Corneal Allograft Survival Research Articles

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.

Decreased active TGF-β2 levels in the aqueous humour during immune reactions following penetrating keratoplasty

The secretion of transforming growth factor (TGF)-beta (2) into the aqueous humour is important for maintaining the immunological privilege of the anterior ocular segment that promotes corneal allograft survival. Levels of total TGF-beta (2), however, were similar in eyes with and without immune reactions following penetrating keratoplasty (PK). In this study, we tested the hypothesis whether the activated form of TGF-beta (2) is decreased in eyes with immune reactions following PK. Anterior chamber puncture was performed in 38 cataract patients without PK (group I), in 10 patients without immune reaction following PK (group II), and in 10 patients following PK with newly diagnosed endothelial immune reaction (group III). About 0.05-0.1 ml of aqueous humour were harvested from each patient. Analysis of active TGF-beta (2) was started via ELISA within 3 h following puncture. Average active TGF-beta (2) concentration was 38.9 pg/ml+/-24.3 in group I, 47.4 pg/ml+/-23.1 in group II, and 12.7+/-26.0 pg/ml in group III. The differences between groups I and III (P=0.005) and groups II and III (P=0.008) were statistically significant. The lowest levels of active TGF-beta (2) were measured in eyes with newly diagnosed endothelial immune reactions. This suggests that TGF-beta (2) has a protective effect on corneal grafts following PK. Further (prospective) studies are being carried out to discover whether active TGF-beta (2)--determined prior to PK--might serve as a predictive parameter for the occurrence of immune reactions.

Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl‐xL

Corneal transplants normally enjoy a high percentage of survival, mainly because the eye is an immune-privileged site. When allograft failure occurs, it is most commonly due to rejection, an immune-mediated reaction that targets the corneal endothelium. While the exact mechanism by which the endothelium is targeted is still unknown, we postulate that corneal endothelial cell loss during allograft failure is mediated by apoptosis. Furthermore, because corneal endothelial cells do not normally regenerate, we hypothesize that suppressing apoptosis in the graft endothelium will promote transplant survival. In a murine model of transplantation, TUNEL staining and confocal microscopy showed apoptosis of the graft endothelium occurring in rejecting corneas as early as 2 weeks posttransplantation. We found that bcl-xL protected cultured corneal endothelial cells from apoptosis and that lentiviral delivery of bcl-xL to the corneal endothelium of donor corneas significantly improved the survival of allografts. These studies suggest a novel approach to improve corneal allograft survival by preventing apoptosis of the endothelium.

Comparative analysis of donor derived, gene modified dendritic cells in keratoplasty

Abstract Purpose: Dendritic cells (DCs) play an essential role during the initiation of immune responses against non‐self antigens. Following organ transplantation, activated donor‐ and recipient‐derived DCs participate actively in graft rejection by sensitising recipient T cells. There is increasing evidence that immature DCs induce antigen‐specific unresponsiveness to self antigens in central lymphoid tissue and in the periphery through different mechanisms. Methods: In this study, we compared the immunoregulatory properties of CTLA4‐Ig and vIL‐10 secreting DC in a high responder corneal transplantation model (BALB/c; H‐2d into C57BL/6; H‐2b). Both the in vitro and in vivo immune response to this approach were monitored. Results: Transduction of DC with AdCTLA4‐Ig prior to LPS stimulation dramatically diminished their allostimulatory capacity in vitro, as demonstrated by an inhibition of proliferation of cocultured allogeneic T cells. Compared to controls, AdvIL‐10 transduced DC also showed a significant decrease in their ability to activate T cells. Whereas adoptive transfer of CTLA4‐Ig transduced DC could not prolong the mean survival time (MST) (MST= 9.7 ± 3.9 days, p = 0.45, n = 6), vIL‐10 expressing DC significantly prolonged corneal allograft survival (MST = 13.4 ± 5.0 days, p = 0.019, n = 7) as compared to controls (MST = 8.3 ± 2.6 days, n = 7). Conclusions: Whereas adoptive transfer of CTLA4‐Ig transduced DC could not prolong the mean survival time (MST) (MST= 9.7 ± 3.9 days, p = 0.45, n = 6), vIL‐10 expressing DC significantly prolonged corneal allograft survival (MST = 13.4 ± 5.0 days, p = 0.019, n = 7) as compared to controls (MST = 8.3 ± 2.6 days, n = 7).

Pharmacologic strategies in the prevention and treatment of corneal transplant rejection

Corneal transplantation remains one of the most successful organ transplantation procedures in humans. The unique structure of the cornea, with its absence of blood vessels and corneal lymphatic, allows the survival of corneal allograft. Recent advances in sutures, storage media, microsurgical instrumentation, and new pharmacological strategies have greatly improved the success of corneal transplantation and the prevention of corneal allograft rejection. Our strategies in the management and prevention of corneal graft rejection can modify and improve the survival of corneal allografts. Preoperative evaluation, understanding the risk factors, and management of ocular surface disorders may greatly improve the survival of the corneal transplant. Early recognition of corneal allograft rejection and aggressive treatment may improve the survival of the corneal graft. Furthermore, patients who undergo corneal transplantation should be maintained under close ophthalmic surveillance and patients should be informed to report immediately whenever symptoms of corneal graft rejection occur. The mainstay of therapy is topical corticosteroids. In severe cases, periocular, intravenous, and oral corticosteroids therapy can be rendered. New therapeutic modalities such as cyclosporine, tacrolimus, daclizumab, mycophenolate mofetil, leflunomide, rapamycin, and others may prove to be of help in the prevention and treatment of corneal graft rejection. Early recognition of corneal graft rejection and prompt treatment are mandatory for the successful survival of the corneal allograft.

The influence of inducible costimulator fusion protein (ICOSIg) gene transfer on corneal allograft survival

The purpose of this paper is to analyse the effects of local or systemic administration of adenovirus type 5 encoding the inducible costimulator fusion protein (AdICOSIg) on its influence on prolonging corneal allograft survival. The ICOSIg chimeric molecule was generated by fusing the murine ICOS to a rat FcIgG portion and a recombinant adenovirus (Ad) was made thereof. A major histocompatibility complex (MHC) class I/II mismatched rat corneal transplant model was used. The recipients were randomly assigned to receive ex vivo gene-modified corneas expressing either ICOSIg or a single i.p. injection (1.0 x 10(9) infectious particles) of AdICOSIg two days after transplantation and graft survival was analysed. Moreover, the influence of ICOSIg fusion protein on anti-adenovirus immunity also was investigated. The ex vivo gene transfer of ICOSIg in cultured corneas resulted in high levels of ICOSIg protein in culture supernatants. However, neither ex vivo nor systemic gene therapy resulted in a significant prolongation of graft survival. Interestingly, the generation of anti-adenovirus antibodies could not be inhibited by systemic ICOSIg fusion protein expression. Unlike CTLA4Ig, sole ICOSIg gene therapy is not a successful strategy for the prevention of allogeneic graft rejection in corneal transplantation.

Effects of a small non-peptidic CD4 inhibitor J2 in prevention of mouse corneal allograft rejection

To investigate the effects of small non-peptidic CD4 inhibitor J2 on the prevention of allograft rejection in mouse corneal transplantation. Twenty-three C57BL/6 mice (donor) and 76 BALB/c mice (recipients) were used to establish corneal allograft model, and were divided into 4 groups randomly. Group A, autograft control; Group B, allograft control (placebo was given in both control groups); Group C and Group D, allograft groups, were treated with orally ciclosporin A and J2, respectively. Drugs were delivered for 12 days beginning on the day of transplantation. Graft survival was assessed by Kaplan-Meier analysis. On day 21 after transplantation, flow cytometry analysis of peripheral blood mononuclear cells and histological examination of graft were performed. No graft rejection was observed in Group A. The average transplant survival time of Group B was (17.8 +/- 2.1) d. In Group D, the transplant survival time showed a statistically prolongation to (40.6 +/- 8.3) d (P = 0.00) as compared with Group B, but the survival time was less than that of Group A (P = 0.04). There was no statistically significant difference between Group D and Group C (38.1 +/- 9.9) d (P = 0.99). The results of flow cytometry analysis showed that CD4(+) cells and CD8(+) cells in peripheral blood mononuclear cells did not increase in Group D. Fewer inflammatory cells were found in the corneal allograft in Group D. J2 could inhibit rejection and prolong corneal allograft survival time in mouse.

J2 prolongs the corneal allograft survival through inhibition of the CD4 + T cell-mediated response in vivo

In our previous report, we described a novel non-peptidic organic ligand of CD4 D1, designated J2, as a potential inhibitor of CD4 D1 and thus CD4-dependent T cell responses in vitro. In this work, we further used a murine model of corneal allograft rejection to determine its in vivo immunosuppressive activities. To mimic the situation in high-risk human eyes, the recipient mice corneas were all induced by intrastromal sutures to serve as neovascularized graft beds. J2 was administrated by mouth 3 h before transplantation and thereafter on consecutive 12 days. The results showed that J2 could significantly prolong the median survival time of the corneal allografts, compared to the untreated control group. And the subsequent functional assays, including T cell phenotype analysis, delayed-type hypersensitivity (DTH) and enzyme-linked immunospot (ELISPOT) assays revealed that the immunosuppressive activity of J2 was associated with its inhibitory effects on the CD4 + T cells and these cells-mediated responses. All these results suggest that J2 is a potential lead for the development of new immunosuppressive agents to prevent the corneal allograft rejection.

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-γ 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-γ 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-γ, TNF-α, 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-γ 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.

Very Late Antigen 1 Blockade Markedly Promotes Survival of Corneal Allografts

To investigate the role of very late antigen 1 (VLA-1) (also known as integrin receptor alpha(1)beta(1)) in corneal transplantation inflammation and allograft survival. Cell infiltration and vasculogenesis (both angiogenesis and lymphangiogenesis) associated with allodisparate corneal transplantation were assessed in VLA-1-deficient conditions and controls by immunofluorescent microscopic studies. Corneal allograft survival was also assessed after anti-VLA-1 antibody treatment and in VLA-1 knockout recipient mice. Anti-VLA-1 antibody treatment leads to a profound reduction in the granulocytic, monocytic, and T-cell infiltration after corneal transplantation. In addition, corneal angiogenesis and lymphangiogenesis were both significantly suppressed in VLA-1 knockout mice. Remarkably, universal graft survival was observed in both anti-VLA-1 antibody treatment and knockout mice. Very late antigen 1 blockade markedly reduces inflammation and inflammation-induced tissue responses, including vasculogenic responses, associated with corneal transplantation and promotes allograft survival. These studies offer insights into important integrin-mediated mechanisms of corneal transplant-related inflammation and provide possible new integrin-based immunotherapies for transplant rejection.

Prolongation of corneal allograft survival by CTLA4-FasL in a murine model

To investigate the therapeutic effect of CTLA4-FasL-B7 costimulatory pathway blockage-on graft survival in a murine model of corneal transplantation. Orthotopic penetrating keratoplasty was performed on BALB/c mice. The mice were randomized into four groups: the isograft group, untreated allograft group, cyclosporine A drug delivery system (CsA DDS)-anterior chamber implanted group, and 10 microg/mL CTLA4-FasL-treated group. Allografts were from C57BL/6 mice. Survival time of corneal grafts was evaluated. Immunohistological method and TdT-mediated dUTP Nick End Labeling (TUNEL) were applied for the detection of CD4+ T cells and apoptotic cells in corneal transplants. To assess whether peripheral immune tolerance appeared after the treatment of CTLA4-FasL, CsA DDS-implanted- and CTLA4-FasL-treated BALB/c mice with clear grafts received skin allografts at 4 weeks after keratoplasty, and the status of corneal transplants were observed when skin grafts were rejected. Allografts in the CTLA4-FasL group (median survival time [MST] = 106 days, p = 0.0042) and the CsA DDS group (MST = 60 days, p = 0.0037) revealed extending survival time, compared with that in the untreated allograft group (MST = 14 days). There were significantly fewer CD4-positive T cells in both the isograft group and the CsA DDS group. In the untreated allograft group, the number of CD4+ T cells gradually increased from day 1 until the final day of observation (day 21). By contrast, it reached a peak on day 7 and then absolutely reduced in the CTLA4-FasL group. Many apoptotic cells were detected on day 7 in the CTLA4-FasL group, but very few were seen in the other groups. Within 30 days of skin-graft rejection, previously healthy and long-standing corneal grafts became rejected in the CsA DDS group but remained clear in the CTLA4-FasL group. CTLA4-FasL can prolong the survival time of corneal allografts in mice, exerting a negative regulation on T-cell activation simultaneously by blocking B7 costimulatory signals and inducing Fas-FasL apoptotic pathway. Due to the adjunctive role of FasL, it also appears to be a potential activity of tolerance induction through T-cell apoptotic pathways.

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.

Effect of the Preservation-to-Surgery Interval on Corneal Allograft Survival in Low-Risk Patients

To investigate the role of the preservation-to-surgery interval on corneal allograft survival in low-risk patients. Eighteen donor corneas obtained from an overseas eye bank were preserved and transported overseas in Optisol-GS solution (Chiron Vision, Irvine, CA) in Group 1. Thirty fresh, young, healthy, and unscreened donor corneas soaked in the same medium were used in Group 2. Average preservation-to-surgery time was more than 8 days in Group 1 and less than 30 hours in Group 2. Corneal allograft survival rates were determined by Kaplan-Meier estimates of the survivor functions. The log-rank test was used to determine statistical significance of the differences between groups. was 233.3 +/- 37.7 hours in Group 1 and 20.8 +/- 4.6 hours in Group 2 (P < .05 for the comparison of average times). There were significant differences between the groups with respect to donors' age and enucleation time, but there were no statistically significant differences between the groups in terms of graft diameter and recipient diameter or for corneal allograft survival in low-risk patients. The graft survival rate was 83.3% in Group 1 and 93.3% in Group 2 at the end of the follow-up period. Preservation-to-surgery time has no effect on corneal allograft survival in low-risk patients. However, prospective, randomized, long-term and large-scale clinical trials are necessary to confirm these findings.

Effects of Spironolactone on Corneal Allograft Survival in the Rat

Purpose: Spironolactone has recently been shown to have suppressive effects on several immunoactive and proinflammatory cytokines. In this study, we investigated the effects of spironolactone on the prevention of corneal allograft rejection in a MHC class I/II mismatch rat corneal transplant model. Methods: Grafted animals for corneal survival analysis were assigned to receive either spironolactone suspension (orally, 100 mg/kg/day, n = 7), phosphate-buffered saline (PBS, orally, same volume as spironolactone, n = 9) or remained untreated (n = 16). Additional grafted rats treated with spironolactone (n = 6) or PBS (n = 8) were sacrificed on day 12 for quantitative RT-PCR analysis for mechanistic studies. Results: Mean (±SEM) graft survival was significantly prolonged in animals receiving spironolactone (14.9 ± 2.0 days) compared with both PBS-treated (12.3 ± 1.2 days, p = 0.007) and untreated controls (13.0 ± 1.0 days, p = 0.01). We found a decrease in corneal neovascularization in spironolactone-treated rats compared with the PBS-treated group, although the difference was not statistically significant. Spironolactone affected both systemic (down-regulation of CD25+ cells in spleen) and local immune response (up-regulation of IL-10 in cornea). Conclusion: We present initial results demonstrating anti-inflammatory effects of spironolactone.

γδ T Cells Promote Anterior Chamber-Associated Immune Deviation and Immune Privilege through Their Production of IL-10

Anterior chamber-associated immune deviation (ACAID) is a form of peripheral tolerance that is induced by introducing Ags into the anterior chamber (AC) of the eye, and is maintained by Ag-specific regulatory T cells (Tregs). ACAID regulates harmful immune responses that can lead to irreparable injury to innocent bystander cells that are incapable of regeneration. This form of immune privilege in the eye is mediated through Tregs and is a product of complex cellular interactions. These involve F4/80+ ocular APCs, B cells, NKT cells, CD4+CD25+ Tregs, and CD8+ Tregs. gammadelta T cells are crucial for the generation of ACAID and for corneal allograft survival. However, the functions of gammadelta T cells in ACAID are unknown. Several hypotheses were proposed for determining the functions of gammadelta T cells in ACAID. The results indicate that gammadelta T cells do not cause direct suppression of delayed-type hypersensitivity nor do they act as tolerogenic APCs. In contrast, gammadelta T cells were shown to secrete IL-10 and facilitate the generation of ACAID Tregs. Moreover, the contribution of gammadelta T cells ACAID generation could be replaced by adding exogenous recombinant mouse IL-10 to ACAID spleen cell cultures lacking gammadelta T cells.

Effects of local and systemic viral interleukin-10 gene transfer on corneal allograft survival

In this study, we explored the immunomodulatory effects of viral interleukin (IL) IL-10 after ex vivo and in vivo gene transfer in experimental corneal transplantation. Wistar-Furth rats were used as donors and major histocompatibility complex class I/II-disparate Lewis rats served as recipients. For ex vivo gene therapy donor corneas were either transfected with liposome/vIL-10 plasmid DNA mixtures or transduced with a vIL-10 expressing adenovirus vector (AdvIL-10). For in vivo studies, recipients were treated with AdvIL-10 intraperitoneally 1 day before transplantation. Graft survival was analysed using the Kaplan-Meier survival method. To monitor the efficacy of the therapy messenger RNA (mRNA) cytokine expression profiles in grafts and draining lymph nodes were analysed by quantitative real-time reverse transcription-polymerase chain reaction. Moreover, anti-adenovirus immunity was also investigated. Neither ex vivo liposome-mediated vIL-10 gene transfer nor ex vivo AdvIL-10 gene transfer led to prolonged corneal allograft survival. In contrast, corneal allograft survival was significantly prolonged in animals receiving systemic AdvIL-10 gene transfer. Moreover, only systemic vIL-10 gene therapy modulated the cytokine mRNA expression profile in draining lymph nodes. Interestingly, systemic AdvIL-10 gene transfer could not inhibit the generation of anti-adenovirus antibodies. Our data indicate systemic expression of the vIL-10 gene is required to modulate the cytokine expression profile in the draining lymph nodes, which might be a pre-requisite for the success of cytokine gene therapy.

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.

Differential Effects of Costimulatory Pathway Modulation on Corneal Allograft Survival

T lymphocytes have a central role in allograft rejection. On engagement of the T cell receptor by antigenic peptide-major histocompatibility complex (MHC) complex, a second "costimulatory" signal is critical to full T-cell activation or downregulation. In this study, the effect on corneal allograft survival of modulation of the costimulatory molecules programmed death-1 (PD-1) and inducible costimulatory (ICOS) molecule was examined. These molecules are known to modulate, respectively, negative or positive T-cell activation signals. A dimeric PD-L1 immunoglobulin (Ig) fusion protein was generated to stimulate the inhibitory receptor PD-1, and a monoclonal antibody was used to block ICOS. The effect of PD-1 engagement and ICOS blockade on lymphocyte activation by in vitro T-cell proliferation and the effect on orthotopic corneal allograft survival in BALB/c mice were determined. Both reagents demonstrated T-cell inhibition in vitro. PD-L1.Ig treatment of BALB/c mice prolonged fully MHC-mismatched C3H donor corneal allograft survival, with a median survival time (MST) of 21 days. This was significantly prolonged compared to isotype control protein-treated recipients (MST 13 days, P < 0.003). Allograft survival in BALB/c recipients treated with anti-ICOS antibody showed no prolongation of survival compared with the isotype control antibody (MST, 12 days in both groups). Augmented ligation of the PD-1 negative costimulatory molecule significantly prolongs corneal allograft survival. However, in contrast to findings in other allograft models, signaling through the positive costimulatory molecule ICOS appears to be less important in allogeneic rejection of cornea.

Anterior chamber–associated immune deviation and its impact on corneal allograft survival

Anterior chamber–associated immune deviation and its impact on corneal allograft survival

Peripheral Tolerance Via the Anterior Chamber of the Eye: Role of B Cells in MHC Class I and II Antigen Presentation

Ags introduced into the anterior chamber (AC) of the eye induce a form of peripheral immune tolerance termed AC-associated immune deviation (ACAID). ACAID mitigates ocular autoimmune diseases and promotes corneal allograft survival. Ags injected into the AC are processed by F4/80(+) APCs, which migrate to the thymus and spleen. In the spleen, ocular APCs induce the development of Ag-specific B cells that act as ancillary APCs and are required for ACAID induction. In this study, we show that ocular-like APCs elicit the generation of Ag-specific splenic B cells that induce ACAID. However, direct cell contact between ocular-like APCs and splenic B cells is not necessary for the induction of ACAID B cells. Peripheral tolerance produced by ACAID requires the participation of ACAID B cells, which induce the generation of both CD4(+) regulatory T cells (Tregs) and CD8(+) Tregs. Using in vitro and in vivo models of ACAID, we demonstrate that ACAID B cells must express both MHC class I and II molecules for the generation of Tregs. These results suggest that peripheral tolerance induced through the eye requires Ag-presenting B cells that simultaneously present Ags on both MHC class I and II molecules.