10 immunoregulatory cytokine [50]. NKT cellderived IL-10 might contribute to the ad hoc immune privileged environment set up in the splenic marginal
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zone when the cells interact to induce Treg cells during ACAID. Additional T cells involved in CD8+ Treg cell development Certainly, the CD4+ T cell is needed for the development of the afferent CD4+ Treg cell in eye-induced tolerance. However contradictory evidence has been reported for the need for CD4+ T cell help in the generation of the efferent CD8+ Treg cells, in vivo [47] versus in vitro [58]. gdT cells contribute to tolerance because a few studies in gd T cell-deficient mice have shown that ACAID does not develop in their absence [59,60]. The actual function of the gd T cells in the generation of Treg cells in immune privilege is not well understood but gd T cells are critical for oral tolerance [61–63]. Unique elements of Treg cells in ACAID Wilbanks and Streilein [64] were the first to describe Treg cells in ACAID. There are at least two subsets of Treg cells that develop into antigens administered to the eye; one functions to suppress induction of immune responses (afferent, CD4+) and the other suppresses the immune effector cells (efferent, CD8+). CD4+ Treg afferent suppression Recent studies have shown that there are two populations of the afferent CD4+ Treg cells: CD4+CD25+ and CD4+CD25- [65]. Both types of CD4+ Treg cells can be generated in the absence of the well-characterized Foxp3+ naturally occurring CD4+CD25+ Treg cells, therefore ruling out the possibility that the afferent regulatory CD4+ T cells in ACAID were merely the omnipresent natural CD4+CD25+ Treg cells. This study reported that both ACAID CD4+ Treg cell populations were suppressive even though the ACAID CD4+CD25+ Treg cells were Foxp3 positive and the ACAID CD4+ CD25- Treg cells were Foxp3 negative. The CD4+ Treg cell in ACAID is reported to express PD-1 (programmed death-1) suppressor molecule but whether these Treg cells also express CD25 was not determined in these studies [66]. Laboratories are just beginning to explore if the same mechanisms that lead to the development of CD8+ Treg cells are involved in the development of the ACAID afferent CD4+ Treg cells. In other words, are the F4/80+ APC and the same mixture of cells observed in the ACAID MZ cell clusters required for the generation of the afferent CD4+ Treg cell in ACAID. CD8+ Treg efferent suppression CD8+ Treg cells generated in vitro by lymphocyte exposure to tolerogenic APC turn on a unique set of genes in the Treg cells [67]. Induced genes included a group known to promote TGFb production or activation of another group that assisted in cellular migration to sites of antigen. Genes that were downregulated were ones known to interfere with TGFb production. The CD8 Treg cells generated during ACAID require expression of the CD103 (aEb7 integrin) adhesion molecule for their suppressor function. Like many other Treg cells these ACAID CD8+ express Foxp3 [68] and produce IL-10 and TGFb [48]. However, it 551
Review remains to be determined how ACAID-induced CD8+ Treg cells mediate suppression. Local immune regulatory mechanisms in the eye The ability of ocular stromal cells to induce regulatory capabilities in T cells was first shown with pigment epithelium of the iris and ciliary body [69,70], but RPE cells in the eye also share the ability to modulate T cells to become Treg cells. The mechanisms of this response include a requirement for cellcell contact and ligation of B7 and CTLA 4 molecules [13]. Pigmented cells within the eye also express FasL and thus are capable of inducing apoptosis of transgressing immune cells [20] in addition to altering their function. Various fluids collected from the eye, including the aqueous humor and the vitreous fluids, are able to induce Treg cells in vitro. Similarly, supernatants from cultures of iris and ciliary body tissue were immune modulatory mainly because of the TGFb content [71]. Treg cells can be generated in vitro by exposure of spleen cells (which include naı¨ve CD4 T cells) to APC previously treated with aqueous humor [39]. Besides TGFb, other components of aqueous humor are able to modulate T cells toward regulation [72,73]. For instance, CD4+CD25+ Treg cells are induced when primed CD4+ T cells expressing melanocortin 5 receptor are exposed to aMSH [74]. aMSH, however, will not substitute for TGFb in the in vitro ACAID protocol and therefore contributes to an aspect of immune privilege in the eye different from ACAID. Commonalities between eye induced and oral tolerance Historically, both ACAID and oral tolerance have been used therapeutically in mice (ACAID, oral tolerance) and humans (oral tolerance). Introduction of antigen into the eye has prolonged corneal grafts [75] and lessened the onset of experimental autoimmune uveitis (EAU) [76]. Oral tolerance has also been used to reduce the symptoms of EAU [77,78] and other autoimmune diseases [79]. However, not until recently did it become apparent that the two locally induced models of peripheral tolerance might share other mechanisms that lead to the tolerance. A role for F4/80+ APC, iNKT, and B cells in oral tolerance Oral tolerance is induced by repeated oral administration of protein antigens or sensitizing haptens to induce a state of active suppression [80]. A well-studied example of oral tolerance is induced by feeding low amounts of ovalbumin (OVA) to mice. Initially, a functional role of immature DC for the induction of Treg cells was elaborated in this model [81]. More recently, iNKT cells were found to contribute to the phenomenon of oral tolerance because mice lacking iNKT cells or CD1d could not be rendered tolerant by OVA [82]. Similarly, mice lacking iNKT cells cannot be rendered tolerant by oral administration of nickel salt because they fail to generate nickel-specific CD8+ Treg cells [83,84]. Thus, like ACAID, the induction phase of oral nickel tolerance involves the interaction of several different cell types, namely antigen-transporting APC, CD1d+ B cells, IL-4 and IL-10-producing CD4+ iNKT cells, DC (F4/80+ APC), and CD8+ Treg cells. In both ACAID and oral tolerance, iNKT cells are needed to produce of IL-4 and IL-10 [48] and TGFb [79]. 552
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In the ACAID model, the B cell is thought to present antigen recovered from the transporting APC to T cells [35,85] as well as interact with iNKT cells through its CD1d [33]. However, in an oral tolerance model, it has been suggested that apoptotic B cells contribute to the development of nickel-induced tolerance. The chronic administration of nickel renders nickel-containing B cells proapoptotic [86], but there is no evidence that other haptenic tolerogens, not to mention proteins such as OVA, have a similar effect. The investigators of nickel tolerance present convincing evidence that the iNKT cells induce B cell apoptosis. Other tolerance systems [32,82,87] suggest iNKT cells are needed for tolerance induction because they produce suppressive cytokines [86]. The model of oral nickel tolerance is so far unique in showing that the tolerogenic action of iNKT cells, in part, is based on the induction of apoptosis of target cells [86]. Intriguingly, the requirement of FasL+IL-4+IL-10+ iNKT cells for induction of nickel oral tolerance can be bypassed by adoptively transferring B cells that were rendered apoptotic (through g-irradiation) before transfer [86]. NKT cell-derived IL-10 might function in the induction of nickel tolerance by maintaining DC in an immature, tolerogenic status [88]. Similarly, in the ACAID model NKT cell-derived IL-10 is essential in part because it downregulates CD40 expression and IL-12 production [39,48] and therefore blocks Th1 cell differentiation. It is most likely that in all models of peripheral tolerance, a role of iNKT cells is mediated by both immunosuppressive cytokine production and Fas-FasLinduced apoptosis. Concluding remarks This review describes, in detail, multiple and overlapping mechanisms of immune regulation in the eye that explain its immune privilege. The mechanisms that contribute to the immune privilege of the eye include the ability to induce local and peripheral tolerance to antigens that cross the barriers of the eye; immunosuppressive molecules that down regulate the immune activating capacity of indigenous APC and convert naı¨ve T cells to Treg cells; and specialized regulatory stromal cells that secure the borders of the eye and direct activated otherwise-inflammatory T cells to convert to Treg cells by cell-cell contact. However, to quote a colleague, ‘no single mechanism truly defines immune privilege,’ ‘none of the mechanisms of immune privilege is unique to the eye,’ and ‘all assigned mediators (e.g. FasL, TGFb, or immune deviation) are found (or happen) elsewhere’ [20]. Acknowledgements We appreciate the excellent talents of Peter Mallen in the preparation of the illustrations. We also thank Ms. Amelia Margolis for her assistance in preparing this manuscript for publication.
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29 Zhang-Hoover, J. et al. (2005) Modulation of ovalbumin-induced airway inflammation and hyperreactivity by tolerogenic APC. J. Immunol. 175, 7117–7124 30 D’Orazio, T.J. and Niederkorn, J.Y. (1998) A novel role for TGF-beta and IL-10 in the induction of immune privilege. J. Immunol. 160, 2089– 2098 31 Lutz, M.B. and Schuler, G. (2002) Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol. 23, 445–449 32 Sonoda, K-H. et al. (1999) CD1 reactive NKT cells are required for development of systemic tolerance through an immune privileged site. J. Exp. Med. 190, 1215–1225 33 Sonoda, K.H. and Stein-Streilein, J. (2002) CD1d on antigentransporting APC and splenic marginal zone B cells promotes NKT cell-dependent tolerance. Eur. J. Immunol. 32, 848–857 34 Ashour, H.M. and Niederkorn, J.Y. (2006) Peripheral tolerance via the anterior chamber of the eye: role of B cells in MHC class I and II antigen presentation. J. Immunol. 176, 5950–5957 35 D’Orazio, T.J. and Niederkorn, J.Y. (1998) Splenic B cells are required for tolerogenic antigen presentation in the induction of anterior chamber-associated immune deviation (ACAID). Immunology 95, 47–55 36 Granstein, R.D. et al. (1990) Aqueous humor contains transforming growth factor-b and a small (<3500 daltons) inhibitor of thymocyte proliferation. J. Immunol. 144, 3021–3027 37 Cousins, S.W. et al. (1991) Immune privilege and suppression of immunogenic inflammation in the anterior chamber of the eye. Curr. Eye Res. 10, 287–297 38 Cousins, S.W. et al. (1991) Identification of transforming growth factorbeta as an immunosuppressive factor in aqueous humor. Invest. Ophthalmol. Vis. Sci. 32, 2201–2211 39 Hara, Y. et al. (1992) Analysis of an in vitro-generated signal that induces systemic immune deviation similar to that elicited by antigen injected into the anterior chamber of the eye. J. Immunol. 149, 1531– 1538 40 Lin, H.H. et al. (2005) The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance. J. Exp. Med. 201, 1615–1625 41 van den Berg, T.K. and Kraal, G. (2005) A function for the macrophage F4/80 molecule in tolerance induction. Trends Immunol. 26, 506–509 42 McKnight, A.J. et al. (1996) Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family. J. Biol. Chem. 271, 486–489 43 McKnight, A.J. and Gordon, S. (1996) EGF-TM7: a novel subfamily of seven-transmembrane-region leukocyte cell-surface molecules. Immunol. Today 17, 283–287 44 Pierce, K.L. et al. (2002) Signalling: seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 45 Schrantz, N. et al. (2007) The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells. J. Exp. Med. 204, 841–852 46 Bendelac, A. et al. (2007) The biology of NKT cells. Annu. Rev. Immunol. 25, 297–336 47 Nakamura, T. et al. (2003) CD4+ NKT cells, but not conventional CD4+ T cells, are required to generate efferent CD8+ T regulatory cells following antigen inoculation in an immune privileged site. J. Immunol. 171, 1266–1271 48 Sonoda, K.H. et al. (2001) NK T cell-derived IL-10 is essential for the differentiation of antigen-specific T regulatory cells in systemic tolerance. J. Immunol. 166, 42–50 49 Sonoda, K.H. et al. (2007) NKT cell-derived urokinase-type plasminogen activator promotes peripheral tolerance associated with eye. J. Immunol. 179, 2215–2222 50 Watte, C.M. et al. (2008) Ly49 C/I-dependent NKT cell-derived IL-10 is required for corneal graft survival and peripheral tolerance. J. Leukoc. Biol. 83, 928–935 51 Ljunggren, G-H. and Karre, K. (1990) In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol. Today 11, 237–244 52 Raulet, D.H. et al. (1997) Specificity, tolerance and developmental regulation of natural killer cells defined by expression of class Ispecific Ly49 receptors. Immunol. Rev. 155, 41–52
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Review 53 Anderson, S.K. et al. (2001) The ever-expanding Ly49 gene family: repertoire and signaling. Immunol. Rev. 181, 79–89 54 Ortaldo, J.R. et al. (1998) The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells. J. Immunol. 160, 1158– 1165 55 Skold, M. et al. (2003) MHC-dependent and -independent modulation of endogenous Ly49 receptors on NK1.1+ T lymphocytes directed by Tcell receptor type. Immunology 110, 313–321 56 Ikarashi, Y. et al. (2001) Dendritic cell maturation overrules H-2Dmediated natural killer T (NKT) cell inhibition: critical role for B7 in CD1d-dependent NKT cell interferon gamma production. J. Exp. Med. 194, 1179–1186 57 Maeda, M. et al. (2001) Regulation of NKT cells by Ly49: analysis of primary NKT cells and generation of NKT cell line. J. Immunol. 167, 4180–4186 58 Skelsey, M.E. et al. (2003) CD25+, interleukin-10-producing CD4+ T cells are required for suppressor cell production and immune privilege in the anterior chamber of the eye. Immunology 110, 18–29 59 Skelsey, M.E. et al. (2001) gd T cells are needed for ocular immune privilege and corneal graft survival. J. Immunol. 166, 4327–4333 60 Xu, Y. and Kapp, J.A. (2000) gd T cells are critical for the induction of ACAID. Investigative Ophthalmology and Visual Science Annual Meeting Ft. Lauderdale, FL, April 30 - May 5, 2000, Abstract Issue 41 (4), S117 61 Kapp, J.A. et al. (2004) Gammadelta T cells play an essential role in several forms of tolerance. Immunol. Res. 29, 93–102 62 Kapp, J.A. et al. (2004) gammadelta T-cell clones from intestinal intraepithelial lymphocytes inhibit development of CTL responses ex vivo. Immunology 111, 155–164 63 Locke, N.R. et al. (2006) TCR gamma delta intraepithelial lymphocytes are required for self-tolerance. J. Immunol. 176, 6553–6559 64 Wilbanks, G.A. and Streilein, J.W. (1990) Characterization of suppressor cells in anterior chamber-associated immune deviation (ACAID) induced by soluble antigen. Evidence of two functionally and phenotypically distinct T-suppressor cell populations. Immunology 71, 383–389 65 Keino, H. et al. (2006) Induction of eye-derived tolerance does not depend on naturally occurring CD4+CD25+ T regulatory cells. Invest. Ophthalmol. Vis. Sci. 47, 1047–1055 66 Meng, Q. et al. (2006) CD4+PD-1+ T cells acting as regulatory cells during the induction of anterior chamber-associated immune deviation. Invest. Ophthalmol. Vis. Sci. 47, 4444–4452 67 Keino, H. et al. (2006) CD8+ T regulatory cells use a novel genetic program that includes CD103 to suppress Th1 immunity in eye-derived tolerance. Invest. Ophthalmol. Vis. Sci. 47, 1533–1542 68 Jiang, L. et al. (2007) Increased expression of Foxp3 in splenic CD8+ T cells from mice with anterior chamber-associated immune deviation. Mol. Vis. 13, 968–974 69 Yoshida, M. et al. (2000) Participation of pigment epithelium of iris and ciliary body in ocular immune privilege. 2. Generation of TGF-betaproducing regulatory T cells. Invest. Ophthalmol. Vis. Sci. 41, 3862– 3870 70 Yoshida, M. et al. (2000) Participation of pigment epithelium of iris and ciliary body in ocular immune privilege. 1. Inhibition of T-cell activation in vitro by direct cell-to-cell contact. Invest. Ophthalmol. Vis. Sci. 41, 811–821
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Trends in Immunology Vol.29 No.11 71 Streilein, J.W. et al. (1996) Immunosuppressive properties of tissues obtained from eyes with experimentally manipulated corneas. Invest. Ophthalmol. Vis. Sci. 37, 413–424 72 Taylor, A.W. et al. (1994) Immunoreactive vasoactive intestinal peptide contributes to the immunosuppressive activity of normal aqueous humor. J. Immunol. 153, 1080–1086 73 Taylor, A.W. et al. (1992) Identification of alpha-melanocyte stimulating hormone as a potential immunosuppressive factor in aqueous humor. Curr. Eye Res. 11, 1199–1206 74 Taylor, A.W. and Namba, K. (2001) In vitro induction of CD25+ CD4+ regulatory T cells by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH). Immunol. Cell Biol. 79, 358–367 75 Niederkorn, J.Y. and Mellon, J. (1996) Anterior chamber-associated immune deviation promotes corneal allograft survival. Invest. Ophthalmol. Vis. Sci. 37, 2700–2707 76 Hara, Y. et al. (1992) Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber-associated immune deviation with interphotoreceptor retinoid-binding protein. J. Immunol. 148, 1685–1692 77 Krause, I. et al. (2000) Immunomodulation of experimental autoimmune diseases via oral tolerance. Crit. Rev. Immunol. 20, 1–16 78 Whitacre, C.C. et al. (1996) Oral tolerance in experimental autoimmune encephalomyelitis. Ann. N. Y. Acad. Sci. 778, 217–227 79 Weiner, H.L. et al. (1994) ORAL TOLERANCE:Immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu. Rev. Immunol. 12, 809–837 80 Tomasi, T.B., Jr (1980) Oral tolerance. Transplantation 29, 353–356 81 Khoury, S.J. et al. (1992) Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor beta, interleukin 4, and prostaglandin E expression in the brain. J. Exp. Med. 176, 1355–1364 82 Kim, H.J. et al. (2006) NKT cells play critical roles in the induction of oral tolerance by inducing regulatory T cells producing IL-10 and transforming growth factor beta, and by clonally deleting antigenspecific T cells. Immunology 118, 101–111 83 Roelofs-Haarhuis, K. et al. (2004) Oral tolerance to nickel requires CD4(+) invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells. J. Immunol. 173, 1043–1050 84 Margenthaler, J.A. et al. (2002) CD1-dependent natural killer (NK1.1 +) T cells are required for oral and portal venous tolerance induction. J. Surg. Res. 104, 29–35 85 D’Orazio, T.J. et al. (2001) Ocular immune privilege promoted by the presentation of peptide on tolerogenic B cells in the spleen. II. Evidence for presentation by Qa-1. J. Immunol. 166, 26–32 86 Nowak, M. et al. (2006) Oral nickel tolerance: Fas ligand-expressing invariant NK T cells promote tolerance induction by eliciting apoptotic death of antigen-carrying, effete B cells. J. Immunol. 176, 4581– 4589 87 Naumov, Y.N. et al. (2001) Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets. Proc. Natl. Acad. Sci. U. S. A. 98, 13838–13843 88 De Smedt, T. et al. (1997) Effect of interleukin-10 on dendritic cell maturation and function. Eur. J. Immunol. 27, 1229–1235
Immune regulation and the eye Joan Stein-Streilein Schepens Eye Research Institute, 20 Staniford Street, Boston, MA02114, USA
The eye is an immune privileged site that is styled to maintain the visual pathway while at the same time provide defense against invading organisms. The eye does this by selecting immune responses that function in the absence of inflammation. Immune regulation by the eye takes the form of several active processes including a local immunosuppressive environment, the contribution of soluble factors, Fas-FasL-induced apoptosis and unique suppressive mechanisms used by pigment epithelial cells in the eye. These processes are so effective that antigens encountered in the eye result in specific systemic tolerization; a phenomenon akin to gut-induced oral tolerance. This review discusses the cellular and molecular basis of tolerance induction by the eye and notes the parallels to gut-induced peripheral tolerance. Regional specialization of immune responses The immune response seems to be customized to the organ in which it is initiated, as well as being specialized for the region in which it has to function. Thus, it is not surprising that there are differences in responses to antigen depending on the route of its administration. Immunologists working in the field know these facts and therefore select the route of immunization to bias the response to their desired outcome. For instance, injection of antigen into the eye or gut is used in animal models to induce tolerance and a subcutaneous route used to produce inflammation. The eye and the gut are immunization routes classically used for inducing peripheral tolerance. However, the eye is far more well-studied as a model of immune privilege. Immune privileged sites are known for their acceptance of foreign tissue grafts. Indeed, in humans, mismatched cadaver cornea grafts are accepted 60–70% of the time, whereas 50% are rejected in eyes with preexisting inflammation [1]. The mechanisms of immune privilege in the eye are multiple and overlapping, but what is not generally recognized is that other tissues share some of the mechanisms used to down-regulate inflammation and induce tolerance in their own environments. Thus, what is learned from ocular immunology studies might be generally applicable to other tissues. Every location within the eye is immune privileged (Figure 1). In other words, antigen injected into the anterior chamber [2], vitreous [3] or sub-retinal space [4] induces peripheral tolerance to that antigen. Furthermore, parts of the eye like the cornea are considered to be intrinsically immune privileged tissues because when transplanted to sites perfectly capable of rejecting foreign antigens, they enjoy acceptance. For instance, a cornea is Corresponding author: Stein-Streilein, J. ([email protected]).
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accepted when transplanted under the kidney capsule, a site that is not immune privileged [5,6]. Mechanisms of immune privilege in the eye To understand the eye’s immune response one has to consider the multiple overlapping mechanisms that contribute to the establishment and maintenance of privilege. First, the eye is filled with immunosuppressive factors including neuropeptides, aMSH (melanocyte stimulating hormone), somatostatin, vasoactive intestinal peptide, calcitonin gene related peptide [7,8], cytokines (e.g. TGFb-2), complement inhibitors [9], and an inhibitor of NK cell activity (macrophage inhibitory factor) [10]. Second, the low expression of MHC class II in the eye limits antigen presentation. Third, stromal cells from the iris, ciliary body and retina of the eye are able to convert immune T cells to regulatory (Treg) cells [11–15] (also see Figure 1). Furthermore retinal pigment epithelial (RPE) cells that line the borders of the eye are able to directly inhibit primed T cells [16]. Fourth, death inducing molecules like PDL-1 and FasL are expressed by stromal cells in the eye and induce apoptosis of immune cells that transgress ocular boundaries [17,18]. We will revisit these mechanisms in more detail below. Fas ligand (FasL) Fas-FasL interactions contribute to immune privilege mechanisms. Unlike cytolysis or necrotic death that destroys tissues, the Fas-FasL system leads to apoptosis or programmed cell death without inflammation [19]. The importance of FasL in immune privilege has been studied in the eye and other immune privilege sites [17]. FasL is expressed at strategic locations throughout the eye including the cornea, retina, iris, and ciliary body [20]. The locations are strategic because they guard the junctions and barriers of the eye that encounter the systemic immune system. The FasL system appears to be poised at these locations to induce apoptosis of transgressing inflammatory or tumor cells. Soluble FasL (sFasL) can inhibit FasL-mediated apoptosis through its competition with the Fas receptor and its presence in the serum and aqueous humor of uveitis patients is indicative of a breakdown in this protective system. It is known that apoptosis (by any mechanism), but not necrosis, contributes to tolerance pathways [21]. Besides eliminating Fas-expressing cells, apoptotic cells making IL-10 induce the phagocytic cells that engulf them to produce IL-10 and express tolerogenic characteristics. Anterior chamber associated immune deviation (ACAID) A well-studied model of immune privilege called ACAID was first developed in the mid-1970s [22]. Many aspects of
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Figure 1. Anatomy of the human eye. The major elements of the eye are shown. All the internal components of the eye are immune privileged including the cornea, anterior chamber, ciliary body, retina, retinal pigmented epithelium and vitreous.
ACAID are important and active in other immune privileged sites and it furthermore shares characteristics with oral tolerance induction (also see the article by Tsuji and colleagues in this issue) [23]. In this model, the placement of antigen into the anterior chamber of the eye (i.e. the region between the cornea and the lens, see Figure 1) induces a characteristic immune response that includes the absence of complement fixing antibodies and Th1 and Th2 immune responses specifically to that antigen. However, the animal remains perfectly capable of responding to the antigen and protecting the eye by means of an immune response without inflammation. Much of our understanding of immune privileged sites and their specialized immune responses comes from studies using the ACAID model in the eye [2]. A popular posit is that during ACAID induction, the antigen that is injected into the eye is picked up by indigenous F4/80+ cells, carried through the trabecular meshwork into the bloodstream, and after a possible detour into the thymus [24–26] the APC associated antigen makes its way into the spleen. Within the spleen the F4/80+ APC interact with T cells, NKT cells and marginal zone B cells to induce antigen-specific Treg cells that limit both local and systemic Th1 and Th2 response (Figure 2). The actual F4/80+ cells that leave the eye have never been tracked, because only a few eye-derived F4/80+ cells seed the periphery. It is thought that eye-derived APC recruit and modulate F4/80+ cells from the periphery toward tolerance and thus increase their tolerance potential. Studies that quantify the numbers of F4/80+ cells in the blood or spleen after anterior chamber inoculation show at least a twofold increase in the normal numbers in these tissues, clearly more than could come from the eye [27]. This increase in
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F4/80+ cells is induced by signals from the eye-derived cells and other cells involved in inducing peripheral tolerance to the antigen [28]. It is difficult to characterize the ocular F4/80+ cell as either a macrophage or dendritic cell (DC) because although the F4/80 protein is generally used as a marker of tissue macrophages, it is also found on a small subset of DC. Phenotypically the F4/80+ APC that can induce ACAID express CD11b and CD11c; have low CD40, low MHC class II, low B7–1 and -2 [29], high CD1d; and produce IL-10, TGFb [30] and MIP-2 [27]. The F4/80+ cells do not express the chemokine receptors CCR7 or CCR6 but do express CXCR4 [29]. If the ACAID tolerogenic APC had to be fitted into the DC Procrustean bed, this F4/80+ APC might be called a semimature DC [31] because it lacks CCR6, a marker of immature DC, but also lacks CCR7, a marker for mature DC. During ACAID induction, many F4/80+ APC accumulate in the marginal zone (MZ) of the spleen with other bone marrow-derived cells (Figure 2). Each of the accumulating cell types is critical for the tolerance outcome of the inoculation. The F4/80+ APC interacts through its CD1d molecule with an invariant NKT (iNKT) cell. The iNKT cell is restricted by CD1d, thus this rare population is absent in CD1d knockout mice. Moreover, CD1d deficient or iNKT cell deficient mice are unable to develop ACAID, showing that iNKT cells are crucial for the development of ACAID [32]. Other cells that are also needed include CD1d+ MZ B cells [33] and, of course, the T cells that differentiate into the Tregs. Previous studies have suggested that B cells were additional APC required to actually present antigen to the T cells [34,35]. It appears that the iNKT cells interact with CD1d on the F4/80+ and MZ B cell APC. Why the CD1d restricted iNKT cell needs to interact with two types of CD1d+ APCs (i.e. MZ B cells and the F4/80+ cells) is not understood. Regional specialization of APC indigenous to the eye The eye is filled with immune-suppressing molecules [8] and the fact that indigenous F4/80+ APC are constantly exposed to this immunosuppressive environment might explain their innate tolerogenic ability. Early ACAID investigators showed that aqueous humor directed in vitro APC to become tolerogenic and a major mediator of this signal was TFGb [36–38]. Thus, TGFb has been used in vitro to generate surrogate tolerogenic ACAID APC [39]. The ACAID-like tolerogenic APC, whether ex vivo or induced in vitro with TGFb and antigen, express low class II, low CD40, nondetectable IL-2 and no CCR7 or CCR6. The ACAID-like APC also express increased levels of CD1d and produce IL-10 and TGFb. Because of the similarities with ACAID in vivo, the in vitro model of ACAID is a valuable tool for studying the cellular and molecular mechanisms of tolerance induction that occur in the marginal zone of the spleen. The ACAID F4/ 80+ APC that leave the eye are absolutely critical to the induction of ACAID and like their in vitro-generated counterparts, can induce ACAID when adoptively transferred intravenously to naı¨ve mice. The cell surface molecules that are involved in the interactions between the APC and the other cells needed for ACAID have been studied both in vitro and in vivo. 549
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Figure 2. The immune tolerance reflex arc. The afferent portion of the arc begins with the entrance of antigen to the eye. Central processing takes place in the marginal zone of the spleen. The efferent phase of the tolerance response occurs in the periphery, which includes both local and systemic regulation of the antigen-specific immune response. The cellular and molecular interactions between cells within the marginal zone aggregates. The F4/80+ APC interacts with iNKT cells, MZ B cells, and T cells in the spleen to generate Treg cells. All molecules shown on the surface of or produced by cells have a role in the efficient production of Treg cells after anterior chamber inoculation of antigen. Most studies were done using an assay for suppression of effector responses and therefore relate to the generation of Foxp3+ CD8+ Treg cells. It is thought that the same collection of cells in the MZ also leads to the generation of the CD4+ Treg after anterior chamber inoculation of antigen, but this hypothesis awaits testing.
Critical molecules expressed by tolerogenic ocular APC The F4/80 protein The F4/80 protein turns out to be not only a marker of tolerogenic APC in the ACAID model but also a critical protein that must be expressed by the APC that transports the antigen from the eye to the spleen [40]. ACAID cannot be induced in F4/80-deficient mice but when F4/80-null APC are treated with TGFb they are perfectly capable of inducing tolerance when transferred to F4/80-sufficient, but not F4/80-deficient, recipient hosts. Thus, TGFb receptors are functional on F4/80-null APC and the cells are able to produce the necessary cytokines and receptors for inducing tolerance in a wild-type mouse. Although the exact function of the F4/80 protein remains unknown, its structure might give a clue to its function (reviewed in Ref. [41]). F4/80 has a hybrid structure (Figure 3) consisting of two different protein superfamilies [42] and is a prototypic member of the epidermal growth factor-seven span transmembrane (EGF-TM7) receptor family [43]. EGF like molecules has been observed in a wide variety of extracellular proteins involved in adhesion, receptor-ligand interactions, and extracellular matrix structure. Because of the extracellular module, it is possible that the F4/80 protein is needed for stabilization of cells to each other or to the MZ stromal cells for the induction of tolerance [27]. However, the TM7/G proteincoupled receptor domain is known to mediate signal transduction of an extensive variety of exogenous stimuli [44]. Because dual action was shown for this molecule in other systems, it is possible that the binding of the outer portion of the F4/80 molecule will be followed by an important signal to the APC during induction of ACAID or low-dose oral tolerance [40]. 550
The CD1d MHC class I-like molecule Although it is textbook information that APC present antigen on MHC class II to CD4+ T cells and MHC class I to CD8+ T cells, it turns out that CD1d, a class I like molecule, is important for tolerogenic APC’s communication with NKT cells. CD1d is an MHC class I-like molecule that presents endogenous lipids to T cells [45]. As a
Figure 3. F4/80 Structure. The F4/80 molecule appears to be a crucial component of tolerogenic function of ocular APC. The F4/80 hybrid structure consists of two different superfamily motifs: epidermal growth factor (EGF) and seven span transmembrane (TM7) like families. The EGF-like domains are located at the extracellular NH2 terminus and are probably involved in adhesive interactions with cells and extracellular matrix, whereas the seven span TM7 portion is similar to class B G-protein-coupled receptors is located at the C-terminus is probably responsible for signal transduction. The molecular details of F4/80 involvement in tolerance induction remain unclear.
Review surrogate ligand many studies load a lipid isolated from a marine sponge (a-galactosylceramide, a-GalCer) onto the CD1d molecule to stimulate CD1d-restricted NKT cells. In our hands, the treatment of the tolerogenic ocular APC with a-GalCer is one of the few ways to abrogate their tolerogenic function. Furthermore, injection of this reagent appears to abrogate ACAID and causes increased cornea graft rejection (unpublished observations). F4/80+ APC and the MZ B cell are required to express CD1d in ACAID, possibly to allow interaction with the iNKT cell. Important molecules on the iNKT cell The iNKT cells involved in peripheral tolerance induction via the eye comprise an even smaller subpopulation of this already rare population of cells. About 85% of the NKT cells in the mouse are restricted by CD1d and express the invariant Va14 Ja18 TCR. The majority of iNKT cells are double negative (i.e. CD4- CD8 ) and only a minor population is CD4+ [46]. The iNKT cell required for ACAID expresses CD4 [47] and produces the immunosuppressive molecules IL-10 and TGFb [32,48], the chemokine RANTES [28] and the serine protease urokinase [49]. Because ACAID can be induced in MHC class II knockout mice and antibodies to CD4 protein removes the ability to induce ACAID in these mice [47], CD4+ iNKT cells but not conventional CD4+ T cell may play a role in this process. The actual function of the CD4 molecule on the iNKT cell in ACAID is unknown. Ly49 C/I NK inhibitory receptor Within this small CD4+ iNKT cell population required for ACAID is another minor subpopulation that express the NK inhibitory molecule, Ly49 C/I. Specifically it is this CD4+ Ly49 C/I+ iNKT cell subpopulation that is required for the development of peripheral tolerance via the eye [50]. Ly49 receptors were initially identified on NK cells where they serve to recognize abnormal or foreign cells that do not express conventional levels of self-MHC class I molecules; a concept put forth as the ‘missing-self’ hypothesis [51]. In addition to NK cells, NKT cells, gd T cells, memory CD8+ T cells, B-1B cells, and a subset of DC also express Ly49 molecules [52,53]. Although the family of Ly49 molecules contains both inhibitory and activating receptors, there are no reports of activating Ly49 receptors being expressed by NKT cells [54]. Ly49 receptor expression on NKT cells is governed by several interacting mechanisms including NKT cell maturity, MHC context, and the nature of the TCR [55]. Similar to their role on NK cells, Ly49 molecules appear to influence the type of responsiveness of NKT cells during innate and adaptive immune responses. Engagement of Ly49 receptors inhibits inflammatory cytokine production by NKT cells [56], NKT cell proliferation [57] and cytotoxic activity [54]. Recent studies have extended the understanding of the role of Ly49 molecules in immune regulation to include the idea that NK inhibitory receptors provide positive signals for the efficient production of the IL-10 immunoregulatory cytokine [50]. NKT cellderived IL-10 might contribute to the ad hoc immune privileged environment set up in the splenic marginal
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zone when the cells interact to induce Treg cells during ACAID. Additional T cells involved in CD8+ Treg cell development Certainly, the CD4+ T cell is needed for the development of the afferent CD4+ Treg cell in eye-induced tolerance. However contradictory evidence has been reported for the need for CD4+ T cell help in the generation of the efferent CD8+ Treg cells, in vivo [47] versus in vitro [58]. gdT cells contribute to tolerance because a few studies in gd T cell-deficient mice have shown that ACAID does not develop in their absence [59,60]. The actual function of the gd T cells in the generation of Treg cells in immune privilege is not well understood but gd T cells are critical for oral tolerance [61–63]. Unique elements of Treg cells in ACAID Wilbanks and Streilein [64] were the first to describe Treg cells in ACAID. There are at least two subsets of Treg cells that develop into antigens administered to the eye; one functions to suppress induction of immune responses (afferent, CD4+) and the other suppresses the immune effector cells (efferent, CD8+). CD4+ Treg afferent suppression Recent studies have shown that there are two populations of the afferent CD4+ Treg cells: CD4+CD25+ and CD4+CD25- [65]. Both types of CD4+ Treg cells can be generated in the absence of the well-characterized Foxp3+ naturally occurring CD4+CD25+ Treg cells, therefore ruling out the possibility that the afferent regulatory CD4+ T cells in ACAID were merely the omnipresent natural CD4+CD25+ Treg cells. This study reported that both ACAID CD4+ Treg cell populations were suppressive even though the ACAID CD4+CD25+ Treg cells were Foxp3 positive and the ACAID CD4+ CD25- Treg cells were Foxp3 negative. The CD4+ Treg cell in ACAID is reported to express PD-1 (programmed death-1) suppressor molecule but whether these Treg cells also express CD25 was not determined in these studies [66]. Laboratories are just beginning to explore if the same mechanisms that lead to the development of CD8+ Treg cells are involved in the development of the ACAID afferent CD4+ Treg cells. In other words, are the F4/80+ APC and the same mixture of cells observed in the ACAID MZ cell clusters required for the generation of the afferent CD4+ Treg cell in ACAID. CD8+ Treg efferent suppression CD8+ Treg cells generated in vitro by lymphocyte exposure to tolerogenic APC turn on a unique set of genes in the Treg cells [67]. Induced genes included a group known to promote TGFb production or activation of another group that assisted in cellular migration to sites of antigen. Genes that were downregulated were ones known to interfere with TGFb production. The CD8 Treg cells generated during ACAID require expression of the CD103 (aEb7 integrin) adhesion molecule for their suppressor function. Like many other Treg cells these ACAID CD8+ express Foxp3 [68] and produce IL-10 and TGFb [48]. However, it 551
Review remains to be determined how ACAID-induced CD8+ Treg cells mediate suppression. Local immune regulatory mechanisms in the eye The ability of ocular stromal cells to induce regulatory capabilities in T cells was first shown with pigment epithelium of the iris and ciliary body [69,70], but RPE cells in the eye also share the ability to modulate T cells to become Treg cells. The mechanisms of this response include a requirement for cellcell contact and ligation of B7 and CTLA 4 molecules [13]. Pigmented cells within the eye also express FasL and thus are capable of inducing apoptosis of transgressing immune cells [20] in addition to altering their function. Various fluids collected from the eye, including the aqueous humor and the vitreous fluids, are able to induce Treg cells in vitro. Similarly, supernatants from cultures of iris and ciliary body tissue were immune modulatory mainly because of the TGFb content [71]. Treg cells can be generated in vitro by exposure of spleen cells (which include naı¨ve CD4 T cells) to APC previously treated with aqueous humor [39]. Besides TGFb, other components of aqueous humor are able to modulate T cells toward regulation [72,73]. For instance, CD4+CD25+ Treg cells are induced when primed CD4+ T cells expressing melanocortin 5 receptor are exposed to aMSH [74]. aMSH, however, will not substitute for TGFb in the in vitro ACAID protocol and therefore contributes to an aspect of immune privilege in the eye different from ACAID. Commonalities between eye induced and oral tolerance Historically, both ACAID and oral tolerance have been used therapeutically in mice (ACAID, oral tolerance) and humans (oral tolerance). Introduction of antigen into the eye has prolonged corneal grafts [75] and lessened the onset of experimental autoimmune uveitis (EAU) [76]. Oral tolerance has also been used to reduce the symptoms of EAU [77,78] and other autoimmune diseases [79]. However, not until recently did it become apparent that the two locally induced models of peripheral tolerance might share other mechanisms that lead to the tolerance. A role for F4/80+ APC, iNKT, and B cells in oral tolerance Oral tolerance is induced by repeated oral administration of protein antigens or sensitizing haptens to induce a state of active suppression [80]. A well-studied example of oral tolerance is induced by feeding low amounts of ovalbumin (OVA) to mice. Initially, a functional role of immature DC for the induction of Treg cells was elaborated in this model [81]. More recently, iNKT cells were found to contribute to the phenomenon of oral tolerance because mice lacking iNKT cells or CD1d could not be rendered tolerant by OVA [82]. Similarly, mice lacking iNKT cells cannot be rendered tolerant by oral administration of nickel salt because they fail to generate nickel-specific CD8+ Treg cells [83,84]. Thus, like ACAID, the induction phase of oral nickel tolerance involves the interaction of several different cell types, namely antigen-transporting APC, CD1d+ B cells, IL-4 and IL-10-producing CD4+ iNKT cells, DC (F4/80+ APC), and CD8+ Treg cells. In both ACAID and oral tolerance, iNKT cells are needed to produce of IL-4 and IL-10 [48] and TGFb [79]. 552
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In the ACAID model, the B cell is thought to present antigen recovered from the transporting APC to T cells [35,85] as well as interact with iNKT cells through its CD1d [33]. However, in an oral tolerance model, it has been suggested that apoptotic B cells contribute to the development of nickel-induced tolerance. The chronic administration of nickel renders nickel-containing B cells proapoptotic [86], but there is no evidence that other haptenic tolerogens, not to mention proteins such as OVA, have a similar effect. The investigators of nickel tolerance present convincing evidence that the iNKT cells induce B cell apoptosis. Other tolerance systems [32,82,87] suggest iNKT cells are needed for tolerance induction because they produce suppressive cytokines [86]. The model of oral nickel tolerance is so far unique in showing that the tolerogenic action of iNKT cells, in part, is based on the induction of apoptosis of target cells [86]. Intriguingly, the requirement of FasL+IL-4+IL-10+ iNKT cells for induction of nickel oral tolerance can be bypassed by adoptively transferring B cells that were rendered apoptotic (through g-irradiation) before transfer [86]. NKT cell-derived IL-10 might function in the induction of nickel tolerance by maintaining DC in an immature, tolerogenic status [88]. Similarly, in the ACAID model NKT cell-derived IL-10 is essential in part because it downregulates CD40 expression and IL-12 production [39,48] and therefore blocks Th1 cell differentiation. It is most likely that in all models of peripheral tolerance, a role of iNKT cells is mediated by both immunosuppressive cytokine production and Fas-FasLinduced apoptosis. Concluding remarks This review describes, in detail, multiple and overlapping mechanisms of immune regulation in the eye that explain its immune privilege. The mechanisms that contribute to the immune privilege of the eye include the ability to induce local and peripheral tolerance to antigens that cross the barriers of the eye; immunosuppressive molecules that down regulate the immune activating capacity of indigenous APC and convert naı¨ve T cells to Treg cells; and specialized regulatory stromal cells that secure the borders of the eye and direct activated otherwise-inflammatory T cells to convert to Treg cells by cell-cell contact. However, to quote a colleague, ‘no single mechanism truly defines immune privilege,’ ‘none of the mechanisms of immune privilege is unique to the eye,’ and ‘all assigned mediators (e.g. FasL, TGFb, or immune deviation) are found (or happen) elsewhere’ [20]. Acknowledgements We appreciate the excellent talents of Peter Mallen in the preparation of the illustrations. We also thank Ms. Amelia Margolis for her assistance in preparing this manuscript for publication.
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