The potential of viral vector-mediated gene transfer to prolong corneal allograft survival.

The cornea is a particularly attractive target for gene therapy designed to improve the outcome of corneal transplantation. First, there is a clear and well-defined clinical need. Second, because donor corneas can be preserved for days if not weeks within an eye bank, ex vivo transduction of a donor cornea can be carried out without the urgency associated with many other forms of transplantation. Finally, the partial sequestration of the eye from the systemic circulation decreases the likelihood of spillover of vector and transgene, and the immune privileged nature of the cornea and anterior segment affords a degree of protection from immune responses directed against the vector. A wide range of vectors has been investigated for gene transfer to the cornea. A number of viral vectors, in particular, have proved to be efficient at transducing the cornea and in association with a variety of transgenes, have been used successfully to prolong corneal allograft survival significantly in animal models. The most suitable such vector for future clinical studies in corneal transplantation has yet to be determined, but the most likely include recombinant adenoviral, adeno-associated viral and lentiviral vectors. In this review, we examine the ability of these viral vectors to transduce the cornea, and summarise those studies in which gene therapy has been used to prolong experimental corneal allograft survival.

Graft rejection episodes after Descemet stripping with endothelial keratoplasty: part two: the statistical analysis of probability and risk factors.

AIM: To investigate risk factors and probability of initial immunological graft rejection episodes after Descemet stripping with endothelial keratoplasty (DSEK). METHODS: Outcomes of 598 DSEK cases from a single tertiary referral centre were reviewed. Risk factors and probability of rejection were assessed by multivariate Cox proportional hazards modelling. RESULTS: Rejection episodes occurred in 54 eyes of 48 patients. Estimated probability of a rejection episode was 7.6% by 1 year and 12% by 2 years after grafting. Relative risk of rejection was five times higher for African-American patients compared with Caucasians (p = 0.0002). Eyes with pre-existing glaucoma (9%) or steroid-responsive ocular hypertension (27%) had twice the relative risk of rejection (p = 0.045) compared with eyes that did not have those problems. Patient age, sex and corneal diagnosis did not significantly influence rejection risk. Risk of rejection was not increased when fellow eyes were grafted within 1 year of the first eye (p = 0.62). CONCLUSIONS: Pre-existing glaucoma or steroid-responsive ocular hypertension and race were the two factors that independently influenced relative risk of rejection after DSEK. Rejection risk was not increased if the fellow eye was grafted within the prior year with DSEK.

Immune modulation in corneal transplantation.

Allograft rejection is the most common reason for corneal transplant failure, despite the immunologic privilege of both the graft and the anterior chamber. To prevent corneal allograft rejection, various immunomodulatory strategies have been used in experimental corneal transplantation. These include (1) anti-T-cell receptor and T-cell depletion therapy; (2) manipulation of costimulatory molecule function, including both down-regulation of positive stimulatory molecules and/or up-regulation of inhibitory molecules and overproduction of tumor necrosis factor-related, apoptosis-induced ligand; (3) modulation of cytokine production by reducing proinflammatory cytokines (tumor necrosis factor alpha, interleukin [IL]-12, and IL-1) and/or increasing immunoregulatory cytokines (IL-10 and IL-4); (4) macrophage depletion; and (5) overexpression of the immunomodulatory molecule indoleamine 2,3-dioxygenase. Although these approaches appear promising in animal corneal transplantation models, there has been very little translation of these immunomodulatory approaches in human corneal transplantation.

Lack of IFN-gamma synthesis in aqueous humor during corneal graft rejection correlates with suppressed nitric oxide production by macrophages.

PURPOSE: To investigate cytokine production by leukocytes in aqueous humor (AH) during corneal graft rejection and nitric oxide (NO) production by macrophages as a potential mediator of graft damage. METHODS: Rats received corneal allotransplants and were killed during acute rejection. Leukocytes in AH that expressed tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-10 were quantified by flow cytometry. Isograft and further allograft recipients were killed, and sectioned corneas with conjunctivae were examined by histology for production of inducible nitric oxide synthase (iNOS), NO, and nitrotyrosine (NT). RESULTS: Between 80% and 90% of T cells, NK cells, and macrophages in AH expressed TNF-alpha, and at least 20% expressed IL-10. However, IFN-gamma was undetectable unless cells were first stimulated in vitro with PMA and ionomycin, which yielded IFN-gamma in 25% of cells. iNOS(+) macrophages were identified in donor cornea and AH, correlating precisely with rejection. Cells producing low levels of NO (NO(dim) cells) were found in donor stroma, but NT(+) cells were rare. Both NT(+) and NO(+) cells were rare in the anterior chamber (AC) or attached to corneal endothelium. NT(+) macrophages that were also NO(bright) were associated with sutures in allograft and isograft recipients and within conjunctivae, either scattered or in leukocyte aggregates. CONCLUSIONS: IFN-gamma synthesis is lacking in the AC during rejection, correlating with lack of NO but not of iNOS expression. NO does not appear to mediate endothelial cell death. NT and high levels of NO production are associated with nonspecific inflammatory cells.

Effect of CXCL-1/KC production in high risk vascularized corneal allografts on T cell recruitment and graft rejection.

BACKGROUND: The survival rate of corneal allografts in high-risk vascularized corneal bed recipients is poor, similar to vascularized solid organ allografts. Although the early induction of selective chemokines in solid organs is required for the optimal recruitment of T cells into rejecting allografts, little is known about the role of these chemokines in high risk corneal allografts. METHODS: Orthotopic corneal allotransplants were performed in low-risk (nonvascularized) and high-risk (vascularized) C57BL/6 (H-2b) recipients using Balb/c (H-2d) donors. Intragraft production of CXC chemokines was measured by Luminex and enzyme-linked immunosorbent assay on corneal transplant extracts at different times after surgery. Rabbit anti-KC serum was used to test its role in high risk corneal allograft survival. RESULTS: Early upregulation of CXCL1/KC occurs 3 days after transplantation in high risk allograft only. Moreover, the T-cell chemoattractants, CXCL9/Mig and CXCL10/IP10, are produced late (day 10) after surgery and their production correlates with the recruitment of CD4 T cells into the graft. Furthermore, in vivo neutralization of CXCL1/KC with anti-KC sera results in increased graft survival and decreased recruitment of T cells into high-risk allografts. CONCLUSION: We propose that a high risk vascularized cornea behaves like a vascularized solid organ transplant. The early production of CXCL1/KC is crucial to the induction of T-cell chemoattractants necessary for the recruitment of allospecific CD4 T cells into the graft. In vivo neutralization of CXCL1/KC represents a potential novel therapy that could be used to increase the survival rate of high-risk vascularized corneal allografts.

Visual outcomes and graft survival following corneal transplants: the need for an Irish National Corneal Transplant Registry.

PURPOSE: To evaluate visual outcomes and corneal graft survival at 12 months postoperatively, and to demonstrate the need for an Irish National Corneal Transplant Registry. METHODS: Retrospective, single surgeon, single center, analysis of 44 consecutive corneal transplants was performed between 2001 and 2005. RESULTS: Forty-four eyes of 41 patients underwent penetrating keratoplasty (n = 37), lamellar keratoplasty (n = 6) or limbal graft (n = 1). Indications for surgery (%) were keratoconus 45.45%; bullous keratopathy 2.7%; graft failure 9%; Fuchs endothelial dystrophy 2 4.5%; Irido corneal endothelial syndrome 2.27% and tectonic 15.9%. Visual outcome at 1 year (%) were as follows: improved by one or more Snellen lines (79.5%); disimproved by one or more Snellen lines (9%); unchanged (11.5%). Graft survival at 1 year was 93%. DISCUSSION: Although our findings compare favourably with those of the Australian Corneal Graft Registry, there is no Irish Corneal Transplant Registry with which to compare our results.

Effects of interleukin-12p40 gene transfer on rat corneal allograft survival.

PURPOSE: Despite the immunologically privileged nature of the cornea, graft rejection remains the major cause of human corneal allograft failure. Gene therapy is an interesting approach to introduce immunoregulatory molecules into the graft or the recipient to prevent rejection. In this study we investigated the immmunomodulatory effects of adenovirus-mediated gene transfer of a Th1 antagonist, interleukin-12p40 (IL-12p40), in vitro and on allogeneic graft survival in a rat experimental keratoplasty model. METHODS: Donor corneas were transduced with an E1/E3 deleted adenoviral (Ad) vector encoding the IL-12p40 gene (AdIL-12p40) and assayed for the expression of the therapeutic gene. Cell culture supernatants containing IL-12p40 protein were generated by transducing human corneal endothelial cells with AdIL-12p40 and analysed for their capacity to inhibit production of IFN-gamma by naive T cells. The effect of both local (ex vivo Ad-mediated gene transfer) and systemic (i.p.-injection) over-expression of IL-12p40 was investigated by analysing the survival of corneal allografts transplanted from Wistar-Furth rats to fully MHC-class I/II incompatible Lewis rats. Moreover, the intra-graft mRNA-expression profile of cytokines and T cell markers was investigated at different time points after gene transfer. RESULTS: Adenovirus-mediated gene transfer in cultured corneas led to significant IL-12p40 protein expression as determined by specific ELISA. Moreover we could show that IL-12p40 protein containing supernatants significantly inhibited the production of IFN-gamma by alloreactive naive T cells. Interestingly, neither ex vivo genetic modification of cultured corneas before transplantation nor systemic AdIL-12p40 treatment of recipients receiving allogeneic corneas did improve corneal allograft survival. Real-time RT-PCR analysis of ex vivo modified cornea allografts on day 7 after transplantation showed significantly higher IL-4 mRNA-expression levels in the AdIL-12p40 group compared to the control group. Other significant differences in mRNA-expression levels of intra-graft CD3, CD25, IFN-gamma, TNF-alpha, and IL-10 could not be detected, neither on day 7 nor on the day of rejection. CONCLUSIONS: Despite the capacity of IL-12p40 protein to inhibit the production of IFN-gamma of naive T cells in vitro and some Th1/Th2 shift in vivo, no prolongation of allogeneic graft survival of both AdIL-12p40 modified rat corneas and systemically treated rats could be obtained after transplantation. The possible binding of Ad-mediated IL-12p40 with ubiquitously expressed IL-12p35 in vivo might therefore limit the application of IL-12p40 for the prevention of transplant rejection.

Effect of allergic conjunctival inflammation on the allogeneic response to donor cornea.

PURPOSE: Immunologic rejection is the most common cause of corneal allograft rejection. Ipsilateral ocular inflammation has been identified as a predictor of future corneal graft failure. This study investigates the effect of perioperative allergic conjunctivitis on corneal allograft survival. METHODS: C57BL6 donor corneas were transplanted into naive A/J mice, A/J mice sensitized to short ragweed (SRW) pollen by intraperitoneal injection and then challenged with topical SRW to induce allergic conjunctivitis (Sens(+)Chall(+)), and A/J mice sensitized to SRW and challenged with topical PBS (Sens(+)Chall(-)). Syngeneic grafts were also performed in eyes with allergic conjunctivitis. Graft survival and infiltrating cell phenotype in rejected grafts were compared between groups. RESULTS: Mice with allergic conjunctivitis (Sens(+)Chall(+)) rejected corneal allografts significantly more quickly than naive mice. Syngeneic grafts in allergic eyes survived indefinitely. The rate of rejection in Sens(+)Chall(-) mice was similar to that in naive mice. There were no significant differences, between groups, in the numbers of infiltrating CD4(+) cells, CD8(+) cells, and macrophages at the time of graft rejection. Eosinophils were seldom observed in rejected grafts in naive and Sens(+)Chall(-) mice but were observed consistently in Sens(+)Chall(+) eyes. Eosinophils were also found consistently in the ciliary body of Sens(+)Chall(+) eyes at the time of graft rejection. CONCLUSIONS: Active allergic conjunctivitis at the time of transplantation accelerates corneal allograft rejection. Local conjunctival inflammation is an important factor in accelerating rejection.

Immunological responses in mice to full-thickness corneal grafts engineered from porcine collagen.

Tissue-engineered (TE) corneas were fabricated from porcine collagen cross-linked with 1-ethyl-3-(3-dimethyl aminoproplyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and were transplanted into BALB/c mice orthotopically using a full-thickness penetrating keratoplasty (PKP) procedure. The biocompatibility was evaluated by assessing both local and systemic immune responses. Myeloid cells including granulocytes and macrophages were the main infiltrating cells in recipient cornea and in retro-TE corneal membrane which developed 7-10 days post surgery. Sodium citrate was found to be effective in reducing fibrin accumulation in anterior chamber post grafting at early time points, but it did not prevent formation of the retro-TE corneal membrane. No significant T cell activation was observed in the submandibular draining lymph nodes (SMDLN) by flow cytometry. Anti-porcine type I collagen IgG antibodies were detected in the serum of grafted mice from 2 weeks post grafting and the concentration of antibodies increased with time. Overall, porcine collagen-EDC/NHS TE corneas were tolerated well in murine recipients, causing mainly a self-limiting local innate immune response and a low-grade humoral response with little evidence of sustained T cell activation. Retro-TE corneal membrane formation was the main complication and barrier to clarity.

[Typing for HLA matching. Advantages for keratoplasty]. / Typisierung beim HLA-Matching. Vorteile für die Keratoplastik.

Matching of human leukocyte antigens of donors and recipients (HLA matching) plays a significant role in kidney, heart, lung, and stem cell transplantation. New data demonstrate that HLA matching also significantly prolongs the survival of corneal grafts. The reasons for the late recognition of the significance of HLA matching in corneal transplantation are on the one hand the immune privilege of the eye, which allows corneal transplantation under certain conditions without immunosuppressive therapy, and on the other hand highly erroneous serological typing. Recent molecular DNA typing is almost without errors, and the selection of grafts on the basis of results obtained using this method will improve the long-time survival of future corneal grafts. Today, there are several reasons arguing for the general practice of HLA matching in keratoplasty.

Blockade of the 4-1BB (CD137)/4-1BBL and/or CD28/CD80/CD86 costimulatory pathways promotes corneal allograft survival in mice.

To explore the roles of 4-1BB (CD137) and CD28 in corneal transplantation, we examined the effect of 4-1BB/4-1BB ligand (4-1BBL) and/or CD28/CD80/CD86 blockade on corneal allograft survival in mice. Allogeneic corneal transplantation was performed between two strains of wild-type (WT) mice, BALB/c and C57BL/6 (B6), and between BALB/c and B6 WT donors and various gene knockout (KO) recipients. Some of the WT graft recipients were treated intraperitoneally with agonistic anti-4-1BB or blocking anti-4-1BBL monoclonal antibody (mAb) on days 0, 2, 4 and 6 after transplantation. Transplanted eyes were observed over a 13-week period. Allogeneic grafts in control WT B6 and BALB/c mice treated with rat immunoglobulin G showed median survival times (MST) of 12 and 14 days, respectively. Allogeneic grafts in B6 WT recipients treated with anti-4-1BB mAb showed accelerated rejection, with an MST of 8 days. In contrast, allogeneic grafts in BALB/c 4-1BB/CD28 KO and B6 CD80/CD86 KO recipients had significantly prolonged graft survival times (MST, 52.5 days and 36 days, respectively). Treatment of WT recipients with anti-4-1BB mAb resulted in enhanced cellular proliferation in the mixed lymphocyte reaction and increased the numbers of CD4(+) CD8(+) T cells, and macrophages in the grafts, which correlated with decreased graft survival time, whereas transplant recipients with costimulatory receptor deletion showed longer graft survival times. These results suggest that the absence of receptors for the 4-1BB/4-1BBL and/or CD28/CD80/CD86 costimulatory pathways promotes corneal allograft survival, whereas triggering 4-1BB with an agonistic mAb enhances the rejection of corneal allografts.

B7-H1-induced apoptosis as a mechanism of immune privilege of corneal allografts.

The programmed death-1 (PD-1) costimulatory pathway has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in establishing an immune privilege status of corneal allografts in mice. B7-H1, but not B7-DC or PD-1, was expressed constitutively in the eye, i.e., cornea, iris-ciliary body, and retina. After corneal allografting, PD-1(+)CD4(+) T cells infiltrated and adhered with B7-H1(+) corneal endothelium. Blockade of PD-1 or B7-H1, but not B7-DC, led to accelerated corneal allograft rejection. In B7-H1-expressing corneal allografts, apoptosis of the infiltrating PD-1(+)CD4(+) or CD8(+) T cells was observed, after which there was allograft acceptance. In contrast, B7-H1 blockade suppressed apoptosis of infiltrating PD-1(+) T cells, which led to allograft rejection. In vitro, destruction of corneal endothelial cells by alloreactive T cells was enhanced when the cornea was pretreated with anti-B7-H1 Ab. This is the first demonstration that the constitutive expression of B7-H1 plays a critical role in corneal allograft survival. B7-H1 expressed on corneal endothelial cells maintains long-term acceptance of the corneal allografts by inducing apoptosis of effector T cells within the cornea.

A historical perspective on immunological privilege.

Intimations of immunological privilege in sites of the body such as the eye and the brain go back in the literature more than a century, to reports of experiments using outbred animals and tumor transplants. The starting points of this review, however, are publications stemming from the transplantation of normal tissues and, as far as possible, the use of inbred animals, exploring the way in which interplay between genetic differences of different degree, from single minor histocompatibility antigens to full-house major histocompatibility complex mismatches, has been reported to affect the 'take' of grafts in putatively privileged sites. While these sites traditionally included the brain, the eye, the pregnancy, and the endocrine tissues such as thyroid, parathyroid, adrenal, and islets of Langerhans, from readings of the literature, it is clear that the eye and the pregnancy have claims to being in the strongest positions of privilege. Even then, the position is precarious, with stirrings of the adaptive immune system poised to attack. Various regulatory mechanisms have now moved center stage and will undoubtedly form a significant part in subsequent chapters in this volume. Perhaps surprisingly, as investigations on these mechanisms have advanced, there is evidence for the convergence of those mechanisms controlling both induced tolerance and immunological privilege.

A new model for limbal transplantation using E-GFP for follow-up of transplant survival.

Limbal transplants in humans show a high rate of rejection even under local and systemic immunotherapy. In order to test immunomodulatory treatments a new limbal transplant model in the rat was developed using enhanced green fluorescent protein (E-GFP) as marker for follow-up. Sixty E-GFP-positive limbal transplants from Sprague-Dawley TgN(act-EGFP)Osb4 rats were transplanted onto 18 wild-type inbred Sprague-Dawley (isografts) rats, six wild-type litter mate Sprague-Dawley (sibling) rats, 18 Fischer 344 (allografts) rats, and 18 Fischer 344 rats depleted from monocytes and macrophages by subconjunctival treatment with clodronate liposomes. All rats were monitored three times a week with fluorescence microscopy, until fluorescence had disappeared. At postoperative days 6, 9, 12, and 15, three rats of all groups were killed for immunohistochemical analysis of infiltrating cells. Using a modified digital fluorescence microscope, we were able to monitor transplant behavior over time without disturbance of the ocular surface. The average days of rejection were 14 days in the isograft group, the sibling group, and the untreated allograft group. However, the average day of rejection in the allogeneic macrophage-depleted group was 27 days. Marked infiltration of macrophages and lymphocytes was seen in the untreated isografts and allografts. In the clodronate liposome-treated allografts infiltration was minor. A successful new limbal transplant model is described. The transplant can be accurately followed up in vivo by E-GFP labeling of the donor tissue without disturbing the corneal surface. Although E-GFP itself proved to be immunogenic, local clodronate liposome injections significantly increased graft survival. So the model seems to be useful for testing immunosuppressive or modulatory agents in limbal transplantation studies.

Keratocyte apoptosis and failure of corneal allografts.

BACKGROUND: Murine models of high-risk and low-risk corneal transplantation were used to determine the role of keratocyte apoptosis in the failure of orthotopic allogeneic corneal transplants. MATERIALS AND METHODS: Normal (low-risk, low-rejecting) and inflamed/vascularized (high-risk, high-rejecting) BALB/c recipient beds received fully mismatched C57BL/6 corneal allografts. Apoptosis was detected in the corneal stroma at various time points using an in situ terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay, and ex vivo via Western analysis for active caspase-3. Apoptosis was also measured in a (donor-type) C57BL/6 keratocyte cell line after stimulation of Fas or via use of various pro-inflammatory cytokines. RESULTS: Significantly more apoptotic cells were present in the stroma of rapidly rejecting high-risk corneal allografts compared with low-risk grafts. Apoptotic cells were shown to be nearly uniformly CD45 and hence of a non-hematopoetic lineage. Apoptosis, however, was not present in highly inflamed but ungrafted corneas. Apoptosis was induced in keratocytes in vitro by dual stimulation with agonistic Fas mAb and either interleukin-1beta or tumor necrosis factor-alpha. CONCLUSION: Apoptosis of resident non-bone marrow-derived fibroblastic cells of the corneal stroma is strongly correlated with the failure of corneal allografts, particularly in the highly inflamed microenvironment of the high-risk allograft.

Differential roles of CD8+ and CD8- T lymphocytes in corneal allograft rejection in 'high-risk' hosts.

We examined the role of perforin and FasL in corneal allograft rejection mediated by CD8+ and CD8 T cells. BALB/c corneas were transplanted orthotopically into vascularized, 'high-risk' graft beds in C57BL/6 mice, perforin knockout mice and FasL-defective gld/gld mice. CD8+ and CD8 T cells were collected following graft rejection and adoptively transferred to SCID mice, which were then challenged with BALB/c corneal allografts. In every case, CD8 T cells could mediate graft rejection when adoptively transferred to SCID mice that received BALB/c corneal allografts. Although CD8+ T cells also mediated graft rejection, the tempo was slower. Moreover, CD8+ T cells collected FasL-defective donors that had rejected corneal allografts, mediated corneal allograft rejection in only 50% of the SCID mice that received the adoptively transferred cells. In some cases, CD8+ T-cell-mediated rejection occurred in the absence of delayed-type hypersensitivity and cytotoxic T-lymphocyte activity, but was associated with CD8+ T-cell-mediated apoptosis of BALB/c corneal cells in vitro. The results demonstrate the redundancy in immune mechanisms of corneal allograft rejection. Either CD8+ or CD8 T cells can produce corneal allograft rejection, however functional FasL is necessary for optimal rejection, even in a high-risk setting.

Function of indoleamine 2,3-dioxygenase in corneal allograft rejection and prolongation of allograft survival by over-expression.

Indoleamine 2,3-dioxygenase (IDO) suppresses T cell responses by its action in catabolising tryptophan. It is important in maintenance of immune privilege in the placenta. We investigated the activity of IDO in the cornea, following corneal transplantation and the effect of IDO over-expression in donor corneal endothelium on the survival of corneal allografts. IDO expression was analysed and functional activity was quantified in normal murine cornea and in corneas following transplantation as allografts. Low levels of IDO, at both mRNA and protein levels, was detected in the normal cornea, up-regulated by IFN-gamma and TNF. Expression of IDO in cornea was significantly increased following corneal transplantation. However, inhibition of IDO activity in vivo had no effect on graft survival. Following IDO cDNA transfer, murine corneal endothelial cells expressed functional IDO, which was effective at inhibiting allogeneic T cell proliferation. Over-expression of IDO in donor corneal allografts resulted in prolonged graft survival. While, on one hand, our data indicate that IDO may augment corneal immune privilege, up-regulated IDO activity following cytokine stimulation may serve to inhibit inflammatory cellular responses. While increasing IDO mRNA expression was found in allogeneic corneas at rejection, over-expression in donor cornea was found to significantly extend survival of allografts.

Thiol redox and immune regulation in corneal transplantation.

Penetrating keratoplasty (PKP) is the most common type of clinical grafting performed in humans. Although PKP has emerged as the most successful form of transplantation, PKP in "high-risk" eyes shows high incidence of allograft rejection. The incidence of epithelial rejection after limbal transplantation (LT) is extremely higher and swifter than PKP rejection, and even intensive systemic immunosuppressive therapy is often of no avail. Because failure of corneal grafts is an important cause of blindness, developing new strategies for suppressing graft rejection is a worthy goal for research. Corneal allograft rejection is mainly mediated by the TH1-type immune response, which leads to a delayed-type hypersensitivity reaction. Because the TH2-type immune response regulates the TH1-type immune response, we have successfully elicited allograft survival after both PKP and LT by inducing systemic TH2-type immune responses. Because intracellular thiol redox status of antigen-presenting cells (APC) reportedly regulates TH1/TH2 balance via distinctive cytokine production by APC, we also investigated the effect of modulating macrophage intracellular thiol redox status on corneal allograft survival. These strategies are quite effective in major histocompatibility complex (MHC) matching in mice, although it is believed that MHC matching has no effect on corneal allograft survival according to many rodent studies. Recently, many laboratories are reconsidering HLA matching for allograft survival in human corneal transplantation. It may be possible that MHC matching improves corneal allograft survival in the context of TH1 suppression. We propose that the suppression of the TH1-type immune response and MHC matching together may promote allograft survival in humans.

Prolongation of sheep corneal allograft survival by transfer of the gene encoding ovine IL-12-p40 but not IL-4 to donor corneal endothelium.

Immunological rejection is the major cause of human corneal allograft failure. We hypothesized that local production of IL-4 or the p40 subunit of IL-12 (p40 IL-12) by the grafted cornea might prolong allograft survival. Replication-deficient adenoviral vectors encoding ovine IL-4 or p40 IL-12 and GFP were generated and used to infect ovine corneas ex vivo. mRNA for each cytokine was detected in infected corneas, and the presence of secreted protein in corneal supernatants was confirmed by bioassay (for IL-4) or immunoprecipitation (for p40 IL-12). Sheep received uninfected or gene-modified orthotopic corneal allografts. Postoperatively, untreated corneas (n = 13) and corneas expressing GFP (n = 6) were rejected at a median of 21 and 20 days, respectively. Corneas expressing IL-4 (n = 6) underwent rejection at 18.5 days (p > 0.05 compared with controls) and histology demonstrated the presence of eosinophils. In contrast, corneas expressing p40 IL-12 (n = 9) showed prolonged allograft survival (median day to rejection = 45 days, p = 0.003). Local intraocular production of p40 IL-12 thus prolonged corneal graft survival significantly, but local production of the prototypic immunomodulatory cytokine IL-4 induced eosinophilia, inflammation, and rejection. These findings have important implications for the development of novel strategies to improve human corneal graft survival.