Corneal immune privilege
The concept of corneal immune privilege is entertained by observations of high survival rates of allogeneic corneal grafts despite HLA mismatching between donor and host tissues. It is the uniquely configured corneal anatomy and physiology of the anterior chamber that evade a host immune response through low immunogenicity and generation of alloantigen tolerance [18,19,43]. The cornea is a uniquely avascular tissue and free of lymphatics, preventing direct access of the immune system to the cornea through lack of vasculature, and barring free transport of antigens and APCs to T cell-rich secondary lymphoid organs through absence of lymph vessels. Further, all layers of the cornea have low constitutive expression of MHC-I and –II antigens, limiting immunogenicity to foreign antigens. Even though DCs are present both in the central and peripheral cornea, they exist in an immature, inactivated state, maintaining immune quiescence in a healthy cornea. The cornea expresses many cell membrane-bound molecules that guard the cornea from immune-mediated inflammation and induce apoptosis of immune effector cells. These molecules include complement regulatory proteins (CRP), Fas ligand (FasL), MHC-Ib and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). FasL (CD95L), a pro-apoptotic molecule, is expressed by the corneal epithelium and endothelium. FasL serves to destroy polymorphonuclear neutrophils (PMNs) and effector T cells that express its receptor Fas/CD95, promoting immune quiescence while protecting against immune-mediated graft rejection [44,45]. The corneal epithelium, stroma and cells of the ciliary body also express programmed death ligand-1 (PD-L1), which upon interaction with its cognate receptor (PD-1) on T cells leads to inhibition of T cell proliferative capacity, induction of apoptosis and suppression of IFN-γ secretion [46], promoting graft survival [47,48]. Expression of PD-1 by T cells is regulated by Notch signaling [49]. PD-1 inhibits T cell proliferation though suppression of Ras and Akt signaling pathways which inhibit transcription of SKP2 leading to upregulation of transforming growth factor-beta (TGF-β)-specific transcription factor Smad3, resulting in cell cycle arrest of T cells [50].
The anterior chamber is rich in soluble immunosuppressive factors such as TGF-β, alpha-melanocyte stimulating hormone (α-MSH), calcitonin gene-related peptide (CGRP), CRP, somatostatin (SOM), indoleamine dioxygenase (IDO), vasointestinal peptide (VIP) and macrophage migration inhibitory factor (MIF), which inhibit T cell and complement activation [18,51]. The most notable contribution is that of anterior chamber-associated immune deviation (ACAID), an alloantigen-specific peripheral immune tolerance to antigens in the anterior chamber, capable of deviating the systemic cytotoxic immune response [52,53]. ACAID suppresses delayed-type hypersensitivity (DTH) response and maintains humoral immunity, promoting graft survival [54,55]. Antigens within the anterior chamber are recognized and processed by F4/80+ APCs that orchestrate allotolerance by upregulating the expression of TGF-β with downregulation of the co-stimulatory molecule CD40/CD40L and interleukin-12 (IL-12) [53,56,57]. Suppression of DTH is brought about by migration of these APCs to the spleen through vascular elements in the trabecular meshwork, and together with splenic accessory immune cells, alloantigen-specific tolerance is achieved [58–60]. While the effect of ACAID on DTH is unequivocal, its impact on the regulation of cytotoxic T lymphocytes (CTL) is more complex, being dictated by the nature of the antigens present in the anterior chamber. Some groups have demonstrated that CTL function remains intact through expression of CTL precursors and effectors in the spleen and lymph nodes of animals inoculated with tumor cells in vitro [60,61], making the hypothesis of a suppressed CTL response an unlikely explanation for tumor growth in ACAID. In contrast, other groups that used a soluble antigen for intracameral inoculation observed inhibition of antigen-specific CD8+ T cell responses, confirming the antigen-dependent effect of ACAID on CTL [62–66]. Since CTL responses contribute to allogeneic corneal graft rejection even though they are not known to be directed against MHC alloantigens [67–70], the effect of ACAID on CTL function against MHC antigens and the involvement of FoxP3+ regulatory T cells (Treg) in modulating CD8+ T cell function during ACAID have been explored [71]. Both CD4+ T and CD8+ T cell populations in the spleen proliferate upon MHC alloantigen-specific ACAID induction, however, once ACAID is expressed, the percentages of these T cells decrease substantially, suggesting ACAID-mediated inhibition of both CD4+ T and CD8+ T cell function. Therefore, we now know that solubility of the antigen is not a necessary determinant of ACAID-mediated CTL immune suppression. Therefore measures to promote ACAID-mediated inhibition of DTH and CTL could prove beneficial in prolonging graft survival. Interestingly, while FoxP3+ regulatory T cells (Treg) increase upon ACAID induction, they have not been shown to be directly involved in the modulation of ACAID-mediated MHC alloantigen-specific T cell function and response [71].
Immunology of Corneal Graft Rejection
Corneal graft rejection occurs when the host immune response is directed toward antigens in the donor corneal button, leading to tissue destruction brought about by cells and mediators of the innate and adaptive immune responses. An immune response may target any of the main layers of the cornea selectively, or, in combination. Compromise of the corneal epithelium and stroma may be reversible, but, rejection of the endothelium invariably results in irreversible endothelial cells loss and may result in permanent graft failure, if not treated judiciously [72]. Sensitization of the host to donor antigens forms the “afferent†arm, also known as the induction phase of corneal allograft rejection. This allorecognition process is orchestrated by APCs presenting donor antigens to naïve T cells in draining lymph nodes in either a direct or indirect fashion [18]. The direct pathway constitutes presentation of donor antigens to naïve T cells directly by donor APCs through non-self MHC-II recognition on their surface, resulting in proliferation of direct alloreactive T effector cells [19]. In contrast, the indirect pathway yields donor antigens to host APCs that travel the cornea, capture donor antigens, and transport them to draining lymph nodes where antigen presentation occurs through recognition of self MHC-II by naïve T cells [19]. While initially believed to be a phenomenon brought about exclusively by the indirect pathway [67], accumulated evidence indicates that both the direct and indirect pathways are implicated in the immune-mediated rejection of orthotopic corneal allografts, especially in high-risk corneal beds with higher immunogenicity and compromised immune privilege [73–78]. The cornea harbors resident populations of the most potent bone marrow-derived epithelial and stromal APCs [22,79], namely, DCs, which are pivotal to the modulation of corneal immunogenicity [80]. These resident DCs are uniformly immature and MHC-II low/negative in the corneal center, but with a change in the microenvironment of the cornea from a quiescent to an inflammatory state, as in corneal transplantation, they express MHC-II and other co-stimulatory molecules, as well as increase in density [22,79,81,82]. More recently additional subpopulations of corneal DCs have been identified, adding to the complexity of the corneal immune system [83–86]. Once DCs undergo maturation, they express co-stimulatory molecules such as CD80, CD86 and CD40 [81], as well as differential adhesion molecules, that activate T cell receptors and induce T cell proliferation through concurrent release of cytokines. Among such cytokines are IL-1, -6 and -12 released by the APCs [80].
Lymph nodes serve as the priming hub for T cell allosensitization and activation, which then drives the subsequent “efferent†arm, or the expression phase, of immune-mediated graft rejection. It is this phase that results in the actual destruction of the graft, making lymph nodes profoundly critical to the process of rejection [87]. In support of the importance of draining lymph nodes in the rejection process, several murine studies have demonstrated that cervical lymphadenectomy prior to orthotopic corneal transplantation yields near complete graft acceptance along with suppressed allospecific DTH response, regardless of the pre-operative risk [77,88]. Following sensitization and activation of naïve T cells, cytokines and chemokines released induce proliferation and trafficking of these alloreactive T cells to the cornea through expression of specific combinations of adhesion molecules [89]. Chemokines (chemotactic cytokines), are small-molecule-weight cytokines that modulate recruitment of leukocytes and immune cells to the inflamed cornea [80,89]. Immune-mediated damage to the graft begins with the release of cytokines, such as tumor necrosis factor-alpha (TNF-α) and IL-1, secondary to the mechanical trauma of surgery. In the setting of high-risk corneal transplantation, cytokines further induce the production of various early chemokines. Overexpression of chemokines monocyte chemotactic protein-1 (MCP-1), chemokine C-C motif ligand 2 (CCL2), regulated on activation normal T cell expressed and secreted (RANTES; CCL5), macrophage inflammatory protein (MIP), MIP-1α (CCL3) and MIP-1β (CCL4) in acute graft rejection leads to additional recruitment of APCs and T cells into the cornea [18,90–92].
Once the graft and infiltrating leukocytes release late chemokines, guidance of alloreactive T cells towards the graft begins [19,93]. Alloreactive T cells then migrate to the cornea where they recognize donor MHC antigens, and also induce the development of memory T cells so that an immune response may be mounted against the same antigens upon re-exposure as in the case of a re-graft [19]. The primary cellular mediators of graft rejection are CD8+ CTL, and CD4+ T-helper (Th) lymphocytes, otherwise known as DTH cells. Even though the role of CTL in corneal graft rejection remains somewhat controversial, they are believed to be sufficient but not necessary for corneal graft rejection [69,94]. Based on the types of cytokines secreted by Th lymphocytes, T cells can be further classified into Th1, Th2 and the more recently discovered Th17 cells [95,96]. Th1 cells are largely considered to be the primary effector cells in corneal graft rejection [19,97]. CD4+ Th1 cells secrete IL-2, IFN-gamma and lymphotoxin, which lead to inflammation as an attack on the inciting antigen. IL-2 is critical to a sustained immune response by its positive feedback on T and B cell activation and proliferation. IFN-γ ensures that macrophages are activated at the site of inflammation, and facilitates further expression of MHC-II antigens in the donor button. Th17 cells on the other hand secrete IL-17, IL-21 and IL-22 [98]. TGF-β is the key differentiation factor for Th17 cells, which acts in concert with IL-6 or IL-21 and IL-23 serves as a stabilizing factor for maintaining the Th17 lineage [99–103]. IL-1 signaling also comes into play via IL-1β-mediated regulation of the dendritic cell-mediated Th17 cell differentiation pathways and maintenance of cytokine expression in Th17 cells [104]. Interestingly, TGF-β is also an inducer of CD4+CD25+Foxp3+ Tregs [105]. Therefore, generation of Th17 cells or CD4+CD25+Foxp3+ Tregs is largely dictated by the cytokine milieu of the tissue microenvironment [99,106,107]. Murine studies demonstrate increased expression of Th17 cells in the early stages of corneal allograft rejection followed by predominance of a Th1 response in the late stage [108]. However, Th17 do not orchestrate the corneal immune response in graft rejection independently. The role of IL-17 in graft rejection is somewhat limited. While some studies using monoclonal antibodies against IL-17 successfully demonstrate a moderate increase in murine corneal allograft survival [109], IL-17 knockout studies in mice failed to show improved graft survival [108]. This has been postulated to be as a result of an emerging Th2 response, which then mediates graft rejection [109,110]. However, there still remains controversy regarding clearly defined pathways given the complex interplay of immune cells in corneal allograft rejection. Murine IFN-γ and IL-17 knockout studies have shown that even MHC-matched corneal allografts are rejected in an IFN-γ- and IL-17-independent manner, suggesting mediators other than the simplistic model of Th1, Th2 or Th17 pathways [111].
