Corneal 6 T.P.A.M.Slegers, M.K. Daly, D.F.P.Larkin

| Core Messages factors for rejection [18, 53]. In one-third of all ∑ Allograft rejection is the commonest single corneal grafts that are deemed failed, signs of a cause of corneal failure destructive attack by the immune system have ∑ Corneal transplant antigen recognition in been observed [61]. most cases is almost exclusively mediated A rejection episode results in loss of donor by recipient antigen-presenting cells. endothelial cells, critical for maintenance of CD4+ T-lymphocytes have the central role corneal transparency. As human endothelial in the alloreactive cell population cells do not repair by mitosis to any meaningful ∑ Endothelial rejection episodes can be extent, the consequence is that donor corneal reversed by intensive topical steroid in transparency is lost if cell density falls below the most patients. Poor outcomes result from threshold necessary for prevention of stromal delay in presentation and/or initiation in swelling. Endothelial decompensation results treatment either (1) from the time of an irreversible ∑ Patients with recipient corneal vascularisa- episode of acute graft rejection or (2) at an in- tion, a previously rejected ipsilateral trans- terval following one or more episodes of rejec- plant and inflammation at the time of tion which have been reversed by therapy. En- transplantation, are at highest risk of rejec- dothelial cells are thus the critical target in the tion and have very poor graft survival allogeneic response. ∑ Little information is available from ran- While, on the one hand, reversal of acute domised trials on prophylaxis by transplan- graft rejection episodes does not present such tation antigen matching or immunosup- challenges in as in other transplanted pression in this patient group tissues, effective prophylaxis in corneal graft re- cipients identified at high risk of rejection is much less evidence-based. Thus the impact of graft rejection continues to justify high priority in corneal research. Although the first success- 6.1 ful penetrating corneal graft was reported in Introduction 1906, it took another half a century before the first description of opacification of a previously Despite the relative immune privilege of the clear corneal graft was published. Paufique cornea as a transplant tissue and both the recip- named this event “maladie du greffon” (disease ient corneal bed and anterior chamber being an of the graft) and suggested that this clinical immune privileged site [35, 49], the most com- finding was caused by sensitisation of the donor mon cause of corneal graft failure in all reports by the recipient [37]. This description followed is allogeneic rejection. In first graft recipients the experiments reported by Medawar a few with no vascularisation of the recipient corneal years previously, in which differences were ob- bed, 2-year survival rates exceed 90%; this de- served between rabbit skin grafts of donor and creases to 35–70% in recipients with high risk recipient origin, giving rise to the term “histo- 74 Chapter 6 Corneal Transplant Rejection

compatibility” [32]. Maumenee subsequently confirmed this suggestion in a rabbit model of in which he showed that donor could induce an immune re- action [31]. The development of corneal trans- plantation models in rat [59] and mouse [46] fa- cilitated study of rejection in inbred donor and recipient animals with a wide range of inves- tigative immunological reagents.

6.2 Fig. 6.1. Subepithelial infiltrates in a penetrating Incidence corneal allograft. The appearance is similar to that seen in adenovirus keratitis, involving the donor In reports from large cohorts of corneal graft cornea only recipients, the proportion undergoing a rejec- tion episode at some stage post-transplant ranges from 18% to 21% [12, 22, 60]. In those Table 6.1. Risk factors for rejection graft recipients in whom rejection occurs, re- ported rates of successful reversal of the rejec- Preoperative Deep vascularisation of two or more quadrants tion episode range from 50% to 90% [21, 34]. Allograft rejection occurs most commonly in Previously rejected ipsilateral graft the second 6 months postgrafting, and it has been reported that more than 10% of the ob- Active corneal inflammation at time of graft served reactions can take place as late as at least 4 years after surgery [23, 34, 39]. This indicates Paediatric graft recipient that all corneal grafts need long-term surveil- Large diameter graft lance and are at risk practically indefinitely. Graft proximity to the limbus

Postoperative Loosening of sutures 6.3 Removal of sutures, wound dehiscence Factors Predisposing to Corneal Graft Rejection Graft inflammation HSV infection recurrence Preoperative characteristics of the graft recipi- Non-viral graft infection ent eye can be clearly identified in many pa- tients to indicate significantly high risk of graft failure. Proposed graft recipient corneas (1) with two or more quadrants of deep vasculari- predisposing to failure due to rejection with ad- sation, (2) bearing a previously rejected graft ditional clinical features that confer significant (Fig. 6.1) and (3) that are inflamed at the time of risk of graft failure due to other complications, transplantation are at significantly higher risk such as glaucoma or ocular surface disease [26, of rejection [2, 6, 30, 57, 63, 62]. There is less 41]. These preoperative clinical features must be robust evidence in the published literature that evaluated carefully in the decision whether to grafts in children, large diameter donor corneas proceed with corneal transplantation. and proximity of donor cornea to the recipient Once transplantation is successfully com- limbus are at higher risk (Table 6.1) [30, 41, 54, pleted, care must be taken to prevent postoper- 56]. Clearly more than one of these factors may ative events which predispose to rejection, such be operational in one patient. There may also be as vascularisation of recipient cornea (Fig. 6.2) association of one or more of the above factors or graft wound, suture loosening, or graft infec- 6.5 Histopathology 75 tion by bacteria or recurrent herpes simplex virus (HSV).

6.4 Clinical Features

Epithelial rejection, diagnosed by a linear opac- ity which stains with fluorescein, comprised up to 10% of all rejection episodes in one series and occurs on average 3 months after grafting [1].Although dead donor epithelial cells are rap- idly replaced by recipient epithelial cells and no Fig. 6.2. Endothelial rejection line, keratic precipi- scarring occurs, the presence of this type of tates and folds in Descemet’s membrane in rejection rejection reflects that the recipient is now sensi- tized to the donor and can progress to stromal and/or endothelial rejection. Stromal rejection is characterised by nummular subepithelial infiltrates (Fig. 6.1), identical to those found in adenovirus keratitis. Patients with both epithe- lial and stromal types of rejection may be asymptomatic or have mild ocular discomfort only. In contrast, patients with endothelial re- jection will usually present with visual distur- bance and iritis symptoms. If examined early after rejection symptom onset, anterior cham- ber cell infiltration without flare or graft abnor- mality will be seen.At later times after symptom Fig. 6.3. Almost total loss of endothelial cells in onset, the signs in succession are (1) aggregated corneal graft specimen removed at graft replacement alloreactive cells adherent to graft endothelium 6 months following rejection onset evident as keratic precipitates, (2) an endo- thelial line with precipitates and (3) localised oedema corresponding to a rejection line or total graft oedema (Fig. 6.2). Visible graft pre- cipitates on slit-lamp biomicroscopy imply focal 6.5 and variable but irreversible endothelial cell Histopathology loss, compromising endothelial pump function and resulting in stroma oedema in those grafts Descriptions of the pathological features of with severe inflammation or low endothelial cell corneal transplant rejection result from exami- density prior to rejection onset. Pachymetry is nation of grafts replaced following irreversible helpful in detecting an increase in oedema and failure. Therefore these specimens illustrate late also deturgescence following the start of steroid changes in end-stage corneal opacification, treatment. In one study it was found that next usually some months at least following treat- to the preoperative diagnosis, graft thickness ment of rejection. Characteristic findings in during rejection, as objectively measured by stroma are vascularisation with mononuclear pachymetry, is a prognostic sign for reversibili- cell infiltration and keratocyte loss; few if any ty of a rejection episode [34]. Risk factors for endothelial cells remain (Fig. 6.3) [28]. Several significant endothelial cell loss are delay in ini- studies have shown increased numbers of HLA tiating anti-rejection treatment more than 1 day class II positive cells infiltrating stroma in sec- and recipient age greater than 60 years [13]. tions of rejected grafts [38, 58]. 76 Chapter 6 Corneal Transplant Rejection

include the paucity of donor-derived major his- 6.6 tocompatibility complex (MHC) class II+ APC, Immunopathological Mechanisms and corneal epithelial and endothelial expres- sion of Fas ligand [7, 16], interaction of which 6.6.1 with Fas on alloreactive effector cells leads to Immune Privilege and Its Breakdown death of the infiltrating leukocyte. Corneal grafts at high risk of rejection are Immune privilege is a dynamic phenomenon identified by several risk factors, most of which in which the destructive effect of a “normal” reflect breakdown of facets of immune privi- immune response to particular antigens is lege. Prospective clinical outcome studies iden- either altered or absent in order to protect the tify the most significant of these to be recipient microanatomy of highly organised tissues in corneal vascularisation, corneal inflammation the eye. In corneal transplantation, both (1) the at the time of transplantation, which induces recipient corneal bed and anterior chamber and APC infiltration in the recipient cornea prior (2) the transplanted tissue have features of to surgery, and a previously rejected ipsilateral immune privilege. graft. Several features of the anterior chamber con- tribute to immune privilege. There are mechan- ical barriers that impair immune cell access to 6.6.2 the anterior chamber and transplanted cornea. Afferent Arm of the Allogeneic Response One barrier is the lack of blood and lymphatic vessels in a normal cornea. While experimental In circumstances where the immune privileged and clinical studies have clearly shown that features of the cornea are bypassed by the im- transplants are much more likely to be rejected mune system, the first stage in rejection is in vascularised corneas, the stimuli to vascular- recognition of the presence of non-self tissue. isation are likely also to induce lymph vessel There are two routes of allorecognition. By the growth. Following transplantation, it is in lymph indirect pathway, recipient APC enter the graft vessels that antigen-presenting cells (APC) mi- to capture and process donor antigens, migrat- grate from the graft to lymphoid organs for pres- ing to the lymphoid system to present the anti- entation of graft antigens to T lymphocytes.An- gen in context with self MHC class II molecules other route for alloreactive cells to reach the to T cells. Most experimental evidence points to anterior chamber and donor corneal endotheli- the neck lymph glands as the location for anti- um is closed by the tight junction barrier formed gen presentation [40, 45, 65]. between non-pigmented epithelial cells and Recent identification in the central cornea non-fenestrated iris vessels [10]. of a population of dendritic cells, which can be- In the event that leukocytes enter the anteri- come MHC II+ and migrate to the draining or chamber, mechanisms are available to either lymph nodes [11, 17, 29], and MHC class II+ deviate or blunt a potentially harmful immune macrophages [9] indicates that direct allore- response. For example the aqueous humour cognition of the corneal graft antigens is possi- contains immunosuppressive molecules as trans- ble. By this pathway, donor APC bearing allo- forming growth factor (TGF)-b,vasoactive antigens migrate from the graft and activate T intestinal polypeptide (VIP), a-melanocyte lymphocytes via their own non-self MHC class stimulating hormone (MSH), and calcitonin II molecules. Direct allorecognition would be gene related protein (CGRP), which contribute more prominent in the occasional clinical cir- to induction by an allograft of deviated sys- cumstance in which a donor cornea is trans- temic delayed-type hypersensitivity [35, 52]. planted which has an increased population of In addition to lack of blood and lymphatic APCs, such as after viral infection. However, in vessels, cornea allografts have been shown in most circumstances it is assumed that corneal laboratory studies to have additional features allorecognition is predominantly by the indi- which contribute to immune privilege. These rect pathway. 6.8 Prevention of Rejection 77

Evidence from cell kinetic studies in murine is likely to be due to delay in recognition and corneal grafts demonstrates that within several initiation of treatment, with resulting signifi- hours of transplantation the graft is infiltrated cant donor endothelial cell loss [13]. In others, by granulocytes and macrophages [27]. From failure to reverse rejection may be due to failure macrophage depleting studies evidence has of topical steroid to reverse effector compo- been provided that these cells play a crucial role nents of the allogeneic response. In respect of in the afferent phase of graft rejection [47]. additional systemic steroid, a single dose of in- travenous methylprednisolone was found to be more effective than oral steroid in patients with 6.6.3 endothelial rejection who presented within Efferent Arm of the Allogeneic Response 8 days of onset [20]. A second pulse of intra- venous methylprednisolone at 24 or 48 h gave When T-helper cells have identified the present- no benefit when compared to a single dose at ed antigen as non-self, effector mechanisms are initial diagnosis [19]. However, a subsequent generated against donor tissue. Cytokines in- randomised trial demonstrated no significant cluding particularly tumour necrosis factor [42] benefit of intravenous methylprednisolone in and interferon-g [25] have been clearly identi- addition to topical steroid, in respect of graft fied in aqueous humour and the cornea prior to survival or interval to a subsequent rejection observed endothelial rejection onset. After episode within a 2-year follow-up period [21].In corneal transplantation it has been shown that the same study, endothelial rejection was re- alloantibody, cytotoxic T lymphocytes and de- versed in 33 of 36 patients treated, indicating layed type hypersensitivity responses are com- that steroid-resistant rejection is uncommon. ponents of the effector response. Experimental Other studies examining the efficacy of topical studies, using CD4+ knockout mice and mono- or oral cyclosporin administered in combina- clonal antibodies directed against CD4+ T cells, tion with intravenous steroid have reported have pointed to the central role of this lympho- similar outcomes, with irreversible rejection in cyte subpopulation [3, 64]. The mechanism by a small proportion of patients [66, 67]. which corneal endothelial cells are killed is not yet clear.At time of graft destruction increasing levels of natural killer (NK) cells, known to be 6.8 able to lyse corneal endothelial cells, are detect- Prevention of Rejection ed in the aqueous humour of grafted rats [14]. There is additional evidence that nitric oxide 6.8.1 could mediate in destruction of donor endo- Immunosuppression thelial cells [8, 44, 51]. In patients without risk factors for graft rejec- tion identified prior to surgery, typical postop- 6.7 erative immunosuppression comprises steroid Treatment of Rejection drops such as dexamethasone 0.1% four times daily for the first 2–3 months, reducing gradual- The objective of treatment is to reverse the re- ly to zero by 6 months post-transplant. Regimes jection episode at the earliest possible time, in vary from centre to centre. There is much less order to minimise donor endothelial cell loss consensus on which additional measures to take and preserve graft function. With the anatomi- as prophylaxis in patients at high risk of rejec- cal advantage that corneal transplants are tion (Table 6.1), in whom topical steroid alone superficial, intensive administration of topical is insufficient to prevent rejection. The result corticosteroid, such as dexamethasone 0.1%, of a continuing shortage of large comparative treatment is successful in reversing most endo- prospective studies is that immunosuppression thelial rejection episodes. In most cases in protocols in current use result from individual which topical steroid fails to reverse rejection, it clinical experience, with some influence from 78 Chapter 6 Corneal Transplant Rejection

experimental evidence and small uncontrolled HLA-A, and -B but not HLA-DR. The possible and/or retrospective clinical studies. However, benefit of planned -DR mismatching in a setting ophthalmologists are cautious about adminis- of known class I histocompatibility is at present tering potentially toxic systemic immunosup- being investigated in an ongoing prospective pressive agents, even in those patients in whom trial, the outcome of which is awaited with in- a surviving graft would allow vision in the only terest. In 1996, a randomised although retro- eye. The subject of immunosuppression in pre- spective study reported a beneficial effect of vention of corneal graft rejection is discussed in DRB1 matching in recipients at high risk on ac- another chapter in this text. count of vascularisation and/or retransplanta- tion [4]. Subsequently a beneficial effect of HLA-DPB1 matching in high-risk corneal trans- 6.8.2 plantation with a significantly higher rate of HLA Matching 1-year rejection-free graft survival compared to those without matching was shown [33]. In vascularised organ there In corneal transplantation therefore the ef- is robust evidence supporting HLA matching of fect of HLA matching is less than clear and the donor and recipient, with the data of Opelz and data are most ambiguous for class II matching. others demonstrating stratification of the risk Resolution of this clinically important issue is of rejection according the number of class I and not simple. In contrast to solid organs, results of especially class II mismatches [36]. HLA match- matching for cornea are likely to be influenced ing is in routine use internationally in cadaver- by the facts that: (1) allorecognition is predomi- ic renal and other . In nantly by the indirect pathway in most patients corneal transplantation by contrast, for recipi- [5], and (2) minor transplantation antigens, ents at high risk of rejection HLA class I and shown to have a significant effect on graft sur- class II -DR matching is routinely done in some vival in untreated rodent recipients [24, 48, 50] countries, whereas in other countries no match- and presented by the indirect pathway, remain ing takes place at all. Roelen suggested a benefit unmatched in HLA-matched recipients. It is for HLA-A and -B matching in high-risk corneal also worth noting here that the effects of HLA allograft recipients based on his findings that matching on corneal graft outcome have not yet primed, donor-specific cytotoxic T cells were been investigated in the setting of systemic im- present in rejected corneas but absent in donors munosuppression prophylaxis. Studies in solid with good graft function [43].However,the ben- organ transplantation have shown that more efit of histocompatibility matching in corneal effective rejection prophylaxis can override an transplantation has been disputed and is cer- HLA matching effect in unsensitised recipients. tainly less clear than for solid organ grafts, even in corneal recipients at perceived high risk of graft rejection. Two large prospective studies on 6.9 HLA-A, -B, or HLA-DR antigen matching high- Future Prospects risk recipients have reported divergent findings. The Collaborative Corneal Transplant Studies Reducing the impact of allograft rejection is a Research Group reported that matching of these major challenge in corneal disease. It can be antigens did not decrease the risk of corneal expected that following developments in tech- graft failure secondary to rejection [53]. In con- niques of lamellar keratoplasty,wider use of this trast the Corneal Transplant Follow-up Study type of surgical procedure,particularly for stro- found there was increased risk of graft rejection mal corneal pathology,will reduce the impact of with mismatch of HLA class I antigens (relative endothelial rejection. However, the presence of risk 1.27 per mismatch), but decreasing risk of a group of patients with no alternative to pene- rejection with -DR mismatches (relative risk trating keratoplasty, a high risk of rejection and 0.58 per mismatch) in high risk patients [55]. no alternative prophylactic intervention justi- This study therefore supported matching at fies clinical trials of novel treatment strategies References 79

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