Mechanisms of Corneal Allograft Rejection and Regional

1 2,3 1 ABIG PTAMLG SYMPOSIUM OPHTHALMOLOGY CAMBRIDGE Mechanisms of DJ Coster , CF Jessup and KA Williams corneal allograft rejection and regional immunosuppression

Abstract corneal graft survival, it continues to improve. There is no evidence that this is so for corneas. Corneal transplantation has not matched the Despite long experienceFthe first penetrating improvements in outcome seen with other corneal graft was performed in 1906 and the clinical transplantation procedures. The first renal graft in the 1960sFcorneal therapeutic strategies, which have improved transplantation has failed to match outcomes in the outcomes of solid vascularised organs are other areas of clinical transplantation. not applicable to corneal transplantation. Developments that have improved the Corneal transplantation is different with outcomes of renal transplants (better systemic respect to relevant transplantation biology and immunosuppression, improved tissue the clinical context in which it is practiced. matching, and the use of living related donors) New approaches need to be developed which have not been applicable to corneal provide regional rather than systemic transplantation. Alternative approaches are immunosuppression. The accessibility of the required which take into account the unique cornea makes it particularly suitable for aspects of the corneal allograft response and the 1Department of topical medication and for gene therapy clinical context in which corneal transplantation Ophthalmology, Flinders approaches. Engineered antibodies, small is performed. The limiting aspect of the clinical University, Adelaide, enough to pass through the cornea, and context is the often-restricted suitability of Australia directed at key molecules in the allograft patients with corneal disease for systemic response have been developed. Gene therapy 2Transplantation immunosuppression. Visual disability and had been developed using viral vectors to Immunology Laboratory, blindness are disabling but this does not justify Queen Elizabeth Hospital, transfect the corneal endothelium with the the potentially life-threatening complications Adelaide, Australia genes for immunosuppressive lymphokines. that go with shutting down the immune system Both approaches show promise. 3 for a prolonged period. Department of Medicine, Eye (2009) 23, 1894–1897; doi:10.1038/eye.2009.17; University of Adelaide, published online 20 February 2009 Adelaide, Australia Regional immunosuppression for corneal Correspondence: DJ Coster, Keywords: corneal transplantation; allograft transplantation Department of rejection; gene therapy; modified antibodies Ophthalmology, Flinders An unusual aspect of corneal allograft rejection Medical Centre, Bedford is the accessibility of the cornea, which raises Park, SA 5042, Australia the possibility of regional rather than systemic Tel: þ 618 8204 4899; immunosuppression. Although the allograft Human corneal graft outcomes Fax: þ 618 8277 0899; response is well conserved throughout E-mail: doug.coster@ flinders.edu.au Corneal graft failure is common, particularly in evolution, differences occur from species to patients who have received grafts for conditions species and from one tissue or organ to another. Received: 11 December 2008 other than keratoconus or stromal dystrophies. Such differences in allograft rejection need to be Accepted: 11 December 2008 The success rate for penetrating corneal grafts is taken into account when using animal models Published online: 20 February as high as 73% at 5 years and 62% at 10 years.1 and when designing new interventions. Both 2009 This is less than the survival of solid the clinical context of corneal transplantation Presented at the Cambridge vascularised organs, for example, the survival and the mechanisms of corneal allograft 2 Ophthalmological of renal transplants is above 90% at 5 years. rejection point to the need to develop regional Symposium, September 2008 Not only is renal graft survival higher than immunosuppression for clinical practice. Regional immunosuppression for corneal grafts DJ Coster et al 1895

Erosion of corneal privilege by inflammation Antigen trafficking is a subject of considerable interest because the exact location of antigen presentation is a Not all corneal grafts have the same tendency to reject. crucial consideration for the development of new Those with undisturbed histology, as is the case with regimens of immunosuppression. In laboratory keratoconus, have a low propensity for rejection. Grafts experiments, antigens delivered to the cornea into recipient corneas that are inflamed reject commonly disseminate widely. Antigens delivered to the cornea and rapidly. Even a history of keratitis years before can be recovered from the conjunctiva, draining lymph transplantation increases the risk of rejection. nodes, and from as far away as the spleen, and Immunological privilege is relative. mesenteric lymph nodes. They travel in soluble form A number of changes in the inflamed cornea contribute or are carried in antigen-presenting cells.14,15 to the erosion of privilege. Normal human corneas carry Antigen presentation is a critical point in the allograft few bone marrow-derived cells capable of processing response because it is the first step in the response which and presenting antigens and initiating the immune is allospecific. Alloantigen is presented in association response.3,4 With inflammation, such cells are matured with MHC molecules to the T-cell receptor (TCR) and recruited into the cornea through the limbal (Figure 1). There are many other interactions between the (peripheral corneal) circulation. Even when the antigen-presenting cell and the T cell, which can inflammation is resolved, these cells persist for months or influence the impact of antigen presentationFincluding years.5 The greater the number of bone marrow-derived coreceptor and costimulatory interactions, which may cells in the host cornea at the time of surgery the higher enhance or impede the impact of presentation. Although the rejection rate. A cornea that has been inflamed is the cell surface molecules involved in TCR triggering are never the same again with regard to immune privilege.6 now largely defined, it is still unclear how the interaction Inflammation upregulates transplantation antigen of the TCR with its cognate peptide/MHC results in the expression making it easier for the host immune system appropriate response for the T cell. A number of models to see the foreign graft.7 Other mechanisms contribute to for TCR triggering exist, and the real situation is the erosion of privilege that complicates corneal probably a combination of these. Regardless of the inflammation. Chronic inflammation induces generation mechanism of receptor triggering it is likely that the final of blood vessels and lymphatics in the normally outcome of T cell–APC interactions is impacted by (1) the avascular cornea.8 These contribute to the connection of temporal expression and density of costimulatory the allograft to the host immune system. Inflammation induces vessels to leak, facilitating ingress of cells, and proteins into the cornea. In an inflamed cornea, macrophages produce factors such as VEGF-C, which in turn induces growth of lymphatics. Pro-inflammatory cytokines gain access to the cornea and anterior chamber and encourage rejection. Normally the aqueous contains immunosuppressive proteins (eg, TGF-b), which contribute to privilege. Thus, inflammation can erode privilege by a number of mechanisms.9

Cellular and molecular events in corneal allograft rejection

The three essential requirements for the initiation of the allograft response are: non-self transplantation antigens, antigen-presenting cells, and host immunocytes. All are present in rejecting corneal allografts. During the indirect Figure 1 Schematic representation of the interaction between a CD4 T-cell and an antigen-presenting cell (APC). Alloantigen pathway of presentation, which is the most important in presented in the groove of host MHC Class II interacts with the corneal allograft rejection, host antigen-presenting cells T-cell receptor (TCR) and associated CD3 chains. Costimulatory process alloantigen, and present it to the host molecules containing positive (CD28) or negative (CTLA-4, PD- immunocyte. Antigen processing is likely to occur in the 1) signalling tyrosine motifs interact with their ligands on APC cornea.10 Where antigen presentation occurs is debated. (CD80, CD86, PD-L1, PD-L2). Src family kinases including Lck, either associated with the coreceptor CD4 or free in the Almost certainly, it can occur at a number of sites, membrane, are important in phosphorylating tyrosine residues including the cornea, the ocular environs, and the in immunoreceptor intracellular tails to facilitate the recruitment draining lymph nodes.11–13 of intracellular messengers and signal transduction.

Eye Regional immunosuppression for corneal grafts DJ Coster et al 1896

molecules, (2) the phosphorylation state of critical location at which antigen presentation was immunoreceptor intracellular tails, (3) the spatial occurring. If antigen presentation occurs remote from the organisation of phosphatases (eg, CD45) and kinases cornea, beyond the reach of locally applied (eg, Lck), and (4) the cytokine milieu at the time of immunosuppression, it will be necessary to seek presentation.16 therapeutic targets more proximal in the afferent limb of As complex as the process is, the interaction between the allograft response. Antigen processing, rather than antigen-presenting cells and host immunocytes offers the antigen presentation may need to be the target. This is prospect of allospecific therapeutic intervention. This has likely to occur in the cornea. Both the antigen and the not been achieved in clinical transplantation. Antibodies antigen-presenting cells are in the cornea and the to key elements of antigen presentation, for example anti- tendency of a cornea to reject is related to the number of CD4 and anti-CD3 antibodies and CTLA4-Ig fusion antigen-presenting cells in the host cornea at the time of protein, have been used clinically with some success, but surgical transplantation. these agents work in a non-specific way to suppress A second approach that we and others have employed immune responses,17–19 because they are administered is to modulate immunological privilege in the cornea by systemically and result in systemic immunosuppression. local over-production of anti-inflammatory cytokines. This approach has not been widely employed in corneal The lymphokine milieu is known to influence the transplantation because systemic immunosuppression establishment of immune responses and antigen cannot usually be justified in this clinical setting. processing. This milieu can be altered with a gene Furthermore, the antibodies employed in this approach therapy approach. Increasing the secretion of are too large to cross into the cornea and therefore cannot anti-inflammatory lymphokines, for example, interleukin be delivered topically. 10 (IL10) might be expected to protect against allograft rejection. Similarly it might be possible to block the pro-inflammatory action of interleukin 12 Alternative therapeutic options (IL12) by over-producing the p40 subunit of IL12, As the therapeutic developments that have contributed which binds the receptor but does not result in to the improvement in outcomes for essential organ immunological activity.21 Indeed an adenoviral gene transplantation are not applicable to corneal therapy approach has been shown to increase graft transplantation, new ways of abrogating the allograft survival in animal experiments in both cases. Although response in the cornea are required. One approach is to these results are interesting and encouraging, it remains interfere with antigen presentation with antibody necessary to develop and test safer, more efficient fragments, antibodies directed at crucial elements of gene therapy vectors. antigen presentation and small enough to get into the eye when delivered topically. Whole antibodies are large molecules, about 150 kDaFtoo large to cross the Conclusions corneal epithelium and enter the stroma and anterior chamber. Not all of the structure of the antibody is The lack of progress in improving the outcome of necessary for specific therapeutic functions. Only the corneal transplantation, particularly in those high-risk antigen-binding site is required to block some responses. cases in whom immunological privilege has been An antibody can be engineered using conventional eroded, needs to be overcome. To achieve this demands molecular biology techniques that are little more than the development of therapies acceptable in the clinical an antigen-binding site, with a molecular weight of context of corneal transplantation. Fortunately the cornea 10–25 kDa. is very accessible and therapeutic agents can be delivered This approach was tested in a rat model of corneal locally by topical medication. It is also particularly transplantation. A mouse anti-rat CD4 scFv was suitable for gene therapy approaches. It is likely that very engineered and investigated as a potential therapy for little progress will be made in improving the outcome of the prevention of corneal allograft rejection. When the corneal transplantation in high-risk patients until antibody fragment was administered systemically by regional immunosuppression can be achieved.3 intraperitoneal injection or by adenoviral expression, a modest prolongation of corneal graft survival was observed. When the fragment was delivered topically, Acknowledgements even though it had been shown to readily enter the cornea, no prolongation of corneal allograft survival was Supported by the National Health & Medical Research observed.20 This suggested that the topically Council and the Ophthalmic Research Institute of administered antibody fragment was not getting to the Australia.

Eye Regional immunosuppression for corneal grafts DJ Coster et al 1897

References aggregates after corneal transplantation in rats. Br J Ophthalmol 2003; 87: 1515–1522. 1 Williams KA, Lowe MT, Bartlett CM, Kelly L, Coster DJ. 13 Yamagami S, Dana MR. The critical role of lymph nodes The Australian Corneal Graft Registry 2007 Report. Flinders in corneal alloimmunization and graft rejection. Invest University Press: Adelaide, 2007. Ophthalmol Vis Sci 2001; 42: 1293–1298. 2 Wolfe RA. Long-term renal allograft survival: a cup half-full 14 Camelo S, Shanley A, Voon AS, McMenamin PG. The and half-empty. Am J Transplant 2004; 4: 1215–1216. distribution of antigen in lymphoid tissues following its 3 Catry L, Van den Oord J, Foets B, Missotten L. Morphologic injection into the anterior chamber of the rat eye. J Immunol and immunophenotypic heterogeneity of corneal 2004; 172: 5388–5395. dendritic cells. Graefes Arch Clin Ex Ophthalmol 1991; 229: 15 Liu Y, Hamrah P, Zhang Q, Taylor AW, Dana MR. Draining 182–185. lymph nodes of corneal transplant hosts exhibit evidence 4 Jager Mj. Corneal Langerhans cells and ocular immunology. for donor major histocompatibility complex (MHC) class II- Reg Immunol 1992; 4: 186–195. positive cells derived from MHC call II-negative grafts. 5 Williams KA, Ash JK, Coster DJ. Histocompatibility antigen J Exp Med 2002; 195: 259–268. and passenger cell content of normal and diseased human 16 Choudhuri K, van der Merwe P. Molecular mechanisms cornea. Transplantation 1985; 39: 265–269. involved in T cell receptor triggering. Semin Immunol 2007; 6 Williams KA, White MA, Ash JK, Coster DJ. Leukocytes in 19: 255–261. the graft bed associated with corneal graft failure. Analysis 17 Casadei DH, del C Rial M, Opelz G, Golberg JC, Argento JA, by immunohistology and actuarial graft survival. Greco G et al. A randomized and prospective study Ophthalmology 1989; 96: 38–44. comparing treatment with high-dose intravenous 7 Pepose JS, Gardner KM, Nestor MS, Foos RY, Pettit TH. immunoglobulin with monoclonal antibodies for rescue of Detection of HLA class I and II antigens in rejected human kidney grafts with steroid-resistant rejection. Transplantation corneal allografts. Ophthalmology 1985; 92: 1480–1484. 2001; 71(1): 53–58. 8 Bill A. The blood-aqueous barrier. Trans Ophthalmol Soc UK 18 Cooperative Clinical Trials in Transplantation Research 1986; 105: 149–155. Group. Murine OKT4A immunosuppression in cadaver 9 Streilein JW. Ocular immune privilege: the eye takes a dim donor renal allograft recipients: a cooperative clinical trials but practical view of immunity and inﬂammation. J Leukoc in transplantation pilot study. Transplantation 1997; 63(9): Biol 2003; 74: 179–185. 1243–1251. 10 Kuffova L, Netukova M, Duncan L, Porter A, Stockinger B, 19 Salomon B, Bluestone JA. Complexities of CD28/B7: Forrester JV. Cross presentation of antigen on MHC Class II CTLA-4 costimulatory pathways in autoimmunity and via the draining lymph node after corneal transplantation in transplantation. Annu Rev Immunol 2001; 19: 225–252. mice. J Immunol 2008; 180: 1353–1361. 20 Theil MA, Takano T, Hawksworth N, Coster DJ, Williams 11 McMenamin PG, Crew J, Morrison S, Holt PG. KA. Low-dose, short-term treatment with anti-CD4 Immunomorphologic studies of macrophages and MHC monoclonal antibody prolongs corneal allograft survival. Class II-positive dendritic cells in the iris and ciliary body of Transp Proc 2001; 33: 635–636. the rat, mouse, and human eye. Invest Ophthalmol Vis Sci 21 Klebe S, Sykes PJ, Coster DJ, Krishnan R, Williams KA. 1994; 35: 3234–3250. Prolongation of sheep corneal allograft survival by transfer 12 Banerjee S, Figueiredo FC, Easty DL, Dick AD, Nicholls SM. of the gene encoding ovine interleukin 10. Transplantation Development of organized conujunctival leukocyte 2001; 71: 1214–1220.

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