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Alloimmunity and Tolerance in Afsaneh Amouzegar, Sunil K. Chauhan and This information is current as J Immunol 2016; 196:3983-3991; ; of September 29, 2021. doi: 10.4049/jimmunol.1600251 http://www.jimmunol.org/content/196/10/3983 Downloaded from References This article cites 116 articles, 55 of which you can access for free at: http://www.jimmunol.org/content/196/10/3983.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Brief Reviews Immunology

Alloimmunity and Tolerance in Corneal Transplantation Afsaneh Amouzegar, Sunil K. Chauhan,1 and Reza Dana1 Corneal transplantation is one of the most prevalent suppressive therapy (7); these outcomes are considerably worse and successful forms of solid tissue transplantation. De- than for kidney, heart, or liver transplants. Interestingly, the spite favorable outcomes, immune-mediated rejec- remarkable rate of success normally seen in low-risk corneal tion remains the major cause of corneal allograft failure. grafts, unlike other solid transplants, is achievable without the Although low-risk graft recipients with uninflamed benefit of HLA matching or profound systemic immune sup- graft beds enjoy a success rate ∼90%, the rejection pression (7). rates in inflamed graft beds or high-risk recipients Despite these favorable outcomes, graft rejection remains the often exceed 50%, despite maximal immune suppres- leading cause of corneal allograft failure (8). Corneal graft rejection can occur in any of the three cell layers of the

sion. In this review, we discuss the critical facets of Downloaded from corneal alloimmunity, including immune and angio- (epithelium, stroma, or endothelium), with endothelial re- genic privilege, mechanisms of allosensitization, cellu- jection being the most prevalent sight-threatening form. This can be attributed to the fact that the endothelial cells of the lar and molecular mediators of graft rejection, and cornea are irreplaceable and perform the critical function of allotolerance induction. The Journal of Immunology, preventing the tissue from getting swollen (9). Graft rejection 2016, 196: 3983–3991. is characterized clinically by graft edema (swelling) and in- flammatory cells that can be seen circulating in the anterior http://www.jimmunol.org/ ore than a century has passed since , chamber of the or attaching as keratic precipitates to the an Austrian ophthalmologist, performed the first graft endothelial cells (Fig. 1A) (10). Several factors have been M partially successful full-thickness human corneal associated with a higher risk for graft failure, with the degree transplant (1). Today, corneal transplantation is one of the of host bed neovascularization being the most significant most prevalent forms of solid tissue transplantation performed prognosticator for earlier and a more severe graft rejection (7, in the world (2). It is estimated that well over 100,000 corneal 11, 12). transplant are performed annually worldwide, with In this review, we discuss the factors involved in ocular nearly 40,000 performed annually in the United States alone inflammation, activation and migration of APCs, pathways of by guest on September 29, 2021 (3). Several trends are notable. First, the number of corneal allosensitization and allotolerance, and mechanisms of graft grafts performed in the developing world, especially Asia, destruction (Fig. 1B). is increasing sharply as the result of enhanced - ing procedures and distribution networks. Second, partial- Immune and angiogenic privilege of the cornea and their implications thickness corneal transplants (lamellar keratoplasty) are in- in transplant creasingly being used in place of full-thickness transplants The term immune privilege was coined by Sir Peter Med- (penetrating keratoplasty) when the entire cornea does not awar in the late 1940s, when he recognized the extended need to be replaced; this trend has led to some decreased risk survival of skin allografts placed in the anterior chamber of for graft rejection, likely because of the decreased load of al- the eye (13). He attributed this unexpected graft survival to logeneic tissue (4, 5). Still, it is critical to emphasize that the what he considered to be immunological ignorance, a pas- most important prognosticator of graft success is the status of sive process of sequestration of foreign Ags in the anterior the recipient bed in which the corneal graft is placed. The 2-y chamber as the result of the absence of draining lymphatic graft survival for penetrating keratoplasty in nonvascularized vessels. However, later in the 1970s, Kaplan and Streilein (14) and uninflamed host beds or low-risk corneal transplants is demonstrated that immune privilege was, in fact, the result ∼90% (6). However, recipients with a history of graft rejec- of an actively maintained and deviant suppressive immune tion or grafts performed in inflamed and vascularized host response to ocular Ags, a phenomenon that was later refer- beds are considered to be at high risk for rejection, with failure red to as anterior chamber–associated immune deviation rates . 50%, despite maximal local and systemic immuno- (ACAID) (15).

Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical addresses: [email protected] (S.K.C.) and [email protected] School, Boston, MA 02114 (R.D.) 1S.K.C. and R.D. contributed equally to this work. Abbreviations used in this article: ACAID, anterior chamber–associated immune devi- ation; CCTS, Collaborative Corneal Transplantation Studies; DC, ; DTH, Received for publication February 10, 2016. Accepted for publication March 10, 2016. delayed-type ; FasL, Fas ligand; IL-1Ra, IL-1R antagonist; LN, lymph This work was supported in part by National Institutes of Health Grants EY24602 (to node; Nrp-1, neuropilin-1; PDL-1, programmed death ligand-1; tolDC, tolerogenic S.K.C.) and EY12963 (to R.D.). DC; Treg, regulatory ; TSP, thrombospondin-1; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Address correspondence and reprint requests to Dr. Sunil K. Chauhan and Dr. Reza Dana, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. E-mail Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600251 3984 BRIEF REVIEWS: ALLOIMMUNITY AND TOLERANCE IN CORNEAL TRANSPLANTATION

FIGURE 1. Immunobiology of corneal transplanta- tion. (A) Clinical manifestation of corneal graft rejec- tion. Infiltrating and T cells attack the graft, often involving a rejection line that marches across the inner endothelial layer of the transplant, leaving an opaque and swollen graft behind. (B) Schematic repre- sentation of corneal alloimmunity. (I) Following trans- plant , upregulation of proinflammatory , Downloaded from adhesion molecules, and proangiogenic factors results in corneal infiltration of immune cells and formation of new blood and lymphatic vessels. (II) APCs, which acquire MHC class II and costimulatory molecules in the in- flammatory environment, egress from the cornea through lymphatic vessels to the draining LNs, where they present alloantigens to naive T cells (Th0). Newly formed lym- http://www.jimmunol.org/ phatic vessels may also contribute to resolution of in- flammation by mediating clearance of inflammatory cells and debris. (III) Primed T cells undergo clonal expansion and differentiate primarily into IFN-g–secreting CD4+ Th1 cells. Tregs modulate induction of alloimmune re- sponses through inhibition of T cell activation or sup- pression of APC stimulatory potential. (IV) Alloreactive Th1 cells migrate through blood vessels and along a chemokine gradient toward the graft, where they mount a by guest on September 29, 2021 DTH response against the allogeneic tissue, resulting in graft opacification and failure.

Immune privilege. ACAID is a form of keratoplasty causes a significant improvement in corneal to alloantigens placed in the anterior chamber of the eye allograft survival(18,19). that results in downregulation of Ag-specific delayed-type In addition to immunological tolerance induced by ACAID, hypersensitivity (DTH) response while promoting humoral several mechanisms contribute to the maintenance of ocular im- immunity and production of noncomplement-fixing Abs muneprivilege(TableI).Thecorneaexpressesanarrayof (16).ThisprocessisthoughttobemediatedbyF4/80+ membrane-bound immunomodulatory molecules that protect APCs in the eye that capture intraocular Ags, enter the it from inflammation and promote immune quiescence. Pro- bloodstream, and migrate to the marginal zone of the grammed death ligand-1 (PDL-1) is a proapoptotic molecule spleen, where their interactions with CD4+ Tcells,gd T that is constitutively expressed at high levels by the cornea (20, cells, B cells, and NKT cells result in the generation of two 21). PDL-1’s interaction with PD-1 on T cells results in inhi- groups of Ag-specific regulatory T cells (Tregs): CD4+ CD25+ bition of T cell proliferation, apoptosis induction, and inhibition Tregs and CD8+ Tregs (17). Studies on the implication of IFN-g production, leading to prolonged allograft survival (20, of ACAID in murine models of corneal transplantation 22). Recent studies established distinct mechanisms for donor- demonstrated that ACAID induction through intraocular and host-derived PDL-1 in promoting corneal allograft survival. injection of allogeneic prior to penetrating Depletion of PDL-1 in graft recipients results in considerably The Journal of Immunology 3985

Table I. Factors involved in immune and angiogenic privilege of the cornea

Factor Function Ref. Angiostatin Inhibits vascular endothelial cell (VEC) proliferation (45) a-MSH Suppresses IFN-g production by T cells, promotes Treg development (33, 38) CGRP Suppresses NO production by (35) CRP Inhibits activation of the (32) Endostatin Promotes VEC apoptosis (46) FasL Promotes apoptosis of PMNs and T cells (26) IDO Promotes T cell apoptosis, suppresses NK cell proliferation (39) IL-1Ra Suppresses APC migration (23) 2 MIF Inhibits NK cell–mediated cytolysis of MHC class I cells (36, 37) PDL-1 Promotes T cell apoptosis, inhibits T cell proliferation and IFN-g production (21, 22) PEDF Suppresses VEGF expression (47) 2 TGF-b Inhibits NK cell–mediated cytolysis of MHC class I cells, suppresses T cell activation (31) TRAIL Promotes T cell apoptosis and proliferation of Tregs (27–29) TSP-1 Inhibits APC maturation and migration, and T cell allosensitization (25) VEGFR-1 Inhibits the mitogenic activity of VEGF-A on VECs (40) VEGFR-2 Inhibits the angiogenic activity of VEGF-C (44) VEGFR-3 Inhibits hemangiogenesis and lymphangiogenesis, decoy (41, 43) nonsignaling receptor for VEGF-C and VEGF-D VIP Suppresses T cell activation and proliferation (34) Downloaded from a-MSH, a-melanocyte stimulating hormone; CGRP, calcitonin gene-related peptide; CRP, complement regulatory proteins; MIF, migration inhibitory factor; PEDF, pigment epithelium-derived factor; VIP, vasoactive intestinal peptide. stronger indirect T cell priming and rapid graft rejection than Angiogenic privilege. The cornea is devoid of vasculature and in wild-type recipients (20). However, elimination of PDL-1 lymphatics, which limits the trafficking of immune cells be- http://www.jimmunol.org/ in the graft donor does not have a significant effect on indirect tween the cornea and the systemic circulation and lym- allorecognition, but it does result in an increased alloreactive phoid organs. This angiogenic privilege is actively maintained T cell infiltration and graft failure (20). IL-1R antagonist (IL- through the expression of several antiangiogenic factors. Corneal 1Ra), which is constitutively expressed by the cornea, pro- epithelial cells constitutively secrete soluble vascular endothelial motes immune privilege through suppression of APC mi- growth factor receptor (VEGFR)-1, which binds VEGF-A and, gration to the cornea (23). Topical administration of IL-1Ra thus, inhibits its mitogenic effect on vascular endothelial cells was shown to promote allograft survival in low-risk and high- (40). Additionally, nonsignaling VEGFR-3, which is constitutively risk graft recipients in a murine model of corneal transplantation expressed by the corneal epithelium, demonstrates antiangiogenic (24). Thrombospondin-1 (TSP-1) is an immunomodulatory activity by acting as a decoy receptor for VEGF-C and VEGF-D by guest on September 29, 2021 glycoprotein expressed by the cornea and immune cells, such (41, 42), thereby suppressing blood and lymphatic vessel growth. as APCs. Studies demonstrated significantly higher rates of graft Studies on a mouse model of corneal transplantation also rejection in TSP-1–null mouse corneal allografts compared demonstrated that blockade of VEGFR-3 or VEGFR-3– with wild-type grafts (25). APC-derived TSP-1 inhibits mat- binding ligands, such as anti–VEGF-C, results in significant uration of APCs during inflammation, regulates their migration inhibition of lymphangiogenesis and, ultimately, prolonged to draining lymph nodes (LNs), and suppresses their capacity in corneal graft survival (43). Corneal epithelial cells additionally direct sensitization of alloreactive T cells (25). Corneal epithelial express soluble VEGFR-2, which inhibits VEGF-C and, thus, and endothelial cells constitutively express the proapoptotic blocks lymphangiogenesis (44). Intracorneal administration molecule Fas ligand (FasL; CD95L). Interaction of FasL with of soluble VEGFR-2 was shown to double allograft survival Fas (CD95)-expressing inflammatory cells, such as in a mouse model of corneal transplantation (44). Other and effector T cells, results in their apoptotic death, thereby antiangiogenic factors that are expressed by the cornea improving allograft survival (26). TRAIL (or Apo2L) is a include angiostatin, which inhibits vascular endothelial cell transmembrane protein highly homologous with FasL that is proliferation and migration (45); endostatin, which blocks constitutively expressed by corneal cells (27). TRAIL has been the mitogenic activity of VEGF on vascular endothelial cells implicated in apoptosis of activated T cells and was found to and promotes their apoptosis (46); pigment epithelium-derived promote the proliferation of Tregs (28). Although no study has factor expressed by corneal epithelium and endothelium that directly associated TRAIL expression with corneal graft survival, exerts its antiangiogenic effect through downregulation of VEGF transfection of mouse donor corneaswithadenoviruscarrying expression (47); and TSP-1, which inhibits hemangiogenesis TRAIL gene significantly improved corneal allograft survival and lymphangiogenesis by induction of vascular endothelial (29). In addition to these membrane-bound proteins, several cell apoptosis and CD36-mediated downregulation of VEGF-C soluble immunoregulatory factors are found in the aqueous (48). Results of a recent study showed that subconjunctival humor of the eye, including TGF-b2, complement regulatory administration of endostatin to mice undergoing corneal proteins, a-melanocyte stimulating hormone, vasoactive intes- transplantation inhibits graft neovascularization and T cell tinal peptide, calcitonin gene-related peptide, somatostatin, macro- infiltration and significantly prolongs corneal graft survival phage migration inhibitory factor, and IDO, which are involved (49). It also was demonstrated recently that PDL-1 has in tolerance induction in macrophages, regulation of dendritic antiangiogenic functions (50). Suture-induced corneal inflam- cells (DCs), and inhibition of complement and NK cell– mation in PDL-1–knockout mice results in a more significant mediated cell lysis and T cell activation (30–39). angiogenic response and higher levels of VEGFR-2 expression 3986 BRIEF REVIEWS: ALLOIMMUNITY AND TOLERANCE IN CORNEAL TRANSPLANTATION compared with wild-type mice (50). This complex interplay Mechanisms of allosensitization: significance of direct versus indirect between antiangiogenic and proangiogenic factors demonstrates allorecognition pathways the importance of regulation of hemangiogenesis and Corneal graft rejection is a complex process during which lymphangiogenesis in the maintenance of corneal immune changes in the corneal microenvironment and the interplay privilege. between cells of the innate and adaptive immune systems result in graft destruction. Early after transplantation, ocular surface Relevance of MHC Ags in corneal alloimmunity inflammation leads to upregulation of proinflammatory cy- All corneal cells, including epithelial, stromal, and endothelial tokines, such as IL-1, IL-6, and TNF-a (59), chemokines cells, express MHC class I Ags. Interestingly, in the healthy including MIP-1a, MIP-1b, MIP-2, and RANTES (60), and cornea, only DCs in the peripheral cornea express MHC class overexpression of adhesion molecules, such as ICAM-1 and II, whereas DCs and other myeloid cell populations residing VLA-1 (61–63). This inflammatory milieu results in the ac- in the center of the cornea express no to minimal levels of quisition of high levels of MHC class II and costimulatory MHC class II (51). In contrast to their kin in the skin, in- molecules, such as CD80 (B7-1), CD86 (B7-2), and CD40, flammatory stresses induce MHC class II expression by the by resident and infiltrating host APCs. Under these circum- significant majority of corneal DCs and corneal epithelial cells stances, donor corneal APCs, which normally lack the ca- (30). pacity to stimulate T cells, become more potent in alloantigen HLA (MHC) matching and systemic immunosuppression presentation and priming of naive T cells into Th1 effectors, are the cornerstones of prophylactic strategies against rejec- the principal mediators of acute corneal graft rejection (see Downloaded from tion for solid organ transplants, such as the kidney; however, below for details of effector mechanisms) (10, 64, 65). Ad- neither is routinely performed in corneal transplantation. A ditionally, inflammation-induced expression of adhesion mol- myriad of studies has been performed since the 1970s on ecules and chemotactic gradient assist in mobilizing host APCs the relative efficiency of HLA matching in corneal allograft from the pericorneal limbal vasculature centripetally toward the survival. The results from these studies have been mixed,

corneal graft (64, 66). http://www.jimmunol.org/ with some studies showing a clear benefit (52, 53). However, The cornea is normally devoid of blood and lymphatic the largest prospective randomized study of HLA matching vessels; however, ocular inflammation leads to formation of in corneal transplantation in the United States, the Collabo- blood and lymphatic neovessels. Inhibition of hemangio- rative Corneal Transplantation Studies (CCTS), showed no genesis and lymphangiogenesis in high-risk recipient mice was benefit for HLA-DR matching (7). Evidence regarding the shown to improve corneal allograft survival (43, 44, 67). The effect of MHC class II/HLA-DR matching remains unclear. pathologic corneal lymphangiogenesis that occurs as a result Many studies demonstrated prolonged survival rates in grafts of inflammation facilitates APC migration to draining LNs, with lower numbers of HLA-DR mismatches, especially in where they prime alloreactive T cells (30). Expression of the high-risk setting (52, 54). This is in contrast with the by guest on September 29, 2021 inflammatory cytokines, adhesion molecule ICAM-1, and CCTS, which reported no significant difference in graft re- CCL2 and CCL20 chemokines facilitate corneal infiltration jection rates between cross-matched and non–HLA-matched groups (7). The failure of CCTS to demonstrate beneficial of innate immune cells, which promote lymphangiogenesis results for HLA matching on graft survival was attributed to through production of VEGF-C and VEGF-D (61, 68–71). various factors, including the possibility of incorrect typing It was demonstrated that APCs, as a part of their matura- (these studies were primarily done in the 1980s before the tion process, also acquire VEGFR-3 expression, which, in advent of DNA-based typing methods) and aggressive im- response to the chemotactic gradient of its principal receptor munosuppressive therapy regimens that can nullify the ben- VEGF-C, mediates APC trafficking to lymphoid tissues (72). eficial effects of HLA matching, in part by suppressing MHC APC migration through afferent lymphatic vessels is also expression (55). However, the CCTS reported that matching dependent on the interaction between the CCR7 receptor and for minor Ags, such as ABO group, may its ligand CCL21, which is expressed by lymphatic endothe- improve graft survival. In humans, donor–recipient matching lial cells during inflammation, and APC interactions with of minor H-Y Ag was recently associated with significantly ICAM-1 and VCAM-1 adhesion molecules (73, 74). The lower graft rejection rates (56). The role of minor histo- importance of lymphatic vessels in allosensitization is under- compatibility Ags is more prominent in low-risk corneal scored by significantly higher APC trafficking and higher re- transplants, where resident corneal APCs express minimal jection rates seen in neovascularized or high-risk corneal graft levels of MHC class II, as described above. However, signif- beds that are lymphatic-rich compared with low-risk hosts (75, icantly increased expression of MHC class II and costimula- 76). Moreover, limiting the access of APCs to draining LNs via tory molecules (e.g., CD80, CD86, CD40) by graft APCs in ipsilateral cervical lymphadenectomy prior to transplantation inflamed host beds leads to activation of the direct pathway was shown to significantly prolong corneal allograft survival in of allosensitization, further underscoring the possible useful- murine models (77), providing proof of concept for the im- ness of MHC matching in the high-risk setting (57, 58). portance of lymphatics in facilitating allosensitization. Despite the preponderancy of data suggesting that MHC Allosensitization or priming of alloreactive T cells occurs via class II matching can enhance corneal allograft survival in two distinct pathways. The direct pathway of allosensitization high-risk grafts, it is likely that, given its high economic costs, involves presentation of donor Ags in the context of nonself- the debate regarding the merits of MHC matching in corneal MHC by donor-derived APCs or passenger leukocytes to transplantation will continue until large randomized clinical host naive T cells (58). However, in the indirect pathway, trials using modern DNA-based typing methods provide more host APCs recruited from the peripheral cornea (recipient definitive answers. bed) present donor Ags in association with self-MHC to naive The Journal of Immunology 3987

T cells in the draining LNs (58). Previously, it was believed demonstrated that 70% of MHC-mismatched grafts and 0% that the indirect pathway of allosensitization was the pre- of MHC-matched grafts undergo rejection, suggesting that dominant, if not exclusive, form of immune response in all Th1 cells are not the sole mediators of graft rejection (87, 88). corneal transplants (78). However, identification of diverse It has long been proposed that skewing the immune sys- populations of bone marrow–derived cells in the cornea that tem toward a Th2 alloimmune response promotes corneal allo- can acquire MHC class II expression under inflammatory graft survival. Yamada et al. (89) found that deviation of conditions and, thus, serve as functional APCs further the alloimmune response toward a Th2 phenotype promotes strengthened the functional role of direct alloreactivity in cor- corneal allograft survival in a murine model of orthotopic neal transplantation (65, 79). Furthermore, accumulating data corneal transplantation. However, recent studies demon- suggest that the relevance of direct or indirect pathways is strated higher rates of corneal allograft rejection in graft re- highly dependent on the graft bed microenvironment (58). In cipients with atopic (allergic) conjunctivitis, a disorder that is the noninflamed setting, in which there is minimal expression primarily mediated through a Th2 immune response (90, 91). of graft-derived MHC molecules, the indirect pathway re- Furthermore, it was demonstrated that corneal graft rejection mains predominant; however, in the high-risk setting, char- in IFN-g–knockout mice is characterized by eosinophilic acterized by graft bed inflammation and acquisition of high corneal infiltrates and is mediated by the Th2 pathway (87). levels of MHC and accessory molecules by graft-borne Thus, in the aggregate, although there is general consensus APCs, the direct pathway is highly functional (Fig. 2) (58, that Th1 cells are the principal effectors of acute corneal graft + 80, 81). It was demonstrated that the use of MHC class II– rejection, it is clear that depletion of CD4 T cells or IFN-g is Downloaded from deficient donor tissue results in considerably prolonged sur- unable to entirely suppress alloreactivity, thereby suggesting vival of high-risk, but not low-risk, corneal grafts (58), further the involvement of myriad effector pathways, including CD8+ underscoring the relevance of the direct pathway in high-risk T cells and Th2 cells. transplantation. Involvement of Th17 cells has been established in the pathogenesis of multiple autoimmune diseases, including Effector immune cells and mechanisms of graft destruction chronic ocular inflammatory conditions (92, 93). In transplant http://www.jimmunol.org/ immunobiology, Th17 cells have been implicated in the de- IFN-g–producing CD4+ Th1 cells are considered the pre- velopment of lung and renal allograft rejection and graft- dominant effector cells in corneal graft rejection (82, 83), but versus-host disease (94–96). Murine models of orthotopic the precise mechanisms by which Th1 cells mediate graft corneal transplantation revealed that the pathogenic role of rejection have not been fully elucidated. Although in vitro + Th17 cells is more evident at the very early stages of graft studies demonstrated that alloreactive CD4 T cells induce g2 apoptosis in corneal endothelial cells (84), in vivo application rejection, whereas IFN- producing Th1 cells are predom- of anti-FasL Ab or Fas-Fc protein does not inhibit CD4+ inantly involved in later stages and are critical for eventual T cell–mediated apoptosis of corneal cells (82). High ex- graft rejection (97). The prolymphangiogenic role of Th17 by guest on September 29, 2021 pression of IFN-g and IL-2 in undergoing rejection cells also was demonstrated in a murine model of autoim- further supports the central role of Th1 cells in corneal allo- mune ocular surface inflammatory disease (98), indicating graft rejection (85, 86). However, 33% of anti-CD4 Ab– that blocking the effect of IL-17 may favor corneal allograft 2/2 treated mice and 45% of CD4-knockout mice still reject their survival. However, ∼90% of corneal allografts in IL-17 corneal allografts, indicating that CD4-independent mecha- mice or wild-type mice treated with anti–IL-17 Ab are still nisms are also involved in graft rejection (83, 85). In addition, rejected (97, 99). Interestingly, IL-17 deficiency retards the studies on the rate of graft rejection in IFN-g–knockout mice development of alloimmune rejection in these hosts and

FIGURE 2. Migration of donor graft–derived APCs and activation of the direct pathway of allosensitization. (A) Ex vivo staining of corneal grafts with Hoechst vital dye tracks the egress of donor APCs posttransplantation. Stained isografts (BALB/c → BALB/c) demonstrate that ex vivo staining [blue (1)] is primarily restricted to the graft and not the host bed. In contrast, stained allografts (C57BL/6, IAb → BALB/c, IAd) evaluated at 24 h posttransplantation demonstrate that exiting donor cells in the host bed are largely CD45+ [red (2)] and express donor IAb [green (3)] (insets show digitally enlarged portions of host beds). Original magnification 360. (B) The frequencies of IFN-g–producing T cells 2 wk after transplantation were assessed using ELISPOT. In high-risk (HR) graft recipients, a significantly greater IFN-g response is generated in directly primed allospecific T cells compared with low-risk (LR) recipients and ungrafted controls (Naive), suggesting that the direct alloresponse is the predominant form of allosensitization in the high-risk graft setting. **p , 0.01. Reprinted with permission from Saban et al. (25) and Huq et al. (58). 3988 BRIEF REVIEWS: ALLOIMMUNITY AND TOLERANCE IN CORNEAL TRANSPLANTATION promotes the expression of Th2-type cytokines, IL-4, IL-5, SCID results in acute corneal allograft rejection (102); how- and IL-13 (97). These data, along with observations on cor- ever, the precise role of these cells as effector cells in mediating neal graft rejection in IFN-g–knockout mice, suggest that graft rejection is yet to be elucidated. elimination of Th1 and Th17 pathways results in a Th2-biased Allotolerance induction: contribution of Tregs to allograft survival immune response and that deviating the host’s immune response One of the primary goals in transplant immunobiology is toward a Th2 phenotype, contrary to previous dogma, may, in donor-specific tolerance induction, which eliminates the need fact, have a deleterious effect on corneal allograft survival (88, 97, for immunosuppressive therapies and promotes graft survival. 99). There is also compelling evidence that IL-17 promotes the Tolerogenic Tregs and DCs are potential candidates that can + + + generation of CD4 CD25 Foxp3 Tregs in corneal allografts be exploited for the induction of allotolerance in corneal and is required for Tregs to exert their immunosuppressive transplantation. Maturation-resistant tolerogenic DCs (tolDCs), function on effector CD4+ T cells (100). which express low levels of MHC class II and costimulatory Studies on the role of CD8+ or cytotoxic T cells in graft molecules, have been implicated in suppression of alloimmunity rejection yielded controversial results, as well. Although some and promotion of graft survival in multiple solid organ transplant murine studies suggested a role for donor-specific CD8+ models (106–108). Studies on murine models of penetrating T cells in high-risk grafts, other studies demonstrated that keratoplasty demonstrated that administration of donor-derived corneal allograft rejection occurs invariably in CD8+ T cell– tolDCs to hosts prior to transplantation increases the frequen- hi

deficient and perforin-deficient mice (101, 102). Overall re- cies of Foxp3 Tregs and significantly improves corneal allograft Downloaded from sults suggest that a CD8+ T cell response is not absolutely survival (109). Results of a recent study indicate that the bene- essential for corneal allograft rejection. Although priming of ficial effect of tolDCs on corneal graft survival is mediated CD8+ T cells occurs, in the absence of costimulatory signals through expansion of CTLA-4 expressing Tregs and downreg- from APCs, CD8+ T cells do not have the ability to reject the ulation of CD28+ Tregs (110). Given that tolDCs induce allo- graft (103). CD8+ suppressor T cells (CD8+ Tregs), which are tolerance primarily through Treg expansion, the majority of

generated during the induction of ACAID, were shown to studies focused on modification of Treg function to promote http://www.jimmunol.org/ suppress allospecific DTH responses via perforin- and FasL- graft survival. independent mechanisms, promoting corneal immune privi- A myriad of studies focused on the role of Tregs in allo- lege (104). Another subset of T cells, double-negative or tolerance induction and in vitro expansion of Tregs to promote 2 2 CD4 CD8 T cells, also was implicated in corneal graft allograft survival (111, 112). Compelling evidence suggests rejection through apoptosis of (102, that corneal allograft–induced donor-specific Tregs are capa- 2 2 105). Adoptive transfer of CD4 CD8 T cells to mice with ble of suppressing the DTH immune response and enhancing by guest on September 29, 2021

FIGURE 3. Treg function and their interactions with APCs in the draining LNs of corneal allograft acceptors versus allograft rejectors. (A) Mean fluorescent intensity and Western blot analysis of Foxp3 expression in CD4+CD25+Foxp3+ Tregs from draining LNs of allograft acceptors and rejectors 3 wk post- transplantation demonstrates significantly higher expression levels of Foxp3 in Tregs from graft acceptors compared with Tregs from grafts undergoing rejection. (B) Tregs isolated from the LNs of graft acceptors are significantly more potent in suppressing the proliferation of CD3-stimulated naive T cells compared with Tregs isolated from LNs of graft rejectors and syngeneic recipients. (C) Confocal photomicrographs of draining LNs show that only Tregs from graft acceptors colocalize with CD11c+ APCs (arrows). (D) Adoptive transfer of CCR7hi Tregs into corneal allograft recipients significantly improves corneal allograft survival. Allograft recipients that receive CCR7lo Tregs demonstrate no improvement in allograft survival. *p = 0.022. Reprinted with permission from Chauhan et al. (116, 118). The Journal of Immunology 3989 allograft survival (99, 100, 112, 113). Generation and ex- more susceptible than Nrp-1+ natural/thymic Tregs to the pansion of Tregs within the corneal allograft is mediated by effects of proinflammatory cytokines expressed in inflamed the glucocorticoid-induced TNFR family–related protein li- host beds, mediating their conversion into exTregs (R. Dana, gand, which is constitutively expressed in the cornea (114). unpublished observations). These data suggest that the patho- Studies revealed that Treg-mediated suppression of effec- logic conversion of Tregs and their impaired function con- tor T cells is primarily contact dependent and mediated by tribute to the loss of corneal immune privilege and allograft membrane-bound glucocorticoid-induced TNFR family–re- rejection. lated protein ligand and CTLA-4 molecules (100). Addi- tionally, IL-17 regulates the expression of Foxp3 and these Conclusions membrane-bound molecules on Tregs (100). As mentioned Corneal allograft rejection is a highly complex process that above, IL-17 is required for the generation of Tregs, and involves an elaborate interplay between cells of the innate and treatment with an anti–IL-17 Ab results in rejection of 90% adaptive immune systems and the lymphovascular system. Our of corneal allografts (100). Although nonocular allograft understanding of the immunology and pathophysiology of survival was previously associated with increased frequencies corneal allograft rejection has improved considerably in recent of Tregs within the graft or the draining LNs (115), a recent years, and many of the recent investigations focused on the study reported no difference in frequencies, but higher levels development of new therapies that could target the afferent of Foxp3 expression in the draining LN Tregs of accepted and efferent arms of immunity at a molecular level, without corneal allografts compared with allografts undergoing rejec- compromising the integrity of the . However, Downloaded from tion (Fig. 3A) (116). The suppressive function of Tregs has the redundancy of cellular and molecular pathways mediating been related to their expression of Foxp3 (Fig. 3B) (116, graft rejection has made this a daunting task. Evolving strat- 117). Moreover, it was demonstrated that Foxp3hi Tregs from egies for allotolerance induction, primarily – accepted grafts are more potent in suppressing naive T cell based therapies, are promising tools that could bring us closer proliferation and secreting IL-10 and TGF-b (116). Studies to safe therapeutic modalities for corneal graft rejection. on homing of Tregs to draining LNs of corneal allograft re- http://www.jimmunol.org/ cipients and their interaction with APCs (Fig. 3C) showed Disclosures that Tregs from allograft acceptors localize in the paracortical The authors have no financial conflicts of interest. region of draining LNs in close proximity with APCs and express higher levels of CCR7, whereas Tregs from graft re- References jectors express lower levels of CCR7 and are less in contact 1. Zirm, E. K. 1989. Eine erfolgreiche totale Keratoplastik (A successful total kera- with APCs; CCR7hi Tregs have a more significant inhibitory toplasty). 1906. Refract. Corneal Surg. 5: 258–261. 2. Dana, M. R., Y. Qian, and P. Hamrah. 2000. Twenty-five-year panorama of effect on T cell proliferation and secrete higher levels of im- corneal immunology: emerging concepts in the immunopathogenesis of microbial

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Differential expression of the complement regulatory proteins in the . 59. Zhu, S., I. Dekaris, G. Duncker, and M. R. Dana. 1999. Early expression of by guest on September 29, 2021 Invest. Ophthalmol. Vis. Sci. 34: 3579–3584. proinflammatory cytokines interleukin-1 and tumor necrosis factor-alpha after 33. Taylor, A. W., J. W. Streilein, and S. W. Cousins. 1994. Alpha-melanocyte- corneal transplantation. J. Interferon Res. 19: 661–669. stimulating hormone suppresses antigen-stimulated T cell production of gamma- 60. Yamagami, S., P. Hamrah, Q. Zhang, Y. Liu, S. Huq, and M. R. Dana. 2005. interferon. Neuroimmunomodulation 1: 188–194. Early ocular chemokine gene expression and leukocyte infiltration after high-risk 34. Taylor, A. W., J. W. Streilein, and S. W. Cousins. 1994. Immunoreactive vaso- corneal transplantation. Mol. Vis. 11: 632–640. active intestinal peptide contributes to the immunosuppressive activity of normal 61. Zhu, S. N., and M. R. Dana. 1999. 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