Orthotopic Corneal Transplantation Research Articles

Corneal Nerve Ablation Abolishes Ocular Immune Privilege by Downregulating CD103 on T Regulatory Cells.

PurposeSevering corneal nerves during orthotopic corneal transplantation elicits the elaboration of the neuropeptide substance P (SP), which induces the generation of CD11c+ contrasuppressor (CS) cells. CS cells disable T regulatory cells (Tregs) that are induced when antigens enter the anterior chamber (AC), either by direct injection or by orthotopic corneal transplantation. This study examined the crucial cell surface molecules on Tregs that are adversely affected by CS cells that are generated by severing corneal nerves.MethodsCS cells were induced by producing shallow 2.0-mm circular incisions in the corneal epithelium in BALB/c mice. CD8+ Tregs were generated by injecting ovalbumin into the AC. The effects of CS cells and SP on the expression and function of two cell surface molecules (CD103 and the receptor of interferon-γ) that are crucial for the induction and function of CD8+ Tregs were analyzed.ResultsSP converted CD11c+, but not CD11c−, dendritic cells (DCs) to CS cells. Severing corneal nerves resulted in a 66% reduction in the expression of CD103 on CD8+ AC-associated immune deviation (ACAID) Tregs, and a 50% reduction in the interferon-γ receptor (IFN-γR). These effects could be mimicked in vitro by coculturing CS cells with CD8+ ACAID Tregs.ConclusionsThe elaboration of SP in response to corneal nerve ablation converts CD11c+ DCs to CS cells. CS cells disable CD8+ ACAID Tregs by downregulating two crucial cell surface molecules, CD103 and IFN-γR, by an SP-dependent pathway. Blocking this pathway may provide a means of restoring ocular immune privilege in corneas subjected to corneal nerve injury.

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10.

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host's alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and interferon-γ and tumor necrosis factor-α-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independently of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.

Effect of Corneal Nerve Ablation on Immune Tolerance Induced by Corneal Allografts, Oral Immunization, or Anterior Chamber Injection of Antigens.

PurposeSevering corneal nerves during corneal transplantation does not affect first corneal transplants, but abolishes immune privilege of subsequent corneal allografts. This abrogation of immune privilege is attributable to the disabling of T regulatory cells (T regs) induced by corneal transplantation. The goal of this study was to determine if severing corneal nerves induces the development of contrasuppressor (CS) cells, which disable T regs that impair other forms of immune tolerance.MethodsEffect of corneal nerve ablation on immune tolerance was assessed in four forms of immune tolerance: anterior chamber–associated immune deviation (ACAID); oral tolerance; corneal transplantation, and intravenously (IV) induced immune tolerance. T regulatory cell activity was assessed by adoptive transfer and by local adoptive transfer (LAT) of suppression assays.ResultsCorneal nerve ablation prevented ACAID and oral tolerance, but did not affect IV-induced immune tolerance. Contrasuppressor cells blocked the action of T regs that were generated by anterior chamber injection, oral tolerance, or orthotopic corneal transplantation. The neuropeptide substance P (SP) was crucial for contrasuppressor activity as CS cells could not be induced in SP−/− mice and the SP receptor inhibitor, Spantide II, prevented the expression of CS cell activity in vivo. Contrasuppressor cells expressed CD11c surface marker that identifies dendritic cells (DC).ConclusionsThe loss of immune privilege produced by corneal nerve ablation following corneal transplantation extends beyond the eye and also affects immune tolerance induced through mucosal surfaces and appears to be mediated by a novel cell population of CD11c+ CS cells that disables T regs.

Oxidative stress and premature senescence in corneal endothelium following penetrating keratoplasty in an animal model.

BackgroundThe purpose of this study was to address the question of how the premature senescence process may affect corneal endothelium after penetrating keratoplasty, because the quality of donor corneal endothelial cells is important for corneal transplant success.MethodsThe cell senescence and induced oxidative stress in corneal endothelium were assessed using a normal-risk orthotopic mice corneal transplantation model. Senescence associated beta-galactosidase (SA-beta-Gal) staining was used to evaluate premature senescence in the endothelium of corneal allografts. Oxidative Stress and Antioxidant Defense RT2-PCR Arrays and in vitro experimental model using H2O2 treatment were used to investigate the possible mechanism.ResultsSA-beta-Gal positivity was observed obviously in mice corneal endothelium of allogenic group and the levels of p16INK4a message and protein increased in endothelium of allogenic group compared to syngenic group. By PCR array, an oxidant-antioxidant imbalance was found in the endothelium of corneal allograft after PKP. The results from mice model were validated using human endothelium samples of corneal allograft after PKP. We also developed an in vitro experimental model using H2O2 treatment to simulate a state of oxidative stress in cultured human corneal endothelial cells (HCECs) and found that elevated ROS levels, the up-regulation of CDK inhibitors and ROS-mediated p16INK4A up-regulation in HCECs occur via the ASK1-p38 MAPK pathway.ConclusionsOur results demonstrate the presence of oxidative stress and premature senescence in the endothelium of corneal allografts following PKP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12886-016-0192-6) contains supplementary material, which is available to authorized users.

Effects of CD25siRNA gene transfer on high-risk rat corneal graft rejection.

Corneal graft rejection is the major cause of corneal failures. Previous studies have shown that the CD25 monoclonal antibody can inhibit corneal graft rejection during the acute phase of rejection in rat models. In the current study, we evaluated the safety and efficacy of the topical Entranster™ vector in rat corneal applications and further investigated the effects of CD25siRNA gene transfer on high-risk rat corneal graft rejection. Fluorescence detection, clinical assessment, hematoxylin and eosin (HE), terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and CD11b assays were used to evaluate the safety and efficacy of CD25siRNA gene transfer in normal SD rat corneas. Orthotopic corneal transplants were performed in alkali burned SD rats. Corneal recipients were divided into four groups that received different treatments. Clinical assessment, western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, HE and transmission electron microscopy (TEM) were performed on all grafts. Low toxicity, no immunogenicity and high transfection efficiency were observed in rat corneas treated with the Entranster™ vector. Reduced endothelial cell apoptosis, inflammatory cell infiltration and graft neovascularisation were observed in the CD25siRNA treatment groups. The graft survival curves showed that CD25siRNA treatment significantly prolonged graft survival time, with better graft transparency and less graft oedema. Lower CD25 and higher IL-10 expression were detected in the CD25siRNA treatment groups during the study period, and a higher FOXP3 level was found in the CD25siRNA group than in the CD25siRNA-twice group on days 14 and 21 post-operation. The Entranster™ vector is an effective vector for corneal gene therapy. CD25 siRNA gene transfer inhibits corneal graft rejection via upregulation of anti-inflammatory molecule expression.

Mechanism of immune tolerance induced by donor derived immature dendritic cells in rat high-risk corneal transplantation.

To study the role of immature dendritic cells (imDCs) on immune tolerance in rat penetrating keratoplasty (PKP) in high-risk eyes and to investigate the mechanism of immune hyporesponsiveness induced by donor-derived imDCs. Seventy-five SD rats (recipient) and 39 Wistar rats (donor) were randomly divided into 3 groups: control, imDC and mature dendritic cell (mDC) group respectively. Using a model of orthotopic corneal transplantation in which allografts were placed in neovascularized high-risk eyes of recipient rat. Corneal neovascularization was induced by alkaline burn in the central cornea of recipient rat. Recipients in imDC group or mDC group were injected donor bone marrow-derived imDCs or mDCs of 1×10(6) respectively 1 week before corneal transplantation via tail vein. Control rat received the same volume of PBS. In each group, 16 recipients were kept for determination of survival time and other 9 recipients were executed on day 3, 7 and 14 after transplantation. Cornea was harvested for hematoxylin-eosin staining and acute rejection evaluation, Western blot was used to detect the expression level of Foxp3. The mean survival time of imDC group was significantly longer than that of control and mDC groups (all P<0.05). The expression level of Foxp3 on CD4(+)CD25(+)T cells of imDC group (2.24±0.18) was significantly higher than that in the control (1.68±0.09) and mDC groups (1.46±0.13) (all P<0.05). Donor-derived imDC is an effective treatment in inducing immune hyporesponsiveness in rat PKP. The mechanism of immune tolerance induced by imDC might be inhibit T lymphocytes responsiveness by regulatory T cells.

Lentivirus‐mediated gene transfer of interleukin 10 to the ovine and human cornea

Gene transfer to a donor cornea ex vivo can modulate corneal graft failure in experimental animal models. We compared a lentiviral vector (LV) carrying the transgene ovine interleukin 10 (IL10) with a comparable adenoviral vector (Ad) for its ability to transduce ovine and human corneas and to modulate ovine corneal allograft survival. The LV carrying the ovine IL10 gene was used to transduce ovine and human corneas in vitro. LV-mediated gene expression in corneal endothelium was assessed by real-time quantitative reverse-transcriptase polymerase chain reaction, at varying doses and duration of transduction. The effect of ex vivo transduction of the donor cornea with LV-SV40-IL10 was assessed following orthotopic corneal transplantation in outbred sheep. Expression of IL10 mRNA in Ad-CMV-IL10-transduced ovine corneas was 10(3)-fold higher than in LV-SV40-IL10-transduced corneas (P < 0.0001), and 10(7)-fold higher than in non-transduced controls. IL10 was secreted rapidly from Ad-CMV-IL10-transduced, organ-cultured corneas, peaking at 13-15 days. IL10 secreted from LV-SV40-IL10-transduced corneas increased 20-fold compared with controls, but had not reached a plateau at 15 days. Gene expression driven by LV-SV40-IL10 varied with vector dose and transduction time, but was less than with Ad-CMV-IL10 at both mRNA and protein levels. Gene expression driven by LV-SV40-IL10 was faster in the human cornea than the ovine cornea. Corneal allograft survival was prolonged by a median of 7 days in the LV-SV40-IL10-treated recipients, compared with the control group (P = 0.026). Although lentiviral vectors show some promise for corneal gene therapy, they are less efficient than adenoviral vectors.

Local treatment with alpha-melanocyte stimulating hormone reduces corneal allorejection.

Corneal grafting is by far the most common form of transplantation. Many grafts suffer from immune rejection and current therapies are associated with many side effects, requiring more effective and safe therapies. alpha-Melanocyte stimulating hormone (alpha-MSH) is a neuropeptide that suppresses host inflammatory defense mechanisms. The purpose of this study was to determine the role of local therapy with alpha-MSH on corneal allograft survival, and the mechanisms by which it may influence graft outcome. Orthotopic corneal transplantation was performed, with recipients receiving subconjunctival alpha-MSH or sham injections twice weekly. Grafts were followed up for 70 days, and graft inflammation/opacification was compared between the two groups in a masked fashion. Graft infiltration and ocular gene expression of select inflammatory cytokines was evaluated at different timepoints. Additionally, allospecific delayed-type hypersensitivity was compared among the groups 3 weeks posttransplantation. Results showed a significant increase in corneal graft survival in alpha-MSH-treated recipients compared with controls. Although 75% of allografts in alpha-MSH-treated hosts survived at 70 days, 43% survived in controls (P=0.04). Graft infiltration studies demonstrated a significant decrease in the number of mononuclear and polymorphonuclear cells in alpha-MSH-treated mice compared with controls at days 7 and 14 after transplantation. Furthermore, allospecific delayed-type hypersensitivity and gene expression of interferon-gamma and interleukin-2 showed a significantly reduced expression in alpha-MSH-treated mice compared with controls. This study provides for the first time, in vivo evidence that treatment with local alpha-MSH may significantly reduce allorejection of orthotopic transplants.

The Role of Cyclosporine and Mycophenolate in an Orthotopic Porcine-to-Rat Corneal Xenotransplantation

We performed this study to investigate the feature of rejection in porcine-to-rat corneal orthotopic transplantation and to evaluate the effect of cyclosporine and mycophenolate on the xeno-rejection. Orthotopic corneal transplantation was done at 91 Sprague-Dawley rats, and they were divided into 10 groups based on the combination of immunosuppressants including dexamethasone, cyclosporine, and mycophenolate mofetil. Graft survival was analyzed and grafted eyes were examined with Hematoxylin & Eosin and CD4 or CD8 staining. Enzyme-linked immunosorbent assays were done for interleukin-2 (IL-2), IL-4, IL-5, IL-10, and interferon (IFN)-γ in cornea, lacrimal gland, and cervical lymph nodes. The longest median survival of the immune suppressant group was 11.00±1.96 days, which showed no statistical differences compared with that of control (8.00±1.52 days). The neutrophils were prominent in the early phase but soon gave way to the monocytes. The number of CD8+ cells was higher than that of CD4+ cells. IL-2 and IFN-γ markedly increased at 10 to13 days in cornea, lacrimal glands, and cervical lymph nodes, which showed a decrease with immunosuppressants except in the cornea. In conclusion, cyclosporine and mycophenolate could not prevent the rejection in porcine to rat orthotopic corneal xenograft associated with infiltraton of CD8+ and innate immune cells.

CD4+CD25+ T cells involved in induction of rat corneal allograft immune tolerance

To explore the expression of CD4+CD25+ regulatory T cells in corneal graft, in the iris and peripheral blood in the high risk rat corneal transplant tolerance induced by superantigen staphylococcal enterotoxin B (SEB). A high risk rat penetrating corneal transplantation model was established, whereas SD rat donor corneas were implanted into Wistar rat recipients. The recipient rats were divided into two groups. Group control and group SEB had intraperitoneal injection of normal saline solution or SEB (75 microg/kg), respectively. Orthotopic corneal transplantation was performed 1 day after the injection. The corneal graft survival time was evaluated. The expression of CD4+CD25+ T cells in corneal grafts and the iris was evaluated by immunohistochemical staining. The percentage of CD4+CD25+ regulatory T cells in peripheral blood was determined by flow cytometry. Corneal graft mean survival time was (11.63+/-2.83) d in group control and (14.13+/-0.99) d in group SEB. The difference of survival time between these two groups was statistically significant (P<0.05). Histopathology showed remarkable inflammatory cells infiltration in the rejected grafts. The intensity of the infiltration was compatible with the severity of rejection reaction. Immunohistochemical staining of grafts showed that the CD4+CD25+ regulatory T cells were expressed in rejected grafts in both groups 20 days after the surgery, but not before the surgery. The iris whole mount staining showed that before the surgery, no expression of CD4+CD25+ regulatory T cells was observed in group control but relatively remarkable expression was observed in group SEB. Twenty days after the transplantation, CD4+CD25+ regulatory T cells were observed in both groups while group SEB had more pronounced expression. Peripheral blood flow cytometry analysis revealed that the mean percentages of CD4+CD25+ regulatory T cells were stable before and after surgery in group control. However, in group SEB, an increase shift appeared and followed by a decrease to normal level 20 d after the surgery. CD4+CD25+ regulatory T cells take part in immune tolerance in rat immune tolerance induced by superantigen SEB in high risk rat corneal transplantation.

Latanoprost does not affect immune privilege of corneal allografts

The present study determined whether topical latanoprost, a prostaglandin (PG) F2α analog, influences the induction of anterior chamber-associated immune deviation (ACAID), corneal neovascularization (NV) or survival of corneal allografts in mice. BALB/c mice received topical latanoprost or PGE2 once or multiple times daily starting 4weeks prior to or the day of anterior chamber injection of C57BL/6 splenocytes. Induction of allo-specific ACAID was assessed by ear challenge with C57BL/6 splenocytes 1week after subcutaneous immunization. In a separate experiment, orthotopic corneal transplantation was performed using C57BL/6 mice as donors and BALB/c mice as recipients. Recipients were randomized in a masked fashion to receive topical latanoprost or PGE2. Graft fate was assessed clinically under surgical microscopy. Presence of MHC class II+ CD11c+ or CD11b+ cells in normal BALB/c mouse eyes following latanoprost or PGE2 administration was assessed immunohistochemically. Control mice received topical 20% dimethyl sulfoxide or no treatment. Allo-specific ACAID was induced after 2 or 6weeks of once daily treatment with latanoprost, and was induced even after 6weeks of multiple treatments with latanoprost. Conversely, mice receiving PGE2 failed to develop ACAID. Opacity and corneal NV scores for allografts treated with latanoprost were statistically indistinguishable from those for control allografts (p>0.05), whereas all allografts treated with PGE2 were rejected. Opacity and NV scores were significantly higher in these allografts than in controls (p<0.05). A number of MHC class II+ CD11c+ cells were present in the central cornea after PGE2 treatment. Topical application of latanoprost does not influence induction of ACAID or graft outcomes including opacity and NV, whereas PGE2 does. Immune privilege of corneal allograft is maintained after topical latanoprost application in mice.

Galα(1‐3)Gal expression of the cornea in vitro, in vivo and in xenotransplantation

To investigate alpha Gal (Gal alpha1-3Gal beta 1-4GlcNAc-R) expression of the porcine cornea in vitro, in vivo and after xenotransplantation. Using the GS-IB4 lectin (Griffonia simplifolia I isolectin B4), the expression of alpha Gal was evaluated in the normal porcine cornea and in cultured corneal stromal and endothelial cells of the second passages. The distribution of alpha Gal epitopes was also evaluated in corneal grafts at 4, 7 and 10 days after pig-to-rat orthotopic corneal transplantation. The expression of alpha Gal was mostly confined to the anterior stromal keratocytes of the normal porcine cornea. However, reactivity for GS-IB4 was markedly increased during cell culture passage (P)-by 18.0% (P1) and 39.0% (P2) in cultivated keratocytes, and by 62.1% (P1) and 87.1% (P2) in cultured endothelial cells. The expression of alpha Gal epitopes was gradually enhanced in all corneal layers of the graft after transplantation. Alpha Gal expression in the cornea was gradually induced during in vitro culture and after xenotransplantation, suggesting a role of putative Gal-related acute humoral rejection in porcine corneal xenotransplantation.

The Function of Donor versus Recipient Programmed Death-Ligand 1 in Corneal Allograft Survival

Programmed death-ligand (PD-L)1 and PD-L2, newer B7 superfamily members, are implicated in the negative regulation of immune responses and peripheral tolerance. To examine their function in alloimmunity, we used the murine model of orthotopic corneal transplantation. We demonstrate that PD-L1, but not PD-L2, is constitutively expressed at high levels by the corneal epithelial cells, and at low levels by corneal CD45+ cells in the stroma, whereas it is undetectable on stromal fibroblasts and corneal endothelial cells. Inflammation induces PD-L1 up-regulation by corneal epithelial cells, and infiltration of significant numbers of PD-L1+CD45+CD11b+ cells. Blockade with anti-PD-L1 mAb dramatically enhances rejection of C57BL/6 corneal allografts by BALB/c recipients. To examine the selective contribution of donor vs host PD-L1 in modulating allorejection, we used PD-L1-/- mice as hosts or donors of combined MHC and minor H-mismatched corneal grafts. BALB/c grafts placed in PD-L1-/- C57BL/6 hosts resulted in pronounced T cell priming in the draining lymph nodes, and universally underwent rapid rejection. Allografts from PD-L1-/- C57BL/6 donors were also significantly more susceptible to rejection than wild-type C57BL/6 grafts placed into BALB/c hosts, primarily as a result of increased T cell infiltration rather than enhanced priming. Taken together, our results identify differential roles for recipient vs donor PD-L1 in regulating induction vs effector of alloimmunity in corneal grafts, the most common form of tissue transplantation, and highlight the importance of peripheral tissue-derived PD-L1 in down-regulating local immune responses.

A New Model of Corneal Transplantation in the Miniature Pig: Efficacy of Immunosuppressive Treatment

Corneal allograft rejection is frequently studied in small rodent or rabbit models. To study mechanisms of rejection in a model that more closely mimics transplantation in humans, we performed orthotopic corneal transplantation in the miniature pig using a 7-mm diameter donor graft. Four groups of recipients were studied: 1) untreated naive, 2) untreated vascularized (high risk), 3) high-risk grafts treated by topical application of prednisolone, or 4) high-risk grafts treated with a combined systemic immunosuppression regime of oral prednisone, cyclosporine A, and mycophenolate mofetil. Both the clinical features and histological assessment of corneal graft rejection showed close similarities to graft rejection in humans. Interestingly, preliminary results indicated that topical steroid treatment was superior to systemic immunosuppression in significantly promoting graft survival. Thus, corneal transplantation in the pig represents an animal model most closely resembling corneal grafting in humans, and offers possibilities for testing various clinically applicable immunosuppressive treatments.

Lentivirus-mediated gene transfer to the rat, ovine and human cornea

Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80-90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and >60 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo.

Keratocyte Apoptosis and Failure of Corneal Allografts

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. 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. 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. 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.

MHC Class I and II Antigens as Targets of Rejection in Penetrating Keratoplasty in Low- and High-Risk Mouse Eyes

To determine the extent to which expression of major histocompatibility complex (MHC) class I and II molecules contributes to rejection of orthotopic corneal transplants in mice. Full-thickness corneas, prepared from eyes of normal C57BL/6 (B6) and BALB/c mice and from B6 mice in which the class II gene I-A or the beta2-microglobulin (beta2mu) gene was disrupted, were placed orthotopically in low- or high-risk eyes of BALB/c (fully incompatible), BALB.B [minor histoincompatible (H) only], and bm12 (class II only disparate) recipients. BALB/c grafts were placed in low-risk eyes of normal B6 and B6 mice with disrupted H-2 DMalpha genes. Graft survival was judged by clinical examination. Recipient-identical class II, but not class I, molecules on fully allogeneic corneas grafted to low-risk beds promoted graft rejection. Allogeneic class II molecules on fully allogeneic corneas placed in high-risk beds promoted graft rejection more strongly than did allogeneic class I molecules. Neither allogeneic class I molecules nor recipient-identical class II molecules on grafts placed in high-risk beds contributed to graft outcome. Mice deficient in H-2 DMalpha failed to reject fully incompatible cornea grafts. On corneal allografts, where minor H antigens are the major barriers to acceptance, allogeneic class II molecules promote rejection if the graft is placed in high-risk eyes, whereas recipient-type class II molecules promote rejection if the graft is placed in low-risk eyes. Allogeneic class I molecules make a minor contribution to rejection only if the grafts are placed in high-risk eyes.

New thoughts on the immunology of corneal transplantation.

Significant advances derived from rodent models of penetrating keratoplasty have transformed our understanding of the pathogenesis of rejection of orthotopic corneal transplants. The high rate of success of corneal allografts placed in low-risk eyes without cover of immunosuppression arises from immune privilege of the cornea graft itself, and of the anterior chamber where it forms the anterior wall. Immune privilege owes its existence in penetrating keratoplasty to an absence of blood and lymph vessels in the graft and its bed, the absence of MHC class II(+) antigen presenting cells in the graft, reduced expression of MHC-encoded alloantigens on graft cells, constitutive expression of T cell-deleting CD95 ligand on corneal graft endothelium, the existence of an immunosuppressive local microenvironment (aqueous humor), and the capacity of the graft to induce anterior chamber associated immune deviation (ACAID). The results of recent experiments provide answers to pertinent questions regarding cornea graft failure: How does the cornea as a graft suppress inflammation and angiogenesis locally? How does the graft promote ACAID to the alloantigens it expresses? and How do corneal cells reduce their vulnerability as targets of effector T cells? The answers offer the possibilities of novel strategies for preventing immune-based corneal allograft failure.

An immunodominant minor histocompatibility alloantigen that initiates corneal allograft rejection.

Murine orthotopic corneal allografts experience immune privilege and have good survival as compared with skin allografts. However, privilege is not complete, and some grafts are still rejected. Unexpectedly, corneas expressing minor histocompatibility (H) alloantigens are rejected at a higher rate than major histocompatibility complex (MHC) disparate grafts. We hypothesize that certain immunodominant minor H alloantigens are extremely immunogenic when expressed in corneal tissue, terminate ocular immune privilege, and initiate corneal allograft rejection. Corneal allograft survival and the role of CD4+ T cells and CD8+ T cells were examined in corneal transplants that expressed genetically defined minor H3 alloantigens. The H3 locus contains at least two minor H genes. H3a is presented by MHC class I and recognized exclusively by CD8+ T cells. H3b is presented by class II and recognized exclusively by CD4+ T cells. Congenic strains that differ from C57BL/10 at (1) H3a, (2) H3b, or (3) H3a+H3b were used for orthotopic corneal and skin transplants. Donor corneas expressing either H3a or H3a+H3b experienced immune privilege and survived longer than skin allografts. By contrast, donor corneas expressing H3b (recognized by CD4+ T cells) experienced vigorous rejection and were eliminated faster than skin allografts. There are minor H alloantigens that terminate ocular immune privilege and initiate corneal allograft rejection. These minor H alloantigens are more immunogenic when expressed in corneal tissue than when they are expressed in skin allografts.

Effect of Locally Administered Anti-CD154 (CD40 Ligand) Monoclonal Antibody on Survival of Allogeneic Corneal Transplants

To determine the effect of ocular administration of anti-CD154 monoclonal antibody on the survival of orthotopic murine corneal transplants. BALB/c mice were used as recipients of multiple minor H- and MHC-mismatched orthotopic corneal transplants. Recipient beds were either avascular (normal-risk) or neovascularized (high-risk) at the time of surgery. Mice were randomized to receive either anti-CD154 antibody or control immunoglobulin by subconjunctival injection. All grafts were evaluated for signs of rejection by slitlamp biomicroscopy until week 20-24 with the therapy tapered and discontinued after week 8 and week 12, respectively. In normal-risk transplantation, the 8-week survival rate improved from 30% in control mice to 90% in anti-CD154 treated mice (p= 0.0061). In high-risk transplantation, the survival rate of anti-CD154-treated mice was enhanced to 55% compared with 0% in control mice at week 8 (p= 0.0184); however, tapering and termination of anti-CD154 led to some loss in graft survival, with a survival rate of 56% in normal-risk recipients, and 22% in high-risk recipients by week 20. Anti-CD40L treated animals displayed lower grades of postoperative corneal neovascularization (p<0.05), in particular in normal-risk recipients. Local ocular administration of anti-CD154 is effective in the prevention of corneal allograft rejection in normal-risk recipients, and in delaying the incidence of rejection in high-risk recipients. Long-term graft survival may not be fully achieved following termination of the CD40-CD154 pathway blockade.