The Neuropeptide α-Melanocyte-Stimulating Hormone Prevents Persistent Corneal Edema following Injury.

Corneal endothelial cells (CEnCs) regulate corneal hydration and maintain tissue transparency through their barrier and pump function. However, these cells exhibit limited regenerative capacity following injury. Currently, corneal transplantation is the only established therapy for restoring endothelial function, and there are no pharmacologic interventions available for restoring endothelial function. This study investigated the efficacy of the neuropeptide α-melanocyte-stimulating hormone (α-MSH) in promoting endothelial regeneration during the critical window between ocular injury and the onset of endothelial decompensation using an established murine model of injury using transcorneal freezing. Local administration of α-MSH following injury prevented corneal edema and opacity, reduced leukocyte infiltration, and limited CEnC apoptosis while promoting their proliferation. These results suggest that α-MSH has a proregenerative and cytoprotective function on CEnCs and shows promise as a therapy for the prevention and management of corneal endothelial dysfunction.

Myeloid-derived suppressor cells promote allograft survival by suppressing regulatory T cell dysfunction in high-risk corneal transplantation.

Highly inflamed and neovascularized corneal graft beds are known as high-risk (HR) environments for transplant survival. One of the primary factors leading to this rejection is reduction in the suppressive function of regulatory T cells (Treg). Our results show that myeloid-derived suppressor cells (MDSC) counteract interleukin-6-mediated Treg dysfunction by expressing interleukin-10. Additionally, MDSC maintain forkhead box P3 stability and their ability to suppress IFN-γ+ Th1 cells. Administering MDSC to HR corneal transplant recipients demonstrates prolonged graft survival via promotion of Treg while concurrently suppressing IFN-γ+ Th1 cells. Moreover, MDSC-mediated donor-specific immune tolerance leads to long-term corneal graft survival as evidenced by the higher survival rate or delayed survival of a second-party C57BL/7 (B6) graft compared to those of third-party C3H grafts observed in contralateral low-risk or HR corneal transplantation of BALB/c recipient mice, respectively. Our study provides compelling preliminary evidence demonstrating the effectiveness of MDSC in preventing Treg dysfunction, significantly improving graft survival in HR corneal transplantation, and showing promising potential for immune tolerance induction.

IL-6 induces Treg dysfunction in desiccating stress-induced dry eye disease.

Regulatory T cells (Tregs) play a critical role in maintaining immune homeostasis, and their dysfunction is implicated in the pathogenesis of various autoimmune disorders, including dry eye disease (DED). Treg dysfunction in DED allows T-helper cell 17 (Th17) mediated chronic inflammation at the ocular surface. In this study, the factors causing Treg dysfunction in DED were investigated. We observed reduced expression of Treg functional markers - FoxP3, CD25, and CTLA-4 in the cells isolated from DED mice (DED Tregs). Additionally, DED Tregs showed increased expression levels of receptors for pro-inflammatory cytokine receptors, namely IL-6R, IL-17RA, and IL-23R. An increased expression level of pro-inflammatory cytokine receptors was observed on exposing Tregs isolated from naïve mice (NTregs) to IL-6 or IL-17, but not IL-23, with a concomitant downregulation of FoxP3, CD25, and CTLA-4 in these cells. Furthermore, among these cytokines, IL-6 induced the most pronounced loss of Treg mediated suppression of Th17 proliferation and IL-10 secretion. In vitro and in vivo blockade of IL-6 effectively restored function in DED Tregs, leading to enhanced suppressive function against proliferating Th17 cells and ameliorating disease severity. In conclusion, this study provides insights into mechanisms of Treg dysregulation in DED, specifically delineating the effect of Th17-associated cytokines, with IL-6 emerging as the critical factor inducing Treg dysfunctionality. These findings highlight the potential for developing novel therapeutic interventions for DED through restoration of immunosuppressive function of Tregs.

Local administration of myeloid-derived suppressor cells prevents progression of immune-mediated dry eye disease.

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature hematopoietic precursors with known immunoregulatory functions. The immunosuppressive role of MDSCs has been highlighted in several inflammatory ophthalmic disorders; however, their therapeutic application in suppressing the immune-mediated changes in dry eye disease (DED) has not been studied. We observed significant reduction in antigen presenting cell (APC) frequencies and their maturation in the presence of MDSCs. Moreover, co-culturing MDSCs with T helper 17 cells (Th17) resulted in reduced Th17 frequencies and their IL-17 expression. On the contrary, MDSCs maintained regulatory T cell frequencies and enhanced their function in-vitro. Furthermore, we delineated the role of interleukin-10 (IL-10) secreted by MDSCs in their immunoregulatory functions. We confirmed these results by flow cytometry analysis and observed that treatment with MDSCs in DED mice effectively suppressed the maturation of APCs, pathogenic Th17 response, and maintained Treg function and significantly ameliorated the disease. The results in this study highlight the potential therapeutic application of MDSCs in treating refractory DED.

The impact of donor diabetes on corneal transplant immunity.

Corneal transplantation is the most common form of solid tissue grafting, with an approximately 80% to 90% success rate. However, success rates may decline when donor tissues are derived from patients with a history of diabetes mellitus (DM). To evaluate the underlying immunopathologic processes that cause graft rejection, we used streptozotocin-induced type 1 DM (DM1) and transgenic Lepob/ob type 2 DM (DM2) diabetic murine models as donors and nondiabetic BALB/c as recipients. DM resulted in an increased frequency of corneal antigen-presenting cells (APCs) with an acquired immunostimulatory phenotype. Following transplantation, recipients that received either type of diabetic graft showed increased APC migration and T helper type 1 alloreactive cells, impaired functional regulatory T cells, and graft survival. Insulin treatment in streptozotocin-induced diabetic mice led to an increased tolerogenic profile of graft APC, lower T helper type 1 sensitization, and a higher frequency of functional regulatory T cells with high suppressive capacity, reflected in increased graft survival. We conclude that both DM1 and DM2 in donors can impact corneal APC functional phenotype, rendering the tissue more immunogenic and thereby increasing the risk of graft failure.

The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critical for Corneal Endothelial Cell Protection and Graft Survival after Transplantation.

Corneal transplantation is the most common form of tissue transplantation. The success of corneal transplantation mainly relies on the integrity of corneal endothelial cells (CEnCs), which maintain tissue transparency by pumping out excess water from the cornea. After transplantation, the rate of CEnC loss far exceeds that seen with normal aging, which can threaten sight. The underlying mechanisms are poorly understood. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide that is constitutively found in the aqueous humor with both cytoprotective and immunomodulatory effects. The curent study found high expression of melanocortin 1 receptor (MC1R), the receptor for α-MSH, on CEnCs. The effect of α-MSH/MC1R signaling on endothelial function and allograft survival in vitro and in vivo was investigated using MC1R signaling-deficient mice (Mc1re/e mice with a nonfunctional MC1R). Herein, the results indicate that in addition to its well-known immunomodulatory effect, α-MSH has cytoprotective effects on CEnCs after corneal transplantation, and the loss of MC1R signaling significantly decreases long-term graft survival in vivo. In conclusion, α-MSH/MC1R signaling is critical for CEnC function and graft survival after corneal transplantation.

Pigment Epithelium-Derived Factor Enhances the Suppressive Phenotype of Regulatory T Cells in a Murine Model of Dry Eye Disease.

Pigment epithelium-derived factor (PEDF) is a widely expressed 50-kDa glycoprotein belonging to the serine protease inhibitor family, with well-established anti-inflammatory functions. Recently, we demonstrated the immunoregulatory role played by PEDF in dry eye disease (DED) by suppressing the maturation of antigen-presenting cells at the ocular surface following exposure to the desiccating stress. In this study, we evaluated the effect of PEDF on the immunosuppressive characteristics of regulatory T cells (Tregs), which are functionally impaired in DED. In the presence of PEDF, the in vitro cultures prevented proinflammatory cytokine (associated with type 17 helper T cells)-induced loss of frequency and suppressive phenotype of Tregs derived from normal mice. Similarly, PEDF maintained the in vitro frequency and enhanced the suppressive phenotype of Tregs derived from DED mice. On systemically treating DED mice with PEDF, moderately higher frequencies and significantly enhanced suppressive function of Tregs were observed in the draining lymphoid tissues, leading to the efficacious amelioration of the disease. Our results demonstrate that PEDF promotes the suppressive capability of Tregs and attenuates their type 17 helper T-cell-mediated dysfunction in DED, thereby playing a role in the suppression of DED.

Thrombospondin-1 Is Necessary for the Development and Repair of Corneal Nerves.

Thrombospondin-1-deficient (TSP-1-/-) mice are used as an animal model of Sjögren's Syndrome because they exhibit many of the symptoms associated with the autoimmune type of dry eye found in primary Sjögren's Syndrome. This type of dry eye is linked to the inflammation of the lacrimal gland, conjunctiva, and cornea, and is thought to involve dysfunction of the complex neuronal reflex arc that mediates tear production in response to noxious stimuli on the ocular surface. This study characterizes the structural and functional changes to the corneal nerves that are the afferent arm of this arc in young and older TSP-1-/- and wild type (WT) mice. The structure and subtype of nerves were characterized by immunohistochemistry, in vivo confocal microscopy, and confocal microscopy. Cytokine expression analysis was determined by Q-PCR and the number of monocytes was measured by immunohistochemistry. We found that only the pro-inflammatory cytokine MIP-2 increased in young corneas of TSP-1-/- compared to WT mice, but tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) all increased in older TSP-1-/- mouse corneas. In contrast, CD11b+ pro-inflammatory monocytes did not increase even in older mouse corneas. Calcitonin gene-related peptide (CGRP)-, but not Substance P (SubP)-containing corneal nerves decreased in older, but not younger TSP-1-/- compared to WT mouse corneas. We conclude that CGRP-containing corneal sensory nerves exhibit distinct structural deficiencies as disease progresses in TSP-1-/- mice, suggesting that: (1) TSP-1 is needed for the development or repair of these nerves and (2) impaired afferent corneal nerve structure and hence function may contribute to ocular surface dysfunction that develops as TSP-1-/- mice age.

Method for single illumination source combined optical coherence tomography and fluorescence imaging of fluorescently labeled ocular structures in transgenic mice.

In vivo imaging permits longitudinal study of ocular disease processes in the same animal over time. Two different in vivo optical imaging modalities - optical coherence tomography (OCT) and fluorescence - provide important structural and cellular data respectively about disease processes. In this Methods in Eye Research article, we describe and demonstrate the combination of these two modalities producing a truly simultaneous OCT and fluorescence imaging system for imaging of fluorescently labeled animal models. This system uses only a single light source to illuminate both modalities, and both share the same field of view. This allows simultaneous acquisition of OCT and fluorescence images, and the benefits of both techniques are realized without incurring increased costs in variability, light exposure, time, and post-processing effort as would occur when the modalities are used separately. We then utilized this system to demonstrate multi-modal imaging in a progression of samples exhibiting both fluorescence and OCT scattering beginning with resolution targets, ex vivo thy1-YFP labeled neurons in mouse eyes, and finally an in vivo longitudinal time course of GFP labeled myeloid cells in a mouse model of ocular allergy.

Neurokinin-1 Receptor Antagonism Reduces Nonallergic Ocular Redness in a Rabbit Model.

Purpose: To evaluate the therapeutic efficacy of topical application of a neurokinin-1 receptor (NK1R) antagonist in a rabbit model of nonallergic ocular redness. Methods: Nonallergic ocular redness was induced in rabbits by a single, topical application of dapiparzole hydrochloride eye drops (0.5%, 1%, 2%, or 5%). The NK1R antagonist L-703,606 was topically applied to the eye at the same time of induction or 20 min after induction, and phosphate buffered saline (PBS) treatment served as the control. Superior bulbar conjunctival images were taken every 30 s for the first 2 min, followed by every 4 min for 8 min, and then every 10 min until 1 h. The severity of ocular redness was evaluated on the images using ImageJ-based ocular redness index (ORI) calculations. Results: The ORI scores were significantly increased after the application of 0.5%, 1%, 2%, or 5% dapiparzole at each time point evaluated, with the most severe redness induced by the 5% dapiprazole that led to a maximal mean increase in ORI score of 14 at 20 min post-induction and thus used for subsequent evaluation of therapeutic efficacy of NK1R antagonism. Topical L-703,606, when applied at the same time as dapiprazole induction, significantly suppressed the increase of ORI scores at all time points (∼40% decrease). Furthermore, when applied at 20 min after dapiprazole induction, L-703,606 rapidly and effectively suppressed the increase of ORI scores at 30, 40, 50, and 60 min (∼30% decrease). Conclusions: Topical blockade of NK1R effectively prevents and alleviates nonallergic ocular redness in a novel animal model.

Effects of Diabetes Mellitus on Corneal Immune Cell Activation and the Development of Keratopathy.

Diabetes mellitus (DM) is one of the most prevalent diseases globally, and its prevalence is rapidly increasing. Most patients with a long-term history of DM present with some degree of keratopathy (DK). Despite its high incidence, the underlying inflammatory mechanism of DK has not been elucidated yet. For further insights into the underlying immunopathologic processes, we utilized streptozotocin-induced mice to model type 1 DM (T1D) and B6.Cg-Lepob/J mice to model type 2 DM (T2D). We evaluated the animals for the development of clinical manifestations of DK. Four weeks post-induction, the total frequencies of corneal CD45+CD11b+Ly-6G- myeloid cells, with enhanced gene and protein expression levels for the proinflammatory cytokines TNF-α and IL-1ß, were higher in both T1D and T2D animals. Additionally, the frequencies of myeloid cells/mm2 in the sub-basal neural plexus (SBNP) were significantly higher in T1D and T2D compared to non-diabetic mice. DK clinical manifestations were observed four weeks post-induction, including significantly lower tear production, corneal sensitivity, and epitheliopathy. Nerve density in the SBNP and intraepithelial terminal endings per 40x field were lower in both models compared to the normal controls. The findings of this study indicate that DM alters the immune quiescent state of the cornea during disease onset, which may be associated with the progressive development of the clinical manifestations of DK.

Acquired Immunostimulatory Phenotype of Migratory CD103+ Dendritic Cells Promotes Alloimmunity Following Corneal Transplantation.

Post-transplantation, T helper 1 (Th1)-mediated immune rejection is the predominant cause of graft failure. Th1 cell sensitization occurs through complex and context-dependent interaction among antigen-presenting cell subsets, particularly CD11b+ dendritic cells (DC2) and CD103+ dendritic cells (DC1). This interaction necessitates further investigation in context of transplant immunity. We use a well-established pre-clinical models of corneal transplantation and identified distinct roles of migratory CD103+ DC1 in influencing the outcomes of the grafted tissue. In recipients with uninflamed corneal beds, migratory CD103+DC1 demonstrate a tolerogenic phenotype that modulate the immunogenic capacity of CD11b+DC2 primarily mediated by IL-10, suppressing alloreactive CD4+Th1 cells via the PD-L1/PD-1 pathway, and enhancing Treg-mediated tolerance via αvß8 integrin-activated TGFß1, thus facilitating graft survival. Conversely, in recipients with inflamed and vascularized corneal beds, IFN-γ produced by CD4+Th1 cells induces migratory CD103+DC1 to adopt an immunostimulatory phenotype, characterized by the downregulation of regulatory markers including αvß8 integrin and IL-10 and the upregulation of IL-12 and costimulatory molecules CD80/86, resulting in graft failure. The adoptive transfer of ex-vivo induced tolerogenic CD103+DC1(iDC1) effectively inhibits Th1 polarization and preserves the tolerogenic phenotype of their physiological counterparts. Collectively, our findings underscore the essential role played by CD103+DC1 in modulating host alloimmune responses.

Assessment of Corneal Graft Outcomes in a Murine Model of Endothelial Keratoplasty.

Objectives: In this study, we establish a protocol for evaluating the outcomes of endothelial keratoplasty, including graft survival, rejection, or failure. Additionally, we also evaluate the alloimmune response in graft recipients. Methods: We performed EK using C57BL/6 (allogeneic) and BALB/c (syngeneic) as donors and BALB/c mice as recipients. Slit-lamp examination and optical coherence tomography were performed for clinical evaluations for 16 weeks post-procedure. Criteria for the assessment of corneal opacity were established and the animals were graded weekly. Additionally, we assessed corneal endothelial cell density by harvesting the corneas and staining with zonula occludens-1 (ZO-1). Lastly, lymph nodes were collected, and CD4+ T cells were MACS-sorted and co-cultured with syngeneic or allogeneic antigen-presenting cells (APCs) to assess the IFN-γ expression levels by alloreactive Th1 cells (ELISPOT) in response to the direct (donor) or indirect (host) pathways of sensitization. Results: We observed graft failure in four animals, including irreversible corneal opacity, graft detachment, and anterior synechiae in the first four weeks. The remaining animals were graded between 0 and 5 as per the established criteria. The total and graft corneal thickness and endothelial cell density progressively worsened with a higher grade of corneal opacity. The direct allosensitization of Th1 cells was significantly higher in mice with a higher grade of corneal opacity. At 16 weeks follow-up, the grafts remained stable with low opacity scores in syngeneic EK recipients; however, the opacity scores were higher and variable in allogeneic EK recipients. Conclusions: These findings establish a standardized protocol to assess the graft outcomes in a murine model of EK. Furthermore, we delineate the underlying immunological pathway that contributes to the immune-mediated rejection of grafts in this model.

Potential applications of mesenchymal stem cells in ocular surface immune-mediated disorders.

We explore the interaction between corneal immunity and mesenchymal stem/stromal cells (MSCs) and their potential in treating corneal and ocular surface disorders. We outline the cornea's immune privilege mechanisms and the immunomodulatory substances involved. In this realm, MSCs are characterized by their immunomodulatory properties and regenerative potential, making them promising for therapeutic application. Therefore, we focus on the role of MSCs in immune-mediated corneal diseases such as dry eye disease, corneal transplantation rejection, limbal stem cell deficiency, and ocular graft-versus-host disease. Preclinical and clinical studies demonstrate MSCs' efficacy in promoting corneal healing and reducing inflammation in these conditions. Overall, we emphasize the potential of MSCs as innovative therapies in ophthalmology, offering promising solutions for managing various ocular surface pathologies.

Effector T Cells Promote Fibrosis in Corneal Transplantation Failure.

Purpose: To evaluate whether fibrosis contributes to corneal transplant failure and to determine whether effector CD4+ T cells, the key immune cells in corneal transplant rejection, play a direct role in fibrosis formation. Methods: Allogeneic corneal transplantation was performed in mice. Graft opacity was evaluated by slit-lamp biomicroscopy, and fibrosis was assessed by in vivo confocal microscopy. Expression of alpha-smooth muscle actin (α-SMA) in both accepted and failed grafts was assessed by real-time PCR and immunohistochemistry. Frequencies of graft-infiltrating CD4+ T cells, neutrophils, and macrophages were assessed using flow cytometry. In vitro, MK/T-1 corneal fibroblasts were co-cultured with activated CD4+CD25- effector T cells isolated from corneal transplant recipient mice, and α-SMA expression was quantified by real-time PCR and ELISA. Neutralizing antibody was used to evaluate the role of interferon gamma (IFN-γ) in promoting α-SMA expression. Results: The majority of failed grafts demonstrated clinical signs of fibrosis which became most evident at week 6 after corneal transplantation. Failed grafts showed higher expression of α-SMA as compared to accepted grafts. Flow cytometry analysis showed a significant increase in CD4+ T cells in failed grafts compared to accepted grafts. Co-culture of activated CD4+CD25- effector T cells with corneal fibroblasts led to an increase in α-SMA expression by fibroblasts. Inhibition of IFN-γ in culture significantly suppressed this increase in α-SMA expression as compared to immunoglobulin G control. Conclusions: Fibrosis contributes to graft opacity in corneal transplant failure and is mediated at least in part by effector CD4+ T cells via IFN-γ.

MMP9 cleavage of the ß4 integrin ectodomain leads to recurrent epithelial erosions in mice.

Integrin α6ß4 is an integral membrane protein within hemidesmosomes and it mediates adhesion of epithelial cells to their underlying basement membrane. During wound healing, disassembly of hemidesmosomes must occur for sheet movement-mediated cell migration. The mechanisms of disassembly and reassembly of hemidesmosomes are not fully understood. The current study was initiated to understand the underlying cause of recurrent corneal erosions in the mouse. Here, we show that in vivo: (1) MMP9 levels are elevated and ß4 integrin is partially cleaved in epithelial cell extracts derived from debridement wounded corneas; (2) the ß4 ectodomain is missing from sites where erosions develop; and (3) ß4 cleavage can be reduced by inhibiting MMP activity. Although ß4, α3 and ß1 integrins were all cleaved by several MMPs, only MMP9 was elevated in cell extracts derived from corneas with erosions. Coimmunoprecipitation studies showed that ß4 integrin associates with MMP9, and protein clustering during immunoprecipitation induced proteolytic cleavage of the ß4 integrin extracellular domain, generating a 100 kDa ß4 integrin cytoplasmic domain fragment. Confocal imaging with three-dimensional reconstruction showed that MMP9 localizes at erosion sites in vivo where the ectodomain of ß4 integrin is reduced or absent. MMP activation experiments using cultured corneal and epidermal keratinocytes showed reduced levels of α6ß4 and ß1 integrins within 20 minutes of phorbol ester treatment. This report is the first to show that ß4 integrin associates with MMP9 and that its ectodomain is a target for cleavage by MMP9 in vivo under pathological conditions.

Descemet Stripping Only Technique for Corneal Endothelial Damage in Mice.

PURPOSE: Descemet stripping only is an emerging surgical technique used to remove central Descemet membrane and corneal endothelial cells in patients with corneal endothelial disease. Here, we describe a murine model of this procedure to help facilitate basic science investigation and evaluation of postoperative outcomes using this surgical technique. METHODS: Slitlamp biomicroscopy, central corneal thickness assessment (by optical coherence tomography), and immunohistochemistry were used to assess the model through 7 weeks of follow-up. RESULTS: Complete removal of the endothelium and Descemet membrane was confirmed by slitlamp biomicroscopy and by histology. Central corneal thickness peaked at day 1 postinjury and then declined over the course of 2 weeks to a stable level of persistent edema. Seven weeks postinjury, immunohistochemical staining for ZO-1 showed the area of Descemet stripping was fully covered by enlarged and dysmorphic corneal endothelial cell. No significant ocular complications were appreciated through the end of the follow-up. CONCLUSIONS: We demonstrate the feasibility of and provide detailed instructions for a murine model of Descemet stripping only. This model provides a potential in vivo platform to investigate the mechanisms and biology of this emerging surgical procedure.

Conventional type I migratory CD103+ dendritic cells are required for corneal allograft survival.

Corneal transplant rejection primarily occurs because of the T helper 1 (Th1) effector cell-mediated immune response of the host towards allogeneic tissue. The evidence suggests that type 1 migratory conventional CD103+ dendritic cells (CD103+DC1) acquire an immunosuppressive phenotype in the tumor environment; however, the involvement of CD103+DC1 in allograft survival continues to be an elusive question of great clinical significance in tissue transplantation. In this study, we assess the role of CD103+DC1 in suppressing Th1 alloreactivity against transplanted corneal allografts. The immunosuppressive function of CD103+DC1 has been extensively studied in non-transplantation settings. We found that host CD103+DC1 infiltrates the corneal graft and migrates to the draining lymph nodes to suppress alloreactive CD4+ Th1 cells via the programmed death-ligand 1 axis. The systemic depletion of CD103+ DC1 in allograft recipients leads to amplified Th1 activation, impaired Treg function, and increased rate of allograft rejection. Although allograft recipient Rag1 null mice reconstituted with naïve CD4+CD25- T cells efficiently generated peripheral Treg cells (pTreg), the CD103+DC1-depleted mice failed to generate pTreg. Furthermore, adoptive transfer of pTreg failed to rescue allografts in CD103+DC1-depleted recipients from rejection. These data demonstrate the critical role of CD103+DC1 in regulating host alloimmune responses.

A Novel Murine Model of Endothelial Keratoplasty.

PURPOSE: The purpose of this study was to establish a murine model of endothelial keratoplasty. METHODS: Endothelial keratoplasty (EK) was performed using C57BL/6 donor and BALB/c recipient mice. The central endothelium and Descemet membrane were removed from the recipient cornea, and a 1.5-mm posterior lamellar donor graft was made adherent to the recipient cornea with a small amount of viscoelastic. Mice were followed through slitlamp microscopy postoperatively, and OCT was used to assess the cornea and anterior chamber and measure central corneal thickness. Histology and immunohistochemistry were performed to confirm graft adherence and endothelial cell morphology. RESULTS: Successfully attached EK grafts were visualized in all transplanted animals. Histology and immunostaining confirmed proper graft orientation and adherence, as well as the presence of donor endothelium on transplanted grafts. We observed maximal corneal edema in all animals at day 1 postoperatively which gradually subsided. EK graft survival was 97% at 8 weeks. CONCLUSIONS: In this study, we describe a novel murine model for EK which we anticipate will enable detailed investigation into the cellular and molecular mechanisms involved in EK pathobiology.

Amplified Natural Killer Cell Activity and Attenuated Regulatory T-cell Function Are Determinants for Corneal Alloimmunity in Very Young Mice.

BACKGROUND: Corneal transplantation outcomes are generally less favorable in young children compared with adults. The purpose of this study was to determine the immunological mechanisms underlying this difference. METHODS: A murine model of allogeneic corneal transplantation was used in the study, and graft survival was determined by evaluating opacity scores for 8 wk. Syngeneic transplantation in the very young host served as a surgical control. The frequencies of total and activated natural killer (NK) cells in cornea posttransplantation were kinetically evaluated using flow cytometry. The regulatory T cell (Treg) frequency and function in naive animals were assessed by flow cytometry and in vitro suppression assays, respectively. Finally, graft survival and immune responses were determined in NK cell-depleted, or adult naive Treg-transferred, young hosts. RESULTS: Corneal allograft survival in the very young recipients was significantly lower than in adult hosts. The frequencies of total NK cells and their interferon gamma-expressing subset in the cornea were significantly higher in the very young mice posttransplantation. In ungrafted mice, frequencies of Treg in draining lymph nodes as well as their capabilities to suppress NK-cell secretion of interferon gamma were lower in the very young compared with adults. In NK cell-depleted or adult Treg--transferred very young recipients, the allograft survival was significantly improved along with the suppressed NK-cell response. CONCLUSIONS: Our data demonstrate that amplified activity of NK cells, together with lower suppressive function of Treg, contributes to early rejection of corneal allografts in very young graft recipients.