Corneal transplantation is one of the most prevalent and successful forms of solid tissue transplantation. Despite favorable outcomes, immune-mediated graft rejection remains the major cause of corneal allograft failure. Although low-risk graft recipients with uninflamed graft beds enjoy a success rate ∼90%, the rejection rates in inflamed graft beds or high-risk recipients often exceed 50%, despite maximal immune suppression. In this review, we discuss the critical facets of corneal alloimmunity, including immune and angiogenic privilege, mechanisms of allosensitization, cellular and molecular mediators of graft rejection, and allotolerance induction.

More than a century has passed since Eduard Zirm, an Austrian ophthalmologist, performed the first partially successful full-thickness human corneal transplant (1). Today, corneal transplantation is one of the most prevalent forms of solid tissue transplantation performed in the world (2). It is estimated that well over 100,000 corneal transplant surgeries are performed annually worldwide, with nearly 40,000 performed annually in the United States alone (3). Several trends are notable. First, the number of corneal grafts performed in the developing world, especially Asia, is increasing sharply as the result of enhanced eye banking procedures and distribution networks. Second, partial-thickness corneal transplants (lamellar keratoplasty) are increasingly being used in place of full-thickness transplants (penetrating keratoplasty) when the entire cornea does not need to be replaced; this trend has led to some decreased risk for graft rejection, likely because of the decreased load of allogeneic tissue (4, 5). Still, it is critical to emphasize that the most important prognosticator of graft success is the status of the recipient bed in which the corneal graft is placed. The 2-y graft survival for penetrating keratoplasty in nonvascularized and uninflamed host beds or low-risk corneal transplants is ∼90% (6). However, recipients with a history of graft rejection or grafts performed in inflamed and vascularized host beds are considered to be at high risk for rejection, with failure rates > 50%, despite maximal local and systemic immunosuppressive therapy (7); these outcomes are considerably worse than for kidney, heart, or liver transplants. Interestingly, the remarkable rate of success normally seen in low-risk corneal grafts, unlike other solid transplants, is achievable without the benefit of HLA matching or profound systemic immune suppression (7).

Despite these favorable outcomes, graft rejection remains the leading cause of corneal allograft failure (8). Corneal graft rejection can occur in any of the three cell layers of the cornea (epithelium, stroma, or endothelium), with endothelial rejection being the most prevalent sight-threatening form. This can be attributed to the fact that the endothelial cells of the cornea are irreplaceable and perform the critical function of preventing the tissue from getting swollen (9). Graft rejection is characterized clinically by graft edema (swelling) and inflammatory cells that can be seen circulating in the anterior chamber of the eye or attaching as keratic precipitates to the graft endothelial cells (Fig. 1A) (10). Several factors have been associated with a higher risk for graft failure, with the degree of host bed neovascularization being the most significant prognosticator for earlier and a more severe graft rejection (7, 11, 12).

FIGURE 1.

Immunobiology of corneal transplantation. (A) Clinical manifestation of corneal graft rejection. Infiltrating monocytes 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 representation of corneal alloimmunity. (I) Following transplant surgery, upregulation of proinflammatory cytokines, 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 inflammatory environment, egress from the cornea through lymphatic vessels to the draining LNs, where they present alloantigens to naive T cells (Th0). Newly formed lymphatic vessels may also contribute to resolution of inflammation by mediating clearance of inflammatory cells and debris. (III) Primed T cells undergo clonal expansion and differentiate primarily into IFN-γ–secreting CD4+ Th1 cells. Tregs modulate induction of alloimmune responses through inhibition of T cell activation or suppression of APC stimulatory potential. (IV) Alloreactive Th1 cells migrate through blood vessels and along a chemokine gradient toward the graft, where they mount a DTH response against the allogeneic tissue, resulting in graft opacification and failure.

FIGURE 1.

Immunobiology of corneal transplantation. (A) Clinical manifestation of corneal graft rejection. Infiltrating monocytes 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 representation of corneal alloimmunity. (I) Following transplant surgery, upregulation of proinflammatory cytokines, 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 inflammatory environment, egress from the cornea through lymphatic vessels to the draining LNs, where they present alloantigens to naive T cells (Th0). Newly formed lymphatic vessels may also contribute to resolution of inflammation by mediating clearance of inflammatory cells and debris. (III) Primed T cells undergo clonal expansion and differentiate primarily into IFN-γ–secreting CD4+ Th1 cells. Tregs modulate induction of alloimmune responses through inhibition of T cell activation or suppression of APC stimulatory potential. (IV) Alloreactive Th1 cells migrate through blood vessels and along a chemokine gradient toward the graft, where they mount a DTH response against the allogeneic tissue, resulting in graft opacification and failure.

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In this review, we discuss the factors involved in ocular inflammation, activation and migration of APCs, pathways of allosensitization and allotolerance, and mechanisms of graft destruction (Fig. 1B).

The term immune privilege was coined by Sir Peter Medawar in the late 1940s, when he recognized the extended survival of skin allografts placed in the anterior chamber of the eye (13). He attributed this unexpected graft survival to what he considered to be immunological ignorance, a passive process of sequestration of foreign Ags in the anterior chamber as the result of the absence of draining lymphatic vessels. However, later in the 1970s, Kaplan and Streilein (14) demonstrated that immune privilege was, in fact, the result of an actively maintained and deviant suppressive immune response to ocular Ags, a phenomenon that was later referred to as anterior chamber–associated immune deviation (ACAID) (15).

Immune privilege

ACAID is a form of immune tolerance to alloantigens placed in the anterior chamber of the eye that results in downregulation of Ag-specific delayed-type hypersensitivity (DTH) response while promoting humoral immunity and production of noncomplement-fixing Abs (16). This process is thought to be mediated by F4/80+APCs in the eye that capture intraocular Ags, enter the bloodstream, and migrate to the marginal zone of the spleen, where their interactions with CD4+ T cells, γδ T cells, B cells, and NKT cells result in the generation of two groups of Ag-specific regulatory T cells (Tregs): CD4+ CD25+ Tregs and CD8+ Tregs (17). Studies on the implication of ACAID in murine models of corneal transplantation demonstrated that ACAID induction through intraocular injection of allogeneic lymphocytes prior to penetrating keratoplasty causes a significant improvement in corneal allograft survival (18, 19).

In addition to immunological tolerance induced by ACAID, several mechanisms contribute to the maintenance of ocular immune privilege (Table I). The cornea expresses an array of membrane-bound immunomodulatory molecules that protect it from inflammation and promote immune quiescence. Programmed death ligand-1 (PDL-1) is a proapoptotic molecule that is constitutively expressed at high levels by the cornea (20, 21). PDL-1’s interaction with PD-1 on T cells results in inhibition of T cell proliferation, apoptosis induction, and inhibition of IFN-γ production, leading to prolonged allograft survival (20, 22). Recent studies established distinct mechanisms for donor- and host-derived PDL-1 in promoting corneal allograft survival. Depletion of PDL-1 in graft recipients results in considerably stronger indirect T cell priming and rapid graft rejection than in wild-type recipients (20). However, elimination of PDL-1 in the graft donor does not have a significant effect on indirect allorecognition, but it does result in an increased alloreactive T cell infiltration and graft failure (20). IL-1R antagonist (IL-1Ra), which is constitutively expressed by the cornea, promotes immune privilege through suppression of APC migration to the cornea (23). Topical administration of IL-1Ra was shown to promote allograft survival in low-risk and high-risk graft recipients in a murine model of corneal transplantation (24). Thrombospondin-1 (TSP-1) is an immunomodulatory glycoprotein expressed by the cornea and immune cells, such as APCs. Studies demonstrated significantly higher rates of graft rejection in TSP-1–null mouse corneal allografts compared with wild-type grafts (25). APC-derived TSP-1 inhibits maturation of APCs during inflammation, regulates their migration to draining lymph nodes (LNs), and suppresses their capacity in direct sensitization of alloreactive T cells (25). Corneal epithelial and endothelial cells constitutively express the proapoptotic molecule Fas ligand (FasL; CD95L). Interaction of FasL with Fas (CD95)-expressing inflammatory cells, such as neutrophils and effector T cells, results in their apoptotic death, thereby improving allograft survival (26). TRAIL (or Apo2L) is a transmembrane protein highly homologous with FasL that is constitutively expressed by corneal cells (27). TRAIL has been implicated in apoptosis of activated T cells and was found to promote the proliferation of Tregs (28). Although no study has directly associated TRAIL expression with corneal graft survival, transfection of mouse donor corneas with adenovirus carrying TRAIL gene significantly improved corneal allograft survival (29). In addition to these membrane-bound proteins, several soluble immunoregulatory factors are found in the aqueous humor of the eye, including TGF-β2, complement regulatory proteins, α-melanocyte stimulating hormone, vasoactive intestinal peptide, calcitonin gene-related peptide, somatostatin, macrophage migration inhibitory factor, and IDO, which are involved in tolerance induction in macrophages, regulation of dendritic cells (DCs), and inhibition of complement and NK cell–mediated cell lysis and T cell activation (3039).

Table I.
Factors involved in immune and angiogenic privilege of the cornea
FactorFunctionRef.
Angiostatin Inhibits vascular endothelial cell (VEC) proliferation (45
α-MSH Suppresses IFN-γ production by T cells, promotes Treg development (33, 38
CGRP Suppresses NO production by macrophages (35
CRP Inhibits activation of the complement system (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
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-γ production (21, 22
PEDF Suppresses VEGF expression (47
TGF-β Inhibits NK cell–mediated cytolysis of MHC class I cells, suppresses T cell activation (31
TRAIL Promotes T cell apoptosis and proliferation of Tregs (2729
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 nonsignaling receptor for VEGF-C and VEGF-D (41, 43
VIP Suppresses T cell activation and proliferation (34
FactorFunctionRef.
Angiostatin Inhibits vascular endothelial cell (VEC) proliferation (45
α-MSH Suppresses IFN-γ production by T cells, promotes Treg development (33, 38
CGRP Suppresses NO production by macrophages (35
CRP Inhibits activation of the complement system (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
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-γ production (21, 22
PEDF Suppresses VEGF expression (47
TGF-β Inhibits NK cell–mediated cytolysis of MHC class I cells, suppresses T cell activation (31
TRAIL Promotes T cell apoptosis and proliferation of Tregs (2729
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 nonsignaling receptor for VEGF-C and VEGF-D (41, 43
VIP Suppresses T cell activation and proliferation (34

α-MSH, α-melanocyte stimulating hormone; CGRP, calcitonin gene-related peptide; CRP, complement regulatory proteins; MIF, macrophage migration inhibitory factor; PEDF, pigment epithelium-derived factor; VIP, vasoactive intestinal peptide.

Angiogenic privilege

The cornea is devoid of vasculature and lymphatics, which limits the trafficking of immune cells between the cornea and the systemic circulation and lymphoid organs. This angiogenic privilege is actively maintained through the expression of several antiangiogenic factors. Corneal epithelial cells constitutively secrete soluble vascular endothelial growth factor receptor (VEGFR)-1, which binds VEGF-A and, thus, inhibits its mitogenic effect on vascular endothelial cells (40). Additionally, nonsignaling VEGFR-3, which is constitutively expressed by the corneal epithelium, demonstrates antiangiogenic activity by acting as a decoy receptor for VEGF-C and VEGF-D (41, 42), thereby suppressing blood and lymphatic vessel growth. Studies on a mouse model of corneal transplantation also demonstrated that blockade of VEGFR-3 or VEGFR-3–binding ligands, such as anti–VEGF-C, results in significant inhibition of lymphangiogenesis and, ultimately, prolonged corneal graft survival (43). Corneal epithelial cells additionally express soluble VEGFR-2, which inhibits VEGF-C and, thus, blocks lymphangiogenesis (44). Intracorneal administration of soluble VEGFR-2 was shown to double allograft survival in a mouse model of corneal transplantation (44). Other antiangiogenic factors that are expressed by the cornea include angiostatin, which inhibits vascular endothelial cell proliferation and migration (45); endostatin, which blocks the mitogenic activity of VEGF on vascular endothelial cells and promotes their apoptosis (46); pigment epithelium-derived factor expressed by corneal epithelium and endothelium that exerts its antiangiogenic effect through downregulation of VEGF expression (47); and TSP-1, which inhibits hemangiogenesis and lymphangiogenesis by induction of vascular endothelial cell apoptosis and CD36-mediated downregulation of VEGF-C (48). Results of a recent study showed that subconjunctival administration of endostatin to mice undergoing corneal transplantation inhibits graft neovascularization and T cell infiltration and significantly prolongs corneal graft survival (49). It also was demonstrated recently that PDL-1 has antiangiogenic functions (50). Suture-induced corneal inflammation in PDL-1–knockout mice results in a more significant angiogenic response and higher levels of VEGFR-2 expression compared with wild-type mice (50). This complex interplay between antiangiogenic and proangiogenic factors demonstrates the importance of regulation of hemangiogenesis and lymphangiogenesis in the maintenance of corneal immune privilege.

All corneal cells, including epithelial, stromal, and endothelial cells, express MHC class I Ags. Interestingly, in the healthy cornea, only DCs in the peripheral cornea express MHC class II, whereas DCs and other myeloid cell populations residing in the center of the cornea express no to minimal levels of MHC class II (51). In contrast to their kin in the skin, inflammatory stresses induce MHC class II expression by the significant majority of corneal DCs and corneal epithelial cells (30).

HLA (MHC) matching and systemic immunosuppression are the cornerstones of prophylactic strategies against rejection for solid organ transplants, such as the kidney; however, neither is routinely performed in corneal transplantation. A myriad of studies has been performed since the 1970s on the relative efficiency of HLA matching in corneal allograft survival. The results from these studies have been mixed, with some studies showing a clear benefit (52, 53). However, the largest prospective randomized study of HLA matching in corneal transplantation in the United States, the Collaborative Corneal Transplantation Studies (CCTS), showed no benefit for HLA-DR matching (7). Evidence regarding the effect of MHC class II/HLA-DR matching remains unclear. Many studies demonstrated prolonged survival rates in grafts with lower numbers of HLA-DR mismatches, especially in the high-risk setting (52, 54). This is in contrast with the CCTS, which reported no significant difference in graft rejection rates between cross-matched and non–HLA-matched groups (7). The failure of CCTS to demonstrate beneficial results for HLA matching on graft survival was attributed to various factors, including the possibility of incorrect typing (these studies were primarily done in the 1980s before the advent of DNA-based typing methods) and aggressive immunosuppressive therapy regimens that can nullify the beneficial effects of HLA matching, in part by suppressing MHC expression (55). However, the CCTS reported that matching for minor histocompatibility Ags, such as ABO group, may improve graft survival. In humans, donor–recipient matching of minor H-Y Ag was recently associated with significantly lower graft rejection rates (56). The role of minor histocompatibility Ags is more prominent in low-risk corneal transplants, where resident corneal APCs express minimal levels of MHC class II, as described above. However, significantly increased expression of MHC class II and costimulatory molecules (e.g., CD80, CD86, CD40) by graft APCs in inflamed host beds leads to activation of the direct pathway of allosensitization, further underscoring the possible usefulness of MHC matching in the high-risk setting (57, 58). Despite the preponderancy of data suggesting that MHC class II matching can enhance corneal allograft survival in high-risk grafts, it is likely that, given its high economic costs, the debate regarding the merits of MHC matching in corneal transplantation will continue until large randomized clinical trials using modern DNA-based typing methods provide more definitive answers.

Corneal graft rejection is a complex process during which changes in the corneal microenvironment and the interplay between cells of the innate and adaptive immune systems result in graft destruction. Early after transplantation, ocular surface inflammation leads to upregulation of proinflammatory cytokines, such as IL-1, IL-6, and TNF-α (59), chemokines including MIP-1α, MIP-1β, MIP-2, and RANTES (60), and overexpression of adhesion molecules, such as ICAM-1 and VLA-1 (6163). This inflammatory milieu results in the acquisition of high levels of MHC class II and costimulatory molecules, such as CD80 (B7-1), CD86 (B7-2), and CD40, by resident and infiltrating host APCs. Under these circumstances, donor corneal APCs, which normally lack the capacity to stimulate T cells, become more potent in alloantigen presentation and priming of naive T cells into Th1 effectors, the principal mediators of acute corneal graft rejection (see below for details of effector mechanisms) (10, 64, 65). Additionally, inflammation-induced expression of adhesion molecules and chemotactic gradient assist in mobilizing host APCs from the pericorneal limbal vasculature centripetally toward the corneal graft (64, 66).

The cornea is normally devoid of blood and lymphatic vessels; however, ocular inflammation leads to formation of blood and lymphatic neovessels. Inhibition of hemangiogenesis and lymphangiogenesis in high-risk recipient mice was shown to improve corneal allograft survival (43, 44, 67). The pathologic corneal lymphangiogenesis that occurs as a result of inflammation facilitates APC migration to draining LNs, where they prime alloreactive T cells (30). Expression of inflammatory cytokines, adhesion molecule ICAM-1, and CCL2 and CCL20 chemokines facilitate corneal infiltration of innate immune cells, which promote lymphangiogenesis through production of VEGF-C and VEGF-D (61, 6871). It was demonstrated that APCs, as a part of their maturation process, also acquire VEGFR-3 expression, which, in response to the chemotactic gradient of its principal receptor VEGF-C, mediates APC trafficking to lymphoid tissues (72). APC migration through afferent lymphatic vessels is also dependent on the interaction between the CCR7 receptor and its ligand CCL21, which is expressed by lymphatic endothelial cells during inflammation, and APC interactions with ICAM-1 and VCAM-1 adhesion molecules (73, 74). The importance of lymphatic vessels in allosensitization is underscored by significantly higher APC trafficking and higher rejection rates seen in neovascularized or high-risk corneal graft beds that are lymphatic-rich compared with low-risk hosts (75, 76). Moreover, limiting the access of APCs to draining LNs via ipsilateral cervical lymphadenectomy prior to transplantation was shown to significantly prolong corneal allograft survival in murine models (77), providing proof of concept for the importance of lymphatics in facilitating allosensitization.

Allosensitization or priming of alloreactive T cells occurs via two distinct pathways. The direct pathway of allosensitization involves presentation of donor Ags in the context of nonself-MHC by donor-derived APCs or passenger leukocytes to host naive T cells (58). However, in the indirect pathway, host APCs recruited from the peripheral cornea (recipient bed) present donor Ags in association with self-MHC to naive T cells in the draining LNs (58). Previously, it was believed that the indirect pathway of allosensitization was the predominant, if not exclusive, form of immune response in all corneal transplants (78). However, identification of diverse populations of bone marrow–derived cells in the cornea that can acquire MHC class II expression under inflammatory conditions and, thus, serve as functional APCs further strengthened the functional role of direct alloreactivity in corneal transplantation (65, 79). Furthermore, accumulating data suggest that the relevance of direct or indirect pathways is highly dependent on the graft bed microenvironment (58). In the noninflamed setting, in which there is minimal expression of graft-derived MHC molecules, the indirect pathway remains predominant; however, in the high-risk setting, characterized by graft bed inflammation and acquisition of high levels of MHC and accessory molecules by graft-borne APCs, the direct pathway is highly functional (Fig. 2) (58, 80, 81). It was demonstrated that the use of MHC class II–deficient donor tissue results in considerably prolonged survival of high-risk, but not low-risk, corneal grafts (58), further underscoring the relevance of the direct pathway in high-risk transplantation.

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 ×60. (B) The frequencies of IFN-γ–producing T cells 2 wk after transplantation were assessed using ELISPOT. In high-risk (HR) graft recipients, a significantly greater IFN-γ 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).

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 ×60. (B) The frequencies of IFN-γ–producing T cells 2 wk after transplantation were assessed using ELISPOT. In high-risk (HR) graft recipients, a significantly greater IFN-γ 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).

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IFN-γ–producing CD4+ Th1 cells are considered the predominant effector cells in corneal graft rejection (82, 83), but the precise mechanisms by which Th1 cells mediate graft rejection have not been fully elucidated. Although in vitro studies demonstrated that alloreactive CD4+ T cells induce apoptosis in corneal endothelial cells (84), in vivo application of anti-FasL Ab or Fas-Fc protein does not inhibit CD4+ T cell–mediated apoptosis of corneal cells (82). High expression of IFN-γ and IL-2 in corneas undergoing rejection further supports the central role of Th1 cells in corneal allograft rejection (85, 86). However, 33% of anti-CD4 Ab–treated mice and 45% of CD4-knockout mice still reject their corneal allografts, indicating that CD4-independent mechanisms are also involved in graft rejection (83, 85). In addition, studies on the rate of graft rejection in IFN-γ–knockout mice demonstrated that 70% of MHC-mismatched grafts and 0% of MHC-matched grafts undergo rejection, suggesting that Th1 cells are not the sole mediators of graft rejection (87, 88).

It has long been proposed that skewing the immune system toward a Th2 alloimmune response promotes corneal allograft survival. Yamada et al. (89) found that deviation of the alloimmune response toward a Th2 phenotype promotes corneal allograft survival in a murine model of orthotopic corneal transplantation. However, recent studies demonstrated higher rates of corneal allograft rejection in graft recipients with atopic (allergic) conjunctivitis, a disorder that is primarily mediated through a Th2 immune response (90, 91). Furthermore, it was demonstrated that corneal graft rejection in IFN-γ–knockout mice is characterized by eosinophilic corneal infiltrates and is mediated by the Th2 pathway (87). Thus, in the aggregate, although there is general consensus that Th1 cells are the principal effectors of acute corneal graft rejection, it is clear that depletion of CD4+ T cells or IFN-γ is unable to entirely suppress alloreactivity, thereby suggesting the involvement of myriad effector pathways, including CD8+ T cells and Th2 cells.

Involvement of Th17 cells has been established in the pathogenesis of multiple autoimmune diseases, including chronic ocular inflammatory conditions (92, 93). In transplant immunobiology, Th17 cells have been implicated in the development of lung and renal allograft rejection and graft-versus-host disease (9496). Murine models of orthotopic corneal transplantation revealed that the pathogenic role of Th17 cells is more evident at the very early stages of graft rejection, whereas IFN-γ−producing Th1 cells are predominantly involved in later stages and are critical for eventual graft rejection (97). The prolymphangiogenic role of Th17 cells also was demonstrated in a murine model of autoimmune ocular surface inflammatory disease (98), indicating that blocking the effect of IL-17 may favor corneal allograft survival. However, ∼90% of corneal allografts in IL-17−/− mice or wild-type mice treated with anti–IL-17 Ab are still rejected (97, 99). Interestingly, IL-17 deficiency retards the development of alloimmune rejection in these hosts and promotes the expression of Th2-type cytokines, IL-4, IL-5, and IL-13 (97). These data, along with observations on corneal graft rejection in IFN-γ–knockout mice, suggest that elimination of Th1 and Th17 pathways results in a Th2-biased immune response and that deviating the host’s immune response toward a Th2 phenotype, contrary to previous dogma, may, in fact, have a deleterious effect on corneal allograft survival (88, 97, 99). There is also compelling evidence that IL-17 promotes the generation of CD4+CD25+Foxp3+ Tregs in corneal allografts and is required for Tregs to exert their immunosuppressive function on effector CD4+ T cells (100).

Studies on the role of CD8+ or cytotoxic T cells in graft rejection yielded controversial results, as well. Although some murine studies suggested a role for donor-specific CD8+ T cells in high-risk grafts, other studies demonstrated that corneal allograft rejection occurs invariably in CD8+ T cell–deficient and perforin-deficient mice (101, 102). Overall results suggest that a CD8+ T cell response is not absolutely essential for corneal allograft rejection. Although priming of CD8+ T cells occurs, in the absence of costimulatory signals from APCs, CD8+ T cells do not have the ability to reject the graft (103). CD8+ suppressor T cells (CD8+ Tregs), which are generated during the induction of ACAID, were shown to suppress allospecific DTH responses via perforin- and FasL-independent mechanisms, promoting corneal immune privilege (104). Another subset of T cells, double-negative or CD4CD8 T cells, also was implicated in corneal graft rejection through apoptosis of corneal endothelium (102, 105). Adoptive transfer of CD4CD8 T cells to mice with SCID results in acute corneal allograft rejection (102); however, the precise role of these cells as effector cells in mediating graft rejection is yet to be elucidated.

One of the primary goals in transplant immunobiology is donor-specific tolerance induction, which eliminates the need for immunosuppressive therapies and promotes graft survival. Tolerogenic Tregs and DCs are potential candidates that can be exploited for the induction of allotolerance in corneal transplantation. Maturation-resistant tolerogenic DCs (tolDCs), which express low levels of MHC class II and costimulatory molecules, have been implicated in suppression of alloimmunity and promotion of graft survival in multiple solid organ transplant models (106108). Studies on murine models of penetrating keratoplasty demonstrated that administration of donor-derived tolDCs to hosts prior to transplantation increases the frequencies of Foxp3hi Tregs and significantly improves corneal allograft survival (109). Results of a recent study indicate that the beneficial effect of tolDCs on corneal graft survival is mediated through expansion of CTLA-4 expressing Tregs and downregulation of CD28+ Tregs (110). Given that tolDCs induce allotolerance primarily through Treg expansion, the majority of studies focused on modification of Treg function to promote graft survival.

A myriad of studies focused on the role of Tregs in allotolerance induction and in vitro expansion of Tregs to promote allograft survival (111, 112). Compelling evidence suggests that corneal allograft–induced donor-specific Tregs are capable of suppressing the DTH immune response and enhancing allograft survival (99, 100, 112, 113). Generation and expansion of Tregs within the corneal allograft is mediated by the glucocorticoid-induced TNFR family–related protein ligand, which is constitutively expressed in the cornea (114). Studies revealed that Treg-mediated suppression of effector T cells is primarily contact dependent and mediated by membrane-bound glucocorticoid-induced TNFR family–related protein ligand and CTLA-4 molecules (100). Additionally, IL-17 regulates the expression of Foxp3 and these membrane-bound molecules on Tregs (100). As mentioned above, IL-17 is required for the generation of Tregs, and treatment with an anti–IL-17 Ab results in rejection of 90% of corneal allografts (100). Although nonocular allograft survival was previously associated with increased frequencies of Tregs within the graft or the draining LNs (115), a recent study reported no difference in frequencies, but higher levels of Foxp3 expression in the draining LN Tregs of accepted corneal allografts compared with allografts undergoing rejection (Fig. 3A) (116). The suppressive function of Tregs has been related to their expression of Foxp3 (Fig. 3B) (116, 117). Moreover, it was demonstrated that Foxp3hi Tregs from accepted grafts are more potent in suppressing naive T cell proliferation and secreting IL-10 and TGF-β (116). Studies on homing of Tregs to draining LNs of corneal allograft recipients and their interaction with APCs (Fig. 3C) showed that Tregs from allograft acceptors localize in the paracortical region of draining LNs in close proximity with APCs and express higher levels of CCR7, whereas Tregs from graft rejectors express lower levels of CCR7 and are less in contact with APCs; CCR7hi Tregs have a more significant inhibitory effect on T cell proliferation and secrete higher levels of immunosuppressive cytokines; and in vitro stimulation of naive Tregs with CCL21 upregulates their CCR7 expression, improves Tregs’ homing ability to draining LNs, and significantly enhances corneal allograft survival (Fig. 3D) (118). These data cumulatively suggest that Tregs become dysfunctional in grafts undergoing rejection. A recent study from our laboratory showed that Treg dysfunction can be prevented by systemic administration of low-dose IL-2. Our data demonstrated that systemic treatment of high-risk recipient mice with low-dose IL-2 results in expansion and improved suppressive function of Tregs, reduced leukocyte infiltration of graft, and significantly improved corneal allograft survival (119).

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 posttransplantation 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).

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 posttransplantation 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).

Close modal

Instability of Tregs or Treg plasticity in the inflammatory environment is a new concept that has recently gained attention. Recent data suggest that, in the inflamed setting, a significant number of Tregs exhibit unstable expression of Foxp3; these ex-Tregs acquire an effector memory T cell phenotype and produce IFN-γ, which may further contribute to the development of autoimmunity (120). Recently, our laboratory used a model of corneal transplantation in Foxp3-lineage reporter–transgenic mice to evaluate the pathologic conversion of Tregs in immune privilege–disrupted (high-risk) hosts. The results suggested that ocular inflammation results in the conversion of Tregs into IFN-γ−producing ex-Tregs. Neuropilin-1 (Nrp-1), a membrane-bound coreceptor that is selectively expressed on natural/thymic Tregs and not on peripherally induced Tregs, was used to further determine the lineage of these exTregs (121). Our results demonstrated that Nrp-1 peripherally induced Tregs are more susceptible than Nrp-1+ natural/thymic Tregs to the effects of proinflammatory cytokines expressed in inflamed host beds, mediating their conversion into exTregs (R. Dana, unpublished observations). These data suggest that the pathologic conversion of Tregs and their impaired function contribute to the loss of corneal immune privilege and allograft rejection.

Corneal allograft rejection is a highly complex process that involves an elaborate interplay between cells of the innate and adaptive immune systems and the lymphovascular system. Our understanding of the immunology and pathophysiology of corneal allograft rejection has improved considerably in recent years, and many of the recent investigations focused on the development of new therapies that could target the afferent and efferent arms of immunity at a molecular level, without compromising the integrity of the immune system. However, the redundancy of cellular and molecular pathways mediating graft rejection has made this a daunting task. Evolving strategies for allotolerance induction, primarily regulatory T cell–based therapies, are promising tools that could bring us closer to safe therapeutic modalities for corneal graft rejection.

This work was supported in part by National Institutes of Health Grants EY24602 (to S.K.C.) and EY12963 (to R.D.).

Abbreviations used in this article:

ACAID

anterior chamber–associated immune deviation

CCTS

Collaborative Corneal Transplantation Studies

DC

dendritic cell

DTH

delayed-type hypersensitivity

FasL

Fas ligand

IL-1Ra

IL-1R antagonist

LN

lymph node

Nrp-1

neuropilin-1

PDL-1

programmed death ligand-1

tolDC

tolerogenic DC

Treg

regulatory T cell

TSP

thrombospondin-1

VEGF

vascular endothelial growth factor

VEGFR

vascular endothelial growth factor receptor.

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The authors have no financial conflicts of interest.