Corneal endothelial cells activate innate and acquired arm of anti-viral responses after cytomegalovirus infection.

Infection of the corneal endothelial cells by human cytomegalovirus (CMV) is an important cause of corneal endotheliitis. CMV endotheliitis is difficult to completely cure and relapses are frequent. This can cause blinding corneal bullous keratopathy. However, the pathogenesis of CMV endotheliitis remains undetermined. To understand the immunopathology of endotheliitis, we examined how corneal endothelial cells prime the anti-viral immunity after CMV infection based on global transcriptional responses. To accomplish this, human corneal endothelial (HCEn) cells were infected with CMV, and the global transcriptional responses were determined by microarray analyses for primary anti-viral responses using network analysis. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and protein array analyses were used to examine whether anti-viral cytokines were induced, i.e., to determine whether innate immune responses were activated. To examine whether priming of acquired immune response was activated, CMV-infected HCEn cells were co-cultured with allogeneic CD8+ T cells from CMV seropositive donors and tested for priming activity for the CD8+ effector T cells by measuring interferon-γ secretion. The CMV-induced responses of HCEn cells were characterized by type I interferon and pattern recognition receptor pathways which represent innate immune priming. The global transcriptional activation was specifically associated with antigen presentation with the antimicrobial response functions. Protein array analyses indicated a significant increase in the secretion of anti-viral inflammatory cytokines including CXCL10 as innate immune responses. When HCEn cells were examined to determine whether CMV infection activated anti-viral acquired immunity, CMV-infected HCEn cells directly stimulated the proliferation of CD8+ T cells from CMV-seropositive donors, and pp65 viral epitope induced interferon-γ secretion from the CD8+ T cells. We conclude that CMV-infected HCEn cells induce innate immune priming along with provisions of acquired immune priming of CD8+ effector T cells. This information should help in the development of useful diagnostic procedures and efficacious therapeutic strategy to treat refractory corneal endotheliitis.

[Impact of the ratio between graft and host corneal size on immune rejection, re-bubbling rate and postoperative endothelial cell loss in 457 eyes after Descemet membrane endothelial keratoplasty (DMEK)]. / Einfluss des Verhältnisses von Transplantatgröße zu Hornhautgröße auf Immunreaktion, Re-Bubbling-Rate und postoperativen Endothelzellverlust bei 457 Augen nach Descemet-Membrane-Endothelial-Keratoplastik (DMEK).

BACKGROUND: The aim of this study was to assess the impact of the ratio between the graft and host corneal size (RGH) on postoperative complications, such as immune reactions, re-bubbling rate and endothelial cell loss (ECL) after Descemet membrane endothelial keratoplasty (DMEK). PATIENTS AND METHODS: Retrospectively, 457 patient eyes were included which had undergone surgery between 2016 and 2019 in the Department of Ophthalmology, Saarland University Medical Center in Homburg/Saar using DMEK or triple DMEK, diagnosed as Fuchs' endothelial dystrophy (n = 431), pseudophakic bullous keratopathy (n = 9) and others (n = 17). The follow-up period extended until the end of 2020. Main outcome measures included immune reaction (IR), re-bubbling rate and the postoperative endothelial cell loss (ECL) at 6 weeks, 6 months and 12 months and whether these measures depended on the RGH. RESULTS: The RGH in this study ranged from 0.35 to 0.62 (0.46 ± 0.04). There were 33 (7.2%) postoperative IRs (DMEK n = 25; triple DMEK n = 8). The average RGH without IR (0.46 ± 0.04) was significantly (p = 0.038) smaller than in the group with IR (0.47 ± 0.05). Re-bubbling was necessary in 159 of 457 (34.8%) patient eyes. The RGH in patient eyes with re-bubbling (0.47 ± 0.04) was significantly (p = 0.014) higher than that in eyes without re-bubbling (0.45 ± 0.04). The mean preoperative endothelial cell count (ECD) was 2603 ± 251 cells/mm2 (min: 2161, max: 3500 cells/mm2). It was shown that a larger RGH had no positive influence on endothelial cell loss (r = 0.001; p = 0.974). CONCLUSION: Our results suggest that a larger graft diameter compared to host corneal size is associated with an increased rate of immune reactions and a higher re-bubbling rate after DMEK. Otherwise, a larger RGH had no positive influence on endothelial cell loss after DMEK. Accordingly, the graft size for DMEK should not be unnecessarily large, especially in eyes with Fuchs' endothelial dystrophy.

Descemet's membrane endothelial keratoplasty surgery: update on the evidence and hurdles to acceptance.

PURPOSE OF REVIEW: Descemet's stripping endothelial keratoplasty (DSEK) is the most popular treatment for endothelial dysfunction, but Descemet's membrane endothelial keratoplasty (DMEK) now provides better vision with lower risk of immunologic rejection. Although DMEK is more challenging, advances in instrumentation and techniques are reducing the learning curve. RECENT FINDINGS: In contrast to DSEK, which includes posterior donor stroma, DMEK consists merely of donor endothelium and Descemet's membrane, so DMEK does not create a stromal interface and induces significantly less posterior surface aberrations, resulting in better vision. Furthermore, multiple centers report remarkably low (<1%) cumulative probability of immunologic graft rejection episodes through 2 years after DMEK. Initially, the biggest challenges were tissue loss in preparation and ensuring attachment. Subsequent improvements have reduced complication rates to levels experienced with DSEK. DMEK/DSEK hybrids and 'thin' DSEK also can provide better vision than standard DSEK; randomized controlled comparisons with DMEK are needed. SUMMARY: DMEK provides an anatomically exact replacement of dysfunctional host endothelium and has set new benchmarks for rejection risk and visual outcomes following endothelial replacement. DMEK is providing new insights into how different corneal layers contribute to immunogenicity and immune tolerance and into the key factors that limit vision after endothelial keratoplasty.

[Immune reactions following excimer laser and femtosecond laser-assisted penetrating keratoplasty]. / Immunreaktionen nach Femtosekunden- und Excimerlaser-Keratoplastik.

In 10-20 % of conventional keratoplasties immunological graft reactions (TPR) occur, which may lead to endothelial cell loss and irreversible transplant rejection. Beside the optical advantages of non-mechanical excimer laser trephination, it does not seem to have immunological disadvantages (13.9 % TPR in keratoconus, 2.9 % in Fuchs' dystrophy after 3 years). The femtosecond laser-assisted keratoplasty has visual and refractive outcomes similar to conventional trephination. However, the "mushroom-shaped" trephination seems to have immunological disadvantages (21.8 % TPR in keratoconus after 14 months) and "top hat-shaped" keratoplasties seem to have no immunological disadvantages (6.6 % TPR in Fuchs' dystrophy after 14 months).

[HLA matching in keratoplasty: lessons learned from lamellar techniques]. / Immunologie der Keratoplastik: Macht HLA-Matching bei lamellären Verfahren Sinn?

BACKGROUND: The immunological mechanisms of graft rejections after penetrating keratoplasty are largely investigated in rodent models. Here, antigens are predominantly processed by host antigen presenting cells (APCs). For this reason, graft rejections are not primarily triggered by mismatches in the major histocompatibility complex (MHC). Consequently, MHC matching (equivalent of HLA matching) does not robustly prevent immunological graft rejections in mice. This, however, may not apply to humans because anatomy and the clinical picture of immune reactions differ vastly. METHODS: Immunological experiments are not feasible in humans for ethical reasons. However, the recent surgical modifications in keratoplasty inadvertently gave rise to several interesting immunological field experiments. We herein discuss the potential insight into human graft rejections from selected clinical observations. On this basis, we have evaluated HLA matching for keratoplasty techniques. RESULTS: Several clinical observations hint towards an active role of donor-derived APCs in graft rejections after human keratoplasty. Additionally, donor-specific anti-HLA antibodies may play a significant role. On this basis we suggest that HLA matching is potentially beneficial in human keratoplasty in contrast to the situation in mice. CONCLUSIONS: Graft rejections are rarely observed after Descemet membrane keratoplasty (DMEK). For this reason, we do not recommend HLA matching here. The same is true for deep anterior lamellar keratoplasty, where graft rejections can usually be treated well. However, HLA matching is a viable option in penetrating keratoplasty. This is especially true for high-risk eyes.

Arginine depletion as a mechanism for the immune privilege of corneal allografts.

The cornea is an immune privileged tissue. Since arginase has been found to modulate T-cell function by depleting arginine, we investigated the expression of arginase in the cornea and its possible role in immune privilege using a murine transplant model. We found that both the endothelium and epithelium of murine corneas express functional arginase I, capable of down-regulating T-cell proliferation in an in vitro culture system. The administration of the specific arginase inhibitor N-hydroxy-nor-L-Arg to recipient mice resulted in an accelerated rejection of allogeneic C57BL/6 (B6) corneal grafts. In contrast, in vivo blockade of arginase activity had no effect in altering the course of rejection of primary skin grafts that express little, if any, arginase. In addition, the inhibition of arginase did not alter systemic T-cell proliferation. These data show that arginase is functional in the cornea and contributes to the immune privilege of the eye, and that modulation of arginase contributes to graft survival.

Analysis of Immune Cells on Donor Corneal Endothelium After Corneal Transplantation Using the HRT-Rostock Cornea Module.

PURPOSE: The aim of this study was to investigate the immune cells on corneal endothelium of the graft in patients who underwent penetrating keratoplasty (PK), Descemet-stripping endothelial keratoplasty (DSEK), and Descemet membrane endothelial keratoplasty (DMEK). METHODS: A total of 43 eyes of 43 patients who underwent PK (17 eyes), DSEK (13 eyes), and DMEK (13 eyes) and who did not show any sign of graft rejection were recruited for the study. Patients who underwent cataract surgery (26 eyes) served as controls. Immune cells on the corneal endothelium were examined with laser in vivo confocal microscopy. The associations between the corneal endothelial cell density, type of keratoplasty, aqueous flare, repeated keratoplasty, and time after surgery versus the density of immune cells were investigated. RESULTS: In vivo confocal microscopy visualized similar numbers of immune cells on the corneal endothelium in the PK, DSEK, and DMEK groups, whereas no immune cells were observed in any of the control patients. The numbers of immune cells tended to be higher in regraft eyes in the PK group (P = 0.00221) and in the DSEK group (P = 0.168) than those in the primary graft eyes. No significant association was found between the density of immune cells and corneal endothelial cell density in the PK, DSEK, and DMEK groups. CONCLUSIONS: Immune cells were observed to a similar extent in the eyes of PK, DSEK, and DMEK subjects even in the absence of any clinical sign of immune rejection. A further prospective longitudinal study will evaluate the effect of immune cells on long-term graft survival and the risk for graft rejection.

Regeneration of corneal endothelium following complete endothelial cell loss in rat keratoplasty.

PURPOSE: Corneal endothelial cells (EC) are crucial for maintaining corneal clarity before and after keratoplasty. Since it is thought that corneal graft rejection leads to irreversible EC loss and transplant failure, we quantified immune mediated EC loss in the rat keratoplasty model and analyzed whether the EC layer would then regenerate. METHODS: Rats were subjected to orthotopic penetrating keratoplasty. We compared endothelial responses to immunological EC loss following allogeneic transplantations between Fisher and Lewis rats (group R) to those following mechanical EC removal in a syngeneic setting between Lewis rats (group S). Animals were followed clinically for corneal opacity for up to one year. Bulbi were excised and prepared for histological examination at different time points: ECs were defined and characterized using Alicarin red S/ DAPI staining on corneal flatmounts. Ki-67/ DAPI staining on flatmount preparations served to detect cell proliferation. Immunohistochemical staining of corneal cryosections was used to characterize infiltrating immune cells. RESULTS: GROUP R: After about two weeks the allografts were completely opaque, which was accompanied by a massive leukocyte infiltration in conjunction with EC destruction, signifying rejection. EC loss without an immune reaction (group S) resulted only in medium opacity levels. In both groups, all grafts regained clarity in the following weeks to months, and a newly-formed endothelial cell layer with irregular and enlarged ECs became apparent on the formerly EC free grafts. Scattered Ki-67 positive cells within the endothelial cell layer were observed during re-endothelialization. In addition to re-endothelialization, the immunological infiltration seen in the allografts at the time of rejection had subsided after one year. CONCLUSIONS: Re-endothelialization following keratoplasty takes place in the rat in vivo and restores graft clarity, following both immunological or surgical destruction of ECs. Following rejection, EC replacement is accompanied by a reduction of immune infiltrates. Peripheral recipient ECs are a sufficient source for graft re-endothelialization, as seen in rats following EC removal. Our results suggest that ECs both proliferate and enlarge during re-endothelialization in the rat keratoplasty model.

VISTA Is Crucial for Corneal Allograft Survival and Maintenance of Immune Privilege.

Purpose: V-domain immunoglobulin suppressor of T cell activation (VISTA) is a novel immune checkpoint receptor and ligand for regulating T cell proliferation and cytokine production. The purpose of the present study was to determine the role of VISTA in the immune privilege of corneal allografts. Methods: Expression of VISTA mRNA in mouse eyes was assessed with reverse-transcription PCR. Corneas of C57BL/6 mice were orthotopically transplanted into the eyes of BALB/c wild-type recipients treated with anti-VISTA mAb, and graft survival was assessed. A separate set of BALB/c mice treated with anti-VISTA mAb or rat IgG received injection of C57BL/6 splenocytes into the anterior chamber, and induction of allospecific anterior chamber-associated immune deviation (ACAID) was assessed. CD4+ and CD8+ T cells in the spleen were assessed with flow cytometry. Results: VISTA mRNA was constitutively expressed in the cornea, and the expression of VISTA was localized to CD11b+ cells on the corneal stroma. Survival of allografts treated with anti-VISTA mAb was less than that of the control. ACAID was induced less efficiently in BALB/c mice treated with VISTA mAb. The proportions of CD8+ T cells and CD8+ CD103+ T cells (CD8+ T regulatory cells) in the spleen of BALB/c mice treated with anti-VISTA mAb were significantly lower than those of the control. Conclusions: VISTA may play an essential role in the acceptance of corneal allografts via involvement with allospecific ACAID, which suppresses T cell infiltration into the cornea.

Immunological features of human corneal endothelial cells.

PURPOSE: To investigate the function of human corneal endothelial cells (HCEC) as immunological cells. METHODS: Expression of HLA-DP, -DQ, -DR, CD40, CD80 and CD86 was determined by immunohistochemical methods. Purified peripheral blood mononuclear cells were cocultured with HCEC, part of which had been pretreated with gamma-interferon (IFN), and activation of lymphocytes was determined by fluorescence-activated cell sorting analysis. RESULTS: In the coculture system, T lymphocytes were activated by corneal endothelial cells; HLA-DP, -DQ, -DR and CD40 expressions were increased by gamma-IFN pretreatment. Costimulatory CD80 was shown on the endothelial cells. CONCLUSIONS: HCEC can be assumed to be involved in the rejection process of corneal transplants as potential antigen-presenting cells.

Does ocular inflammation play a role in xeroderma pigmentosum with endothelial dysfunction: an immunological study.

We report a case of xeroderma pigmentosum (XP) with endothelial dysfunction where the analysis of tears revealed elevated levels of proinflammatory cytokines, even in the absence of active inflammation and neovascularisation of the ocular surface. Although the role of ultraviolet (UV) radiation-induced inflammation in the occurrence of ocular manifestations of XP is known, little is published on the molecular mechanisms and there are no reports quantifying the presence of inflammatory cytokines in the tears of patients with ocular involvement of XP. Tear analysis demonstrated an increase in inflammatory cytokines and chemokines, especially interleukin-8 (2.38 ng/µg), tumour necrosis factor alpha (0.87 ng/µg) and granulocyte monocyte colony stimulating factor (0.44 ng/µg) as compared with the control eye. Effective management of the underlying UV-induced inflammation and promoting DNA repair may play a vital role in managing ocular manifestations and its sequelae in patients of XP.

Immune Cells on the Donor Corneal Endothelium After Corneal Transplantation.

PURPOSE: The aim of this study was to investigate the existence of presumed immune cells observed by contact specular microscopy in patients who underwent penetrating keratoplasty (PK) and Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: This cross-sectional study was conducted on consecutive patients who underwent follow-up visits between January and March 2015 for previously performed PK or DSAEK. Presumed immune cell-suspected "cell-like white dots" were evaluated by scanning slit contact specular microscopy. The association between the grading of presumed immune cells with clinical parameters, such as corneal endothelial cell density, time after surgery, and the titer of steroid administration, was also investigated. RESULTS: A total of 54 eyes of 54 patients who underwent PK (32 eyes/32 patients) and DSAEK (22 eyes/22 patients) were evaluated, and suspected immune cells were observed in all patients. In the PK and DSAEK groups, the number of patients in the presumed immune cell grades 1, 2, 3, and 4 were 19, 10, 2, and 1 and 10, 8, 2, and 2, respectively (P = 0.663). No statistically significant association was found between the immune cell grades and the clinical parameters. CONCLUSIONS: Immune cells were observed on the corneal endothelial grafts in all 54 patients who underwent PK or DSAEK. Although the number of immune cells varied between patients and showed no correlation with clinical parameters, it would be beneficial to conduct a future prospective study to analyze the effect of immune cells on postoperative corneal endothelial cell loss.

Immune Privilege and Eye-Derived T-Regulatory Cells.

Certain cellular components of the eye, such as neural retina, are unable to regenerate and replicate after destructive inflammation. Ocular immune privilege provides the eye with immune protection against intraocular inflammation in order to minimize the risk to vision integrity. The eye and immune system use strategies to maintain the ocular immune privilege by regulating the innate and adaptive immune response, which includes immunological ignorance, peripheral tolerance to eye-derived antigens, and intraocular immunosuppressive microenvironment. In this review, we summarize current knowledge regarding the molecular mechanism responsible for the development and maintenance of ocular immune privilege via regulatory T cells (Tregs), which are generated by the anterior chamber-associated immune deviation (ACAID), and ocular resident cells including corneal endothelial (CE) cells, ocular pigment epithelial (PE) cells, and aqueous humor. Furthermore, we examined the therapeutic potential of Tregs generated by RPE cells that express transforming growth factor beta (TGF-ß), cytotoxic T lymphocyte-associated antigen-2 alpha (CTLA-2α), and retinoic acid for autoimmune uveoretinitis and evaluated a new strategy using human RPE-induced Tregs for clinical application in inflammatory ocular disease. We believe that a better understanding of the ocular immune privilege associated with Tregs might offer a new approach with regard to therapeutic interventions for ocular autoimmunity.

Expression of SV40 virus large T antigen by recombinant adenoviruses activates proliferation of corneal endothelium in vitro.

Infection with the Ad5-SVR4 virus was used to introduce the large T antigen encoding region of the SV40 virus into bovine and human corneal endothelial cells. Expression of large T antigen occurred in 40% of bovine corneal endothelial cells after a 24-h incubation time versus 12% after 8 h of incubation. By 48 h after infection, almost all (92.8%) bovine corneal endothelial cells expressed large T antigen. Bovine and human corneal endothelial cells which expressed large T antigen proliferated and the characteristic morphologic features of corneal endothelium were maintained. This method may enable growth of enough corneal endothelium to perform studies to elucidate the biochemical mechanisms involved in regulating endothelial cell function.

Will Descemet's stripping with automated endothelial keratoplasty (DSAEK) lower the rates of allograft rejection in corneal transplants for endothelial failure?

BACKGROUND: Allograft corneal rejection occurs in a substantial number of full thickness transplants in spite of the relative immune privilege enjoyed by the cornea. Compared to other layers of the cornea, endothelial rejection has most disastrous consequences on graft survival. In the last few years, a new technique, Descemet's stripping with automated endothelial keratoplasty (DSAEK) is being used of selective transplantation of the endothelium. It involves stripping diseased endothelium (and Descemet's) and replacing it by a small lamella fashioned from a cadaveric donor cornea, which consists of endothelium, Descemet's membrane and a part of posterior stoma. HYPOTHESIS: We hypothesize that DSAEK might substantially reduce the incidence of allograft immune rejection in corneal transplant done for cases with endothelial failure. EVALUATION OF THE HYPOTHESIS: In published reports of consisting of more than 300 surgeries and three years experience with DSAEK, no case of graft rejection has been reported. In our opinion, this advantage of DSAEK compared to conventional full thickness keratoplasty could be due to four factors: (a) The transplanted tissue is placed in the anterior chamber and has no exposure to the surface, where the antigen presenting cells (APC) and antibodies are present. (b) Significant reduction in the number of sutures connecting the host and donor tissue may lead to lesser suture related rejection episodes. (c) Absence of direct contact between the host stroma vessels and the transplanted tissue disrupts the immune affecter and effecter arcs. (d) Reduced immunogenicity of the donated tissue due to absence of epithelium. CONSEQUENCES OF THE HYPOTHESIS: If this hypothesis stands true in subsequent studies, it could lead to substantial reduction in the socioeconomic resources involved in management of graft rejection. Even if this hypotheses fails the test of well controlled studies, this would broaden the current understanding of the ocular immunology and the immune privilege with which the anterior chamber of the eye is normally associated with.

[Intracameral application of corticosteroids for treating severe endothelial rejection after penetrating keratoplasty]. / Intrakamerale Applikation von Kortikosteroiden zur Therapie der schweren endothelialen Abstossung nach perforierender Keratoplastik.

Immune reaction is the main cause for graft failure following penetrating keratoplasty. Endothelial immune reaction is the most frequent and most dangerous subtype of rejection because destruction of the graft endothelium can lead to graft failure. "Acute" endothelial rejection is treated by administration of topical and systemic steroids. Intracameral application of corticosteroids by means of an anterior chamber flush is an adjunctive measure that can stop the immune reaction immediately. This measure is thus recommended in all intermediate and severe endothelial rejections.

Immune Cells on the Corneal Endothelium of an Allogeneic Corneal Transplantation Rabbit Model.

Purpose: Corneal endothelial cell density undergoes a progressive decrease for many years after transplantation, eventually threatening patients with late endothelial failure. The purpose of this study was to investigate the possibility of an immunologic response in successfully grafted corneal endothelium. Methods: The corneal endothelium of patients who had undergone corneal transplantation was evaluated by specular microscopy. Rabbit models were subjected to penetrating keratoplasty (PK) with either syngeneic or allogeneic corneal transplants and Descemet's stripping endothelial keratoplasty (DSEK) with allogeneic corneal transplants. The presence of immune cells and expression of proinflammatory cytokines were determined by immunostaining. The corneal endothelium and immune cells were also evaluated by scanning electron microscopy. Results: Scanning slit contact specular microscopy of patients with no features of graft rejection revealed cell-like white dots on the grafted corneal endothelium. The corneal endothelium of the allogeneic PK and DSEK rabbit models displayed the presence of immune cells, including CD4+ T-helper cells, CD8+ cytotoxic T cells, CD20+ B lymphocytes, CD68+ macrophages, and neutrophils, but these immune cells were rarely observed in the syngeneic PK model. These immune cells also produced proinflammatory cytokines. Notably, some of the corneal endothelial cells situated near these immune cells exhibited features of apoptosis. Conclusions: T lymphocytes, B lymphocytes, macrophages, and neutrophils are present on the grafted corneal endothelium in both PK and DSEK allogeneic rabbit models. The potential involvement of immune cells as an underlying pathophysiology for late endothelial failure deserves further examination.