Rethinking immunological privilege: implications for corneal and limbal stem cell transplantation

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Rethinking immunological privilege: implications for corneal and limbal stem cell transplantation Immunological privilege operates within the normal eye by multiple passive and active mechanisms, including antigen sequestration, maintenance of an immunosuppressive local environment and induction of apoptotic death in infiltrating cells of the immune system. Ocular privilege might have developed to protect the eye from the collateral damage associated with an inflammatory response to invading pathogens. Nevertheless, corneal grafts do undergo irreversible immunological rejection and, furthermore, corneal graft rejection is very similar at a histological level to the rejection processes that operate in vascularized organ grafts. Ocular privilege is thus relative. The question arises as to how corneal grafts are rejected in the face of so many mechanisms designed to prevent immune responses from operating inside the eye - a question that is still essentially unanswered. IT is with the eye that this brief review is concerned. The structure of the normal mammalian eye is shown in Fig. 1. It is clear that, for an image to be formed on the retina, light must first pass unimpeded through various transparent ocular media (cornea, .aqueous humour, lens and vitreous humour). Loss of transparency or abnormal shape of an ocular structure anywhere along the path of incident light will result in loss of vision. The cornea is the transparent, refracting surface at the front of the eye (Fig. 1). The bulk of the cornea, the corneal stroma, comprises a regular array of collagen fibrils interspersed with fibroblastic-like keratocytes together with occasional macrophages and interstitial dendritic cells. The anterior surface is bounded by a nonkeratinized epithelium and the posterior surface by a monolayer of metabolically active corneal endothelial cells, which continuously pump water from the stroma into the anterior chamber t. Human corneal endothelial cells have almost no capacity for regeneration and, because corneal transparency depends upon the corneal stroma being maintained in a relatively dehydrated state, it follows that any damage to the endothelial monolayer resulting in the loss of the cells' pump function will lead to loss of corneal transparency. Major histocompatibility complex antigens are expressed to at least some extent on all cells of the cornea, and expression can be upregulated by pro-inflammatory cytokines2.

Corneal transplantation in the treatment of corneal disease Corneal transplantation is a surgical procedure that can restore sight to an eye blinded from corneal opacity arising from infection, accidental trauma, or inherited or acquired disease 2. Active research is being carried out into the development of an artificial cornea, but no such device is currently available for clinical use. In consequence, corneal transplantation depends upon the availability of healthy m

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cal locations, including the brain and the anterior chamber of the eye5. Rabbit orthotopic skin allografts underwent rapid rejecRetina Conjunctiva tion, but similar grafts placed in the anterior chamber of the rabbit eye survived for Iris Choroid lengthy periods, even if they were grafted into specifically presensitized animals, provided that the grafts did not become vascu- Cornea Sclera larized. Locations such as the brain and anterior chamber of the eye that apparently Optic support long-term allograft survival are reLens nerve ferred to as 'immunologically privileged sites '6. Barker and Billingham, in an exhausAnterior c h a m b e r tive and classic early review of the literature 7, (contains aqueous noted that many reports of immunological humour) privilege existed before 1948, and they described an extraordina[y number of priviLimbus leged locations, including the central nervous Vitreous system, the testis, the eye and subcutaneous humour Sclera fat pads (Box 1). Immunological privilege can be defined as a state in which, unexpectedly, an immune Figure 1. Structure of the normal eye. The anterior segment includes cornea, conjunctiva, aqueoushumour-~lled anterior chamber and iris. The posterior segment includes vitreous humour, retina and response does not appear to o c c u r 7. The term choroid,The limbus is the junctional area betweenthe cornea and the sclerawith adjoiningconjunctiva. is usually used in the context of specific tissue sites in which a histoincompatible tumour, allograft or xenograft enjoys unexpectcorneal tissue from cadaveric human donors. Within the constraints edly long, or even indefinite, survival. It can also be used to describe imposed by availability of human tissue, corneal transplantation is a a situation in which the immune response to an invading pathogen common procedure, with approximately 30 000-40 000 grafts in the (e.g. a virus) is markedly attenuated. USA, 1500 grafts in the UK and 1000-1500 grafts in Australia being performed each year3. In developed countries, common indications Immune privilege and the eye for corneal transplantation include keratoconus, bullous keratopathy, The cornea and anterior segment are accepted to be privileged sites, corneal scarring, any one of a number of corneal dystrophies and (1) because grafts of tissue into the anterior chamber can enjoy procorneal infection. In the developing world, the indications are similar longed survival5, and (2) because corneal allografts placed into norbut the sequelae of inflammatory eye diseases are relatively more im- mal, avascular corneal beds in experimental animals (such as the portant. Accurate figures for the prevalence of blindness on a global rabbit) survive indefinitely in unsensitized recipients s. Corneal neoscale are difficult to obtain, but the World Health Organization has vascularization is a frequent complication of inflammatory eye disestimated that at least 38 million people are blind 4, of whom perhaps ease. As a consequence, corneal allografts in humans are placed into one-quarter have corneal opacities. A combination of a chronic short- completely avascular beds only in certain well-defined situations age of human corneas for transplantation, coupled with the fact that for example, in most cases of corneal transplantation for keratoconus. not all corneal grafts are successful, means that corneal transplan- Under such circumstances, the survival of human corneal allografts is excellent, although, as will be described later in this review, the situtation has not yet reached its potential as a cure for blindness. ation with respect to corneas placed in vascularized beds is rather different. Immune privilege also appears to operate in the posterior Immunological privilege In 1948, because of his interest in the immune response to trans- segment of the eye because allografts placed in the subretinal space planted tissue, Peter Medawar transplanted skin to various anatomi- or vitreous cavity show prolonged survival 9. Why should the eye enjoy immunological privilege at all? One of the major roles of the immune response is to protect the host against microbial pathogens and, to this end, engagement of cells of the immune system with foreign antigen will often lead to a full-blown specific inflammatory response designed to clear the invading microor• Brain ganism. One of the mechanisms used by the irrtmune system to clear • Testis pathogens is the delayed hypersensitivity response, a cell-mediated s humour) inflammatory response controlled by sensitized, antigen-specific T cells, which then recruit other cell types to the lesion. Another mecha@ nism is the cytotoxic T-cell response in which antigen-specific, cytoIll toxic T cells kill virus-infected host cells. Delayed-type hypersensitivity responses, in particular, can often cause substantial bystander damage to surrounding tissues, but even a cytotoxic T-cell response

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directed, for example, against a specific virus will necessarily kill the infected host cell as an integral part of the mechanism for clearing the virus. The successful functioning of the mammalian eye as an organ of vision depends primarily on the maintenance of transparency to light. An inflammatory response within the eye might harm rather than protect the host. This is because any collateral damage associated with a specific immune response within the confines of a small and fragile organ might result in clearance of the pathogen at the expense of permanent tissue destruction, loss of transparency and consequent blindness TM. Immune privilege might, therefore, have evolved as a compromise between maintenance of ocular function and protection against infectious disease.

Mechanisms for the maintenance of privilege in the eye Some of the mechanisms that have been proposed for the maintenance of immunological privilege are listed in Box 2 and are shown schematically in Fig. 2. In essentially 'passive' mechanisms such as immunological ignorance, either the afferent arm of the immune response is not triggered because of physical sequestration of antigen or the afferent arm is triggered centrally but the effector arm is ineffective because of lack of access to the target tissue. Various 'active' mechanisms also operate, and these generally involve some degree of active suppression of the immune response, such as immune deviation or induction of apoptosis in infiltrating cells.

blockade. It is often assumed that the eye is immunologically privileged because it lacks lymphatic drainage within the: orbit. However, the human conjunctiva has a rich lymphatic drainage system H and there is some evidence that antigen introduced into the anterior chamber can, in fact, reach the ipsilateral cervical lymph nodes I2. The anterior chamber is probably drained by multiple routes because antigen introduced into the anterior chamber of experimental animals can drain directly into the venous circulation and track to the spleen 13. The cornea enjoys immunological privilege because it is normally Passive mechanisms of priviIege Ocular privilege, at least in a normal eye, might simply reflect a com- avascular, thereby preventing immune cells from gaining access to bination of the blood--eye barrier (analogous to the blood-brain bar- foreign antigen. However, the situation with inflamed or vascularized rier) and the failure of antigen to reach a draining lymph node. corneas is different. Intracorneal injection of either Indian ink into Certainly, presensitization of the host to donor antigens can break im- the prevascularized rabbit cornea I4 or iodinated albumin into the premunological privilege in some circumstances, a finding that indicates vascularized rat cornea 15 leads to the accumulation of antigen in the that efferent immune mechanisms can operate normally within the ipsilateral regional lymph nodes. Labelled material can diffuse eye s, and suggests that immunological privilege involves an afferent through the limbus into the conjunctival drainage, but such accumulation does not occur after injection of antigen into the awtscular cornea. Vascularization of allografts placed in the anterior chamber leads to rejection in preBlood-eye barrier sensitized rabbits 5, indicating that the presence of a blood supply might allow the ingress of immune effector cells and High levels of TGF-13 molecules into a hitherto inaccessible site, thereby overcoming immunological privilege. Immunological surveillance and lymphatic or vascular access are inNormal cornea is extricably linked in the eye. avascular

Active mechanisms of Frivilege Introduced antigen drains to spleen

Constitutive expression of CD95L

Figure 2. The site of action of activeand passivemechanismsof immunologicalprivilegein the eye.

Immune deviation is a term that is used under two sets of circumstances. (1) It is often employed to describe the skewing of a T-cell population towards the production of a set of immunomodulatory cytokines that favour antibody production 16 a so-called Th2 response. (2) It is also used in a more specific sense - in the context of the immunological tolerance that can follow oral administration of antigen. In this case, immune deviation l

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Glossary AIIograft - Transplant between genetically dissimilar members of the same species,

Intracemeral - Into the eye.

Apeptosis - Physiological or programmed cell death, characterized by rapid cleavage of genomic DNA into 180 bp fragments and multiples thereof, corresponding to internucleosomal spacing.

eye.

CD4* and CD8 ÷ T cells - Phenotypically distinct populations of mature,

peripheral T cells bearing either the CD4 or CD8 cell surface antigen. CD4* cells function primarily, but not exclusively, as helper cells and control delayed-type hypersensitivity responses; CD8* cells function pdmarily, but not exclusively, as cytotoxic T cells. Contralateral - On the opposite side to; in context, the other eye. Delayed-type hypersensitivity response - A cell-mediated inflammatory response that typically takes some time to develop. The response is controlled by sensitized, antigen-specific T cells, which recruit other cell types to the inflammatory lesion. DNA microsatellites - Highly polymorphic genetic loci, widely distrib-

uted in the human genome. DNA dinucleotide repeats or 'microsatellites' are readily elaborated by PCR, even from DNA extracted from small numbers of cells. A sub-set of such loci quickly becomes individual-specific and their detection has application in forensic settings. Fas -Also known as APO-1 or CD95, a cell-membrane-expressed mol-

ecule, ligation of which by its specific receptor, Fas ligand (CD95L), usually transduces an apoptotic signal to the Fas-bearing cell. Impression cytology- A method of collecting cells from the surface of a tissue by blotting a suitable support (e.g. a piece of filter paper)

against the surface.

describes the induction of a population of regulatory Th3 cells that mediate direct suppression of immune responsiveness through secretion of the immunomodulatory cytokine transforming growth factorbeta (TGF-{3)17. The elegant experiments of Streilein and co-workers over many years 1°,13,z8have demonstrated that an interesting form of immune deviation operates in the eye. The acronym ACAID - anterior chamber associated immune deviation - has been coined to describe a situation in which intracameral injection of antigen not only fails to elicit a strong local cell-mediated immune response to itself, but also induces partial systemic unresponsiveness to antigen ~3. Humoral responses are unaffected: ACAID represents a skewing of the immune response towards a Th2-type immune response, perhaps through aberrant processing of antigen. An intact spleen 13 and localized high levels of TGF-[3 (Ref. 19)in the aqueous humour appear to be necessary for induction of ACAID. The aqueous humour is plainly an immunosuppressive milieu because, apart from relatively high concentrations of TGF-[3, it also contains a variety of other potentially immunomodulatory molecules such as vasoactive intestinal peptide and et-melanocyte stimulating hormone 18. A third possible mechanism of privilege relates to the induction of apoptosis in leukocytes infiltrating the eye. Apoptosis is a morphologically distinct, highly regulated form of cell death 2° that normally occurs in the absence of overt inflammation. Ligation of Fas (CD95) n

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Ipeilateral - On the same side as; in context, the index eye or same Keratoconus - Corneal thinning, resulting in a conical rather than a

hemispherical corneal shape. Llmbal stem cell - Specialized corneal epithelial stem cell, believed to

be located at the limbus. Limbus - Junctional area between sciera and cornea. Orthotopic graft - Graft placed in the anatomical location appropriate for the tissue or organ being transplanted. A heterotopic graft, in contrast, is placed in an anatomically inappropriate location. Thl ceils - T cells defined functionally by production of pro-inflamma-

tory cytokines and active potentiation of cell-mediated immune responses. Th2 cells - T cells defined functionally by production of immunoregula-

tory cytokines and active potentiation of humoral immune responses. Th3 cells - T cells defined functionally by the secretion of the im-

munomodulatory cytokine TGF-~ and active suppression of Thl-type immune responses. Transforming growth factor 13(TGF-13)-An immunomodulatory cyto-

kine that inhibits T- and B-cell proliferation, downregulates natural killer cell activity and regulates macrophage function amongst other functions. Xenotransplentation - Transplantation across a species barrier.

by Fas ligand leads to apoptotic death of the Fas-bearing cell. The eye2~and the testis 22 are reputedly the only organs in the body that constitutively express Fas ligand. In a murine model of ocular herpes simplex virus type I infection, expression of Fas ligand on epithelial and endothelial cells of the cornea, iris, ciliary body and retina was associated with the apoptotic death of infiltrating T cells. This occurred within 24 h of infection and was associated with little bystander damage to ocular tissues 21. In mutant g/d mice (mice lacking CD95L), the viral infection was more invasive, caused more tissue damage, and did not induce T-cell apoptosis when compared with infection in wild-type control mice 21. The outcome of corneal allografts, limbal stem.cell allografts and xenografts How effective is immunological privilege of the eye in preventing unwanted inflammatory-type immune responses? A partial answer can be found by a closer examination of the outcome of various transplants of ocular tissue into the anterior segment of the eye.

Outcome of corneal transplantation In many large series, Kaplan-Meier survival of full-thickness corneal grafts has been reported to be of the order of 90% at 12 months -'3,24. However, ten-year survival falls to 60%. In almost all series, irreversible rejection is the most common cause of failure23,24, accounting

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for 10-40% of all failures, and this incidence of rejection occurs despite universal prophylaxis with topical glucocorticosteroids 25. Corneal graft survival differs from vascularized organ graft survival in that graft attrition tends to be much slower in the former and can occur late after surgery23. In contrast, vascularized organ grafts are often lost within the first 3-6 months after surgery. Despite these differences, corneal grafts undergo a rejection process that is histologically very similar to the process in other tissues 2. Recipients of corneal grafts fall into two relatively well-defined sub-groups. The first group, characterized by patients suffering from keratoconus or one of the corneal dystrophies, comprises those in whom the cornea is avascular and the anterior segment has never been inflamed. Such recipients typically enjoy excellent graft survival, which can be as high as 95% at ten years. In developed countries, about one-third of recipients will fall into this low-risk category. The second group, characterized by patients with a history of ocular infection, trauma, or other acquired disease, comprises those with vascularized corneas and a history of anterior segment inflammation. Corneal graft survival in this group is of the order of only 55% at ten years. Two-thirds of patients in developed countries will fall into this latter, high-risk group z3.

Outcome of timbal stem-cell transplantation Corneal transplantation cannot help all patients with damaged corneas. Some patients suffer disease restricted almost entirely to the epithelium of the ocular surface. Such ocular surface disease is believed to result from an insufficiency of corneal epithelial stem cells - cells that are physically located at the limbus (the junctional area between the cornea and sclera) z6. Transplantation of partial-thickness pieces of limbal tissue containing putative stem cells has resulted in the regeneration of a stable ocular surface both in experimental animals27,zsand in some patients suffering from ocular surface disease 29. Although limbal autotransplantation using donor limbus collected from the normal contralateral eye has been very successful in reestablishing a relatively normal ocular surface in patients with uniocular disease 3°, the use of an autograft is not possible for patients suffering from bilateral disease. Furthermore, there has been at least one recorded instance 31 in which collection of limbal autograft tissue from an apparently normal other eye resulted in subsequent damage to that eye. For patients with bilateral disease, allotransplantation using tissue collected from a donor eye is the only option. Partial-thickness limbal allotransplantation is still a relatively uncommon procedure and its long-term potential is unclear, but immunological rejection has been reported both in humans 32,33 and in experimental animal models 2s,34.~5. We investigated the survival of donor-derived epithelial cells on the ocular surface following limbal allotransplantation in a patient with bilateral limbal stem..cell failure induced by wearing contact lenses 33. Epithelial cells were harvested by impression cytology from the ocular surface of the grafted eye at various times after transplantation. DNA was extracted and amplified by polymerase chain reaction (PCR), and short tandem-repeat DNA polymorphisms were used to distinguish donor cells from recipient cells. Cells of donor phenotype were detected over the grafts at the time of surgery and in the central cornea 12 weeks later, but by 20 weeks only recipient-type cells were detected, despite systemic immunosuppression of the recipient with azathioprine and cyclosporin A. Interestingly, the grafts became oedematous and haemorrhagic at about one month after transplantation; in a rabbit model, :such an appearance was correlated with ongoing rejection 28. Histologically,

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rejecting limbal allografts become infiltrated with activated "CD4÷ and CD8 +T cells.

Outcome of experimental corneal xenotransplantation Interest in experimental corneal xenotransplantation 36-38has been fuelled by a world-wide shortage of human corneas for transplantation. Corneal xenografts undergo acute accelerated rejection within several days of transplantation, probably as a result of deposition of preformed donor-specific natural antibody on the graft, followed by activation of the complement cascade. This process, which occurs at the same time in both normal and athymic animals, occurs significantly faster in corneas that have been deliberately prevascularized 37. The histopathology of xenograft rejection in normal recipients is characterized by (1) early corneal epithelial and corneal endothelial cell damage, (2) granulocytic infiltration and (3) interstitial haemorrhage from recipient corneal and iris capillaries, (4) followed at 1-2 weeks by infiltration with T cells, macrophages and eosinophils. Thus, orthotopic corneal xenografts undergo accelerated damage mediated by pre-existing antibody within days of transplantation, followed by a late cell-mediated response that causes further destruction. This picture is quite similar to that seen in vascularized organ grafts, except that the initial damage in the latter occurs within the space of minutes to hours rather than the 2-3-day time-frame seen in the cornea. O c u l a r privilege o v e r w h e l m e d ?

Experimental and clinical corneal allograft rejection2, experimental limbal allograft rejection 28,35and late corneal xenograft rejection37 are all marked by an influx of both CD4 + and CD8 ÷ T cells',, together with a heterogeneous mixture of other leukocytes. Furthern~ore, monoclonal antibodies to CD4 can prevent corneal allograft rejection in rodent models39,4°. Not surprisingly, T cells have been assumed to initiate and control the damage observed in acute allograft rejection ~tnd, by inference, to be involved in limbal allograft rejection and in the late xenograft response. In this respect, rejection of tissues at the front of the eye is very similar to the processes seen in vascularized organ grat~s. The constitutive expression of Fas ligand on ocular tissue would be expected to induce apoptosis in any T cells infiltrating corneal grafts. Stuart et al. recently demonstrated 41 that, whereas orthotopic corneal allografts were accepted at a rate of 45% in normal mice, grafts from gld mutant donors (CD95L- mice) into normal recipients, or normal donor corneas transplanted to lpr mutant recipients (CD95mice), were rejected at a rate of 100%. These authors concluded that CD95L-induced apoptosis of infiltrating T cells is actually necessary for corneal graft survival: in the absence of a functional CD95-CD95L interaction, rejection is inevitable. Using the terminal transferase-mediated dUTP-biotin nick-end labelling (TUNEL) 1

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technique42, which allows visualization of apoptotic nuclei in tissue sections by the staining of fragmented chromatin, we examined (1) rejecting allografts in the rat, (2) time-matched rat isografts and (3) normal rat corneas for positively stained (i.e. apoptotic) nuclei. At the time of acute rejection and even earlier, most infiltrating cells were found to be TUNEL+, indicative of the induction of apoptotic cell death (Williams et al., unpublished). These findings are consistent with the expected sequelae of local Fas ligand expression, but quite inconsistent with the long-standing observations from many laboratories that corneal grafts often fail as a result of rejection. Either a very small percentage of infiltrating leukocytes is able to induce a very substantial amount of damage, or the cells that infiltrate ocular grafts can cause damage very quickly, before they themselves die in situ. Support for the latter possibility comes from the work of Lowin et al. 43, who showed that Fas ligand-based killing of target cells is triggered by Tcell receptor occupancy. Perhaps infiltrating T cells can kill cells of the cornea before they themselves are induced to undergo suicide. Recently, Vaux et al. made transgenic mice expressing CD95L on pancreatic islet cells 44. Fetal pancreases from these transgenic animals were then transplanted under the kidney capsule in normal allogeneic mouse recipients. Not only did CD95L expression fail to protect the transplanted pancreases from rejection, but mice of the CD95Ltransgenic donor strain developed an unexpected inflammatory infiltration in their own pancreatic tissue. The implication is that anomalous expression of CD95L can actually induce inflammation. These data do not support the hypothesis22that the induction of CD95L expression on tissue allografts will provide a mechanism for the circumvention of graft rejection. Concluding remarks Despite the operation of multiple mechanisms of immunological privilege in the anterior segment of the eye, corneal graft rejection is a real and relatively common phenomenon. It seems that the mechanisms by which the eye maintains transparency in the face of microbial infection are insufficient to overcome the very strong and essentially nonphysiological stimuli produced by either alloantigen or xenoantigen. This is especially true where past or active ocular inflammation has resulted in corneal neovascularization. At a molecular level, the ways in which invading T cells damage corneal grafts before they themselves are killed are quite uncertain. However, it can be predicted with some certainty that the beguiling possibility of deliberate Fas ligand-mediated immunosuppression 22 will prove insufficient to prevent T-cell-dependent graft rejection. Acknowledgements. We thank Scott Standfield for expert assistance. This work was funded by the National Health & Medical Research Council of Australia and by the Ophthalmic ResearchInstituteof Australia. References 1 Tuft, S.J. and Coster, D.J. (1990) The corneal endothelium, Eye 4, 389424 2 Williams, K.A. and Coster, D.J. (1993) Clinical and experimental aspects of corneal transplantation, Transplant. Rev. 7, 44-64 3 Coster, D.J. and Williams, K.A. (1992) Donor cornea procurement, some special problems in Asia, Asia Pac. J. Ophthalmol. 4, 7-12 4 Thylefors, B., Negrel, A.D., Pararajasegaram, R. and Dadzie, K.Y. (1995) Global data on blindness, Bull. WHO 73, 115-121 5 Medawar, P.B. (1948) Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye, Br. J. Exp. Pathol. 29, 58~9 m

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6 Griffith,TS. and Fergnson,T.A. (1997) The role of Fas L-induced apoptosis in immune privilege, Immunol. Today 18, 240-244 7 Barker, C.E and Billiagham, R.E. (1977) Immunologieadly privileged sites, Adv. lmmunol. 25, 1-54 8 Silverstein, A.M. and Khodadoust, A~A. (1973) Transplantation biology of the cornea, Ciba Found. Syrup. 15 (new series), 105-125 9 Jiang, L.Q., Jorquera, M. and Streilein, J.W. (1993) Subretinal space and vitreous cavity as immunologicaily privileged sites for retinal aliografts, Invest. Ophthalmol. Vts. Sci. 34, 3347-3354 10 Streilein, J.W. (1990) Anterior chamber associated immune deviation, the privilege of immunity in the eye, Surv. Ophthalmol. 35, 67-73 11 Rao, N.A. et al. (1983) The role of the penetrating wound in the development of sympathetic ophthalmia - experimental observations, Arch. Ophthalmol. 101, 102-104 12 Niedcrkorn, J.Y. and Lynch, M.G. (1989) Reconsidering the immunologic privilege and lymphatic drainage of the anterior chamber of the eye, Transplant. Proc. 21, 25%260 13 Streilein, J.W. and Niederkorn, J.Y. (1981) Induction of anterior chamberassociated immune deviation requires an intact, functional spleen, J. Exp. Med. 153, 1058-1067 14 Smolin, G. and Hyndiuk, R.A. (1971) Lymphatic drainage from vascularized rabbit cornea, Am. J. Ophthalmol. 72, 147-151 15 Collin, H.B. (1970) Lymphatic drainage of eq-albumin from the vascularized cornea, Invest. Ophthalmol. 9, 146-155 16 Luccy, D.R., Clerici, M. and Shearer, G.M. (1996) 1~pe 1 and type 2 cytoldne dysregulation in human infectious, neoplastic, and inflammatory diseases, Clin. MicrobioL Rev. 9, 532-562 17 Fukaura, H. et al. (1996) Induction of circulating myelin basic protein and proteolipid protein-spedfic transforming growth factor-131-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients, J. Clin. Invest. 98, 70-77 18 Stleilein, J.W. (1995) Immunological non-responsiveness and acquisition of tolerance in relation to immune privilege in the eye, Eye 9, 236-240 19 Wilbanks, G.A., Mammolenti, M. and Streilein, J.W. (1992) Studies on the induction of anterior chamber-associated immune deviation (ACAID). m . Induction of ACAID depends upon intraocular transforming growth factor-J3, Eur. J. lmmunol. 22,165-173 20 Vanx, D.L. and Strasser,A. (1996) The molecular biolot~ of apoptosis, Proc. Natl. Acad. Sci. U.S.A. 93, 2239-2244 21 Griffith, TS. et al. (1995) Fas ligand-induced apoptnsis as a mechanism of immune privilege, Science 270,118%1192 22 Bellgrau, D. et al. (1995) A role for CD95 ligand in preventing graft rejection, Nature 377, 630-632 23 Williams, K.A., Muchlberg,S.M., Wing, SJ. and Coster, D.J. (1993) The Australian Corneal Graft Registry, 1990-1992 report, Aust. New ZealundJ. Ophthalmol. 21 (Suppl.), 148 24 Vail,A. et al. (1994) Influence of donor and histocompatibility factors on corneal graft outcome, Transplantation 58, 1210-1216 25 Rinne, J.R. and Stalting, R.D. (1992) Current practices in the prevention and treatment of corneal graft rejection, Cornea 11, 326-32.8 26 Kruse, EE. (1994) Stem ceils and corneal epithelial regeneration, Eye 8, 170-183 27 Chen,J.J.Y. and Tseng, S.C.G. (1990) Corneal epithelial wound healing in partial limbal deficiency, Invest. Ophthalmol. Vts. Sci. 31, 1301-1314 28 Swift, G.J. et al. (1996) Survival of rabbit limbal stem cell aliografts, Transplantation 62, 568-574 29 Tsai, R.J.E and Tseng, S.C.G. (1994) Human allograft limbal transplantation for corneal surface reconstruction, Cornea 13, 389-400 30 Kenyon, K.R. and Tseng, S.C.G. (1989) Limbal autograft transplantation for ocular surface disorders, Ophthalmology," 96, 70%723

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31 Jenkins, C., Tuff, S., Liu, C. and Buckley, R. (1993) Limbal transplantation in the management of chronic contact*lens-associated epitheliopathy, Eye 7, 629-633 32 Thnfl, R.A. and Sugar, J. (1993) Graft failure in keratoepithelioplasty, Cornea 12, 362-365 33 Williams, K.A. et al. (1995) Use of DNA polymorphisms and the polymerase chain reaction to examine the survival of a human limbal stem cell allograft, Am. J. OphthalmoL 120, 342-350 34 Weise, R.A. et al. (1985) Conjunctival transplantation. Autologous and homologous grafts, Arch. Ophthalmol. 103, 1736-1740 35 Yao, Y-F. et al. (1995) Ocular resurfacing and alloepithelial ~jection in a murine keratoepithelioplasty model, Invest. Ophthalmol. T~s. Sci. 36, 2623-2633 36 Ross, J.R., Howell, D.N. and Sanfilippo, EP. (1993) Characteristics of corneal xenograft rejection in a discordant species combination, Invest. Ophthalmol. Vis. Sci. 34, 2469-2476 37 Larkin, D.E, Takano, T., Standfield, S.D. and Williams, K.A. (1995) Experimental orthotopic corneal xenotransplantation in the rat, mechanisms of graft rejection, Transplantation 60, 491-497

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38 Larkin, D.EP. and Williams, K.A. (1995) The host response in experimental corneal xenotransplantation, Eye 9, 254-260 39 Ayliffe, W. et al. (1992) Prolongation of rat corneal graft survival by treatment with anti-CD4 monoclonai antibody, Br: J. Ophthalmol. 76, 602-606 40 He, Y.G., Ross, J. and Niederkorn, LY. (1991) Promotion of marine orthotopic corneal allograft survival by systemic administration of anti-CD4 monoclonal antibody, Invest. Ophthalmol. V'ts. Sci. 32, 2723-2728 41 Stuart, 17. et al. (1997) CD95 iigand (FasL).induced apoptosis is necessary for corneal allograft survival, J. Clin. Invest. 99, 396-402 42 Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation, J. Cell Biol. 119, 493-501 43 Lowin, B., Hahne, M., Mattmann, C. and Tschopp, J. (19'94) Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lyti¢ pathways, Nature 370, 650--652 44 Allison, J., Georgiou, H.M., Strasser, A. and Vaux, D.L. (1997) Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts, Proc. Natl. Acad. Sci. U. S. A. 94, 3943-3947

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