Targeted delivery of autoantigen to dendritic cells prevents development of spontaneous uveitis.

Restoration of immunological tolerance to self antigens has been a major drive in understanding the mechanisms of, and developing new treatments for, autoimmune and autoinflammatory disease. Sessile dendritic cells (DC) are considered the main instruments underpinning immunological tolerance particularly the CD205+ (DEC205+) cDC1 subset in contrast to DCIR2+ cDC2 which mediate immunogenicity. Targeting DC using autoantigen peptide-antibody fusion proteins has been a well explored methodology for inducing tolerance. Here we show that subcutaneous (s.c.) inoculation of hen-egg lysozyme (HEL)-DEC205 Ig fusion prevents the development of spontaneous uveoretinitis (experimental autoimmune uveoretinitis, EAU) in a transgenic mouse model generated by crossing interphotoreceptor retinol binding protein (IRBP)-HEL (sTg HEL) with HEL specific TCR (sTg TCR) mice. Prolonged suppression of EAU required injections of HEL-DEC205 Ig once weekly, reflecting the half life of s.c. DC. Interestingly, HEL-DCIR2 Ig also had a suppressive effect on development of EAU but less so than DEC205 Ig while it had minimal effect on preventing the retinal atrophy associated with EAU. In addition, HEL-DEC205 Ig was only effective when administered s.c. rather than systemically and had no effect on EAU induced by adoptive transfer of HEL-activated T cells. These data demonstrate the importance of systemic (lymph node) rather than local (eye) antigen presentation in the development of EAU as well as suggest a potential therapeutic approach to controlling sight-threatening immune-mediated uveitis provided relevant antigen(s) can be identified.

The atypical chemokine receptor-2 fine-tunes the immune response in herpes stromal keratitis.

Herpes stromal keratitis (HSK) is a blinding corneal disease caused by herpes simplex virus-1 (HSV-1), a common pathogen infecting most of the world's population. Inflammation in HSK is chemokine-dependent, particularly CXCL10 and less so the CC chemokines. The atypical chemokine receptor-2 (ACKR2) is a decoy receptor predominantly for pro-inflammatory CC chemokines, which regulates the inflammatory response by scavenging inflammatory chemokines thereby modulating leukocyte infiltration. Deletion of ACKR2 exacerbates and delays the resolution of the inflammatory response in most models. ACKR2 also regulates lymphangiogenesis and mammary duct development through the recruitment of tissue-remodeling macrophages. Here, we demonstrate a dose-dependent upregulation of ACKR2 during corneal HSV-1 infection. At an HSV inoculum dose of 5.4 x 105 pfu, but not at higher dose, ACKR2 deficient mice showed prolonged clinical signs of HSK, increased infiltration of leukocytes and persistent corneal neovascularization. Viral clearance and T cell activation were similar in ACKR2-/- and wild type mice, despite a transient diminished expression of CD40 and CD86 in dendritic cells. The data suggest that ACKR2 fine-tunes the inflammatory response and the level of neovascularization in the HSK.

A Role for Folate in Microbiome-Linked Control of Autoimmunity.

The microbiome exerts considerable control over immune homeostasis and influences susceptibility to autoimmune and autoinflammatory disease (AD/AID) such as inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes (T1D), psoriasis, and uveitis. In part, this is due to direct effects of the microbiome on gastrointestinal (GI) physiology and nutrient transport, but also to indirect effects on immunoregulatory controls, including induction and stabilization of T regulatory cells (T reg). Secreted bacterial metabolites such as short-chain fatty acids (SCFA) are under intense investigation as mediators of these effects. In contrast, folate (vitamin B9), an essential micronutrient, has attracted less attention, possibly because it exerts global physiological effects which are difficult to differentiate from specific effects on the immune system. Here, we review the role of folate in AD/AID with some emphasis on sight-threatening autoimmune uveitis. Since folate is required for the generation and maintenance of T reg , we propose that one mechanism for microbiome-based control of AD/AID is via folate-dependent induction of GI tract T reg , particularly colonic T reg, via anergic T cells (T an). Hence, folate supplementation has potential prophylactic and/or therapeutic benefit in AID/AD.

The Role of Inflammation in Diabetic Retinopathy.

Inflammation is central to pathogenic processes in diabetes mellitus and the metabolic syndrome and particularly implicates innate immunity in the development of complications. Inflammation is a primary event in Type 1 diabetes where infectious (viral) and/or autoimmune processes initiate disease; in contrast, chronic inflammation is typical in Type 2 diabetes and is considered a sequel to increasing insulin resistance and disturbed glucose metabolism. Diabetic retinopathy (DR) is perceived as a vascular and neurodegenerative disease which occurs after some years of poorly controlled diabetes. However, many of the clinical features of DR are late events and reflect the nature of the retinal architecture and its cellular composition. Retinal microvascular disease is, in fact, an early event pathogenetically, induced by low grade, persistent leukocyte activation which causes repeated episodes of capillary occlusion and, progressive, attritional retinal ischemia. The later, overt clinical signs of DR are a consequence of the retinal ischemia. Metabolic dysregulation involving both lipid and glucose metabolism may lead to leukocyte activation. On a molecular level, we have shown that macrophage-restricted protein tyrosine phosphatase 1B (PTP1B) is a key regulator of inflammation in the metabolic syndrome involving insulin resistance and it is possible that PTP1B dysregulation may underlie retinal microvascular disease. We have also shown that adherent CCR5+CD11b+ monocyte macrophages appear to be selectively involved in retinal microvascular occlusion. In this review, we discuss the relationship between early leukocyte activation and the later features of DR, common pathogenetic processes between diabetic microvascular disease and other vascular retinopathies, the mechanisms whereby leukocyte activation is induced in hyperglycemia and dyslipidemia, the signaling mechanisms involved in diabetic microvascular disease, and possible interventions which may prevent these retinopathies. We also address a possible role for adaptive immunity in DR. Although significant improvements in treatment of DR have been made with intravitreal anti-VEGF therapy, a sizeable proportion of patients, particularly with sight-threatening macular edema, fail to respond. Alternative therapies targeting inflammatory processes may offer an advantage.

Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

Immune Privilege: The Microbiome and Uveitis.

Immune privilege (IP), a term introduced to explain the unpredicted acceptance of allogeneic grafts by the eye and the brain, is considered a unique property of these tissues. However, immune responses are modified by the tissue in which they occur, most of which possess IP to some degree. The eye therefore displays a spectrum of IP because it comprises several tissues. IP as originally conceived can only apply to the retina as it contains few tissue-resident bone-marrow derived myeloid cells and is immunologically shielded by a sophisticated barrier - an inner vascular and an outer epithelial barrier at the retinal pigment epithelium. The vascular barrier comprises the vascular endothelium and the glia limitans. Immune cells do not cross the blood-retinal barrier (BRB) despite two-way transport of interstitial fluid, governed by tissue oncotic pressure. The BRB, and the blood-brain barrier (BBB) mature in the neonatal period under signals from the expanding microbiome and by 18 months are fully established. However, the adult eye is susceptible to intraocular inflammation (uveitis; frequency ~200/100,000 population). Uveitis involving the retinal parenchyma (posterior uveitis, PU) breaches IP, while IP is essentially irrelevant in inflammation involving the ocular chambers, uveal tract and ocular coats (anterior/intermediate uveitis/sclerouveitis, AU). Infections cause ~50% cases of AU and PU but infection may also underlie the pathogenesis of immune-mediated "non-infectious" uveitis. Dysbiosis accompanies the commonest form, HLA-B27-associated AU, while latent infections underlie BRB breakdown in PU. This review considers the pathogenesis of uveitis in the context of IP, infection, environment, and the microbiome.

Activation of dendritic cells by crosslinked collagen hydrogels (artificial corneas) varies with their composition.

Activated T cells are known to promote fibrosis, a major complication limiting the range of polymeric hydrogels as artificial corneal implants. As T cells are activated by dendritic cells (DC), minimally activating hydrogels would be optimal. In this study, we evaluated the ability of a series of engineered (manufactured/fabricated) and natural collagen matrices to either activate DC or conversely induce DC apoptosis in vitro. Bone marrow DC were cultured on a series of singly and doubly crosslinked hydrogels (made from recombinant human collagen III [RHCIII] or collagen mimetic peptide [CMP]) or on natural collagen-containing matrices, MatrigelTM and de-cellularised mouse corneal stroma. DC surface expression of major histocompatibility complex Class II and CD86 as well as apoptosis markers were examined. Natural matrices induced low levels of DC activation and maintained a "tolerogenic" phenotype. The same applied to singly crosslinked CMP-PEG gels. RHCIII gels singly crosslinked using either N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide with the coinitiator N-hydroxy succinimide (EDC-NHS) or N-cyclohexyl-N-(2-morpholinoethyl)carbodiimide metho-p-toulenesulfonate with NHS (CMC-NHS) induced varying levels of DC activation. In contrast, however, RHCIII hydrogels incorporating an additional polymeric network of 2-methacryloyloxyethyl phosphorylcholine did not activate DC but instead induced DC apoptosis, a phenomenon observed in natural matrices. This correlated with increased DC expression of leukocyte-associated immunoglobulin-like receptor-1. Despite low immunogenic potential, viable tolerogenic DC migrated into and through both natural and manufactured RHCIII gels. These data show that the immunogenic potential of RHCIII gels varies with the nature and composition of the gel. Preclinical evaluation of hydrogel immunogenic/fibrogenic potential is recommended.

TGF-ß1-activated type 2 dendritic cells promote wound healing and induce fibroblasts to express tenascin c following corneal full-thickness hydrogel transplantation.

We showed previously that 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide hydrochloride (EDC) cross-linked recombinant human collagen III hydrogels promoted stable regeneration of the human cornea (continued nerve and stromal cell repopulation) for over 4 years. However, as EDC cross linking kinetics were difficult to control, we additionally tested a sterically bulky carbodiimide. Here, we compared the effects of two carbodiimide cross linkers-bulky, aromatic N-cyclohexyl-N0-(2-morpholinoethyl)-carbodiimide (CMC), and nonbulky EDC-in a mouse corneal graft model. Murine corneas undergoing full-thickness implantation with these gels became opaque due to dense retro-corneal membranes (RCM). Corneal epithelial cytokeratin 12 and alpha smooth muscle actin indicative of functional tissue regeneration and wound contraction were observed in RCM surrounding both hydrogel types. However, quantitatively different levels of infiltrating CD11c+ dendritic cells (DC) were found, suggesting a hydrogel-specific innate immune response. More DC infiltrated the stroma surrounding EDC-N-hydroxysuccinimide (NHS) hydrogels concurrently with higher fibrosis-associated tenascin c expression. The opposite was true for CMC-NHS gels that had previously been shown to be more tolerising to DC. In vitro studies showed that DC cultured with transforming growth factor ß1 (TGF-ß1) induced fibroblasts to secrete more tenascin c than those cultured with lipopolysaccharide and this effect was blocked by TGF-ß1 neutralisation. Furthermore, tenascin c staining was found in 40- to 50µm long membrane nanotubes formed in fibroblast/DC cocultures. We suggest that TGF-ß1 alternatively activated (tolerising) DC regulate fibroblast-mediated tenascin c secretion, possibly via local production of TGF-ß1 in early wound contraction, and that this is indirectly modulated by different hydrogel chemistries.

Retinoic acid-induced autoantigen-specific type 1 regulatory T cells suppress autoimmunity.

Regulatory T (Treg) cells help to maintain tolerance and prevent the development of autoimmune diseases. Retinoic acid (RA) can promote peripheral conversion of naïve T cells into Foxp3+ Treg cells. Here, we show that RA can act as an adjuvant to induce antigen-specific type 1 Treg (Tr1) cells, which is augmented by co-administration of IL-2. Immunization of mice with the model antigen KLH in the presence of RA and IL-2 induces T cells that secrete IL-10, but not IL-17 or IFN-γ, and express LAG-3, CD49b and PD-1 but not Foxp3, a phenotype typical of Tr1 cells. Furthermore, immunization of mice with the autoantigen MOG in the presence of RA and IL-2 induces Tr1 cells, which suppress pathogenic Th1 and Th17 cells that mediate the development of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease of the CNS. Furthermore, immunization with a surrogate autoantigen, RA and IL-2 prevents development of spontaneous autoimmune uveitis. Our findings demonstrate that the induction of autoantigen-specific Tr1 cells can prevent the development of autoimmunity.

Transmission Electron Microscopy Data on drusen-like deposits in the retinal degeneration sTg-IRBP: HEL mouse model.

Histology (H&E) and transmission electron microscopy (TEM) data are provided showing age-related changes in the retinal structure of sTg-IRBP:HEL mice. These include substantial photoreceptor loss, atrophy of the retinal pigment epithelium, Bruch׳s membrane disruption and thickening, along with the presence of drusenoid deposits and changes in basal laminar infoldings. These features resemble some of those key characteristics found in the course of human dry (atrophic) age-related macular degeneration (AMD), particularly with regard to drusen. Hence, we believe the sTg-IRBP:HEL mouse model represents a useful and promising archetype for future study of the mechanism of drusen formation in AMD.

Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice.

Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2'-deoxyuridine (IdU), 5-chloro-2'-deoxyuridine (CldU) and 5-ethynyl-2'-deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ±â€¯0.2 days) compared to 10 week old mice (4.97 ±â€¯0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ±â€¯1.4% of LESCs were shown to enter S phase in a 24 h period and were estimated to divide every 2-3 weeks. Within 24 h of corneal injury this rose significantly to 32.8 ±â€¯10.0% of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24-48 h period after wounding in young adults, LESC activation continued to increase (86.5 ±â€¯8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ±â€¯9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.

The atypical chemokine receptor-2 does not alter corneal graft survival but regulates early stage of corneal graft-induced lymphangiogenesis.

PURPOSE: To re-evaluate the role of the atypical chemokine receptor-2 (ACKR2) in corneal graft rejection and investigate the effect of ACKR2 on inflammation-associated lymphangiogenesis using murine orthotopic corneal transplantation. METHODS: Corneal grafts were performed and evaluated in the settings of syngeneic, allogeneic and single antigen (HY-antigen) disparity pairings. Corneal vessels were quantified in whole mounts from WT, ACKR2-/- and F4/80-/-ACKR2-/- mice that received syngeneic or allogeneic grafts using anti-CD31 and anti-Lyve-1 antibodies. RESULTS: Syngeneic corneal grafts in WT and ACKR2-/- mice were 100% accepted. Fully histo-incompatible allogeneic grafts were rapidly rejected (100%) with similar tempo in both WT and ACKR2-/- hosts. Around 50% of single-antigen (HY) disparity grafts rejected at a slow but similar tempo (60 days) in WT and ACKR2-/- mice. Prior to grafting, F4/80-/-ACKR2-/- mice had lower baseline levels of limbal blood and lymphatic vessels compared to ACKR2-/- mice. Syngeneic grafts, but not allogeneic grafts, in ACKR2-/- and F4/80-/-ACKR2-/- mice induced higher levels of lymphatic sprouting and infiltration of Lyve-1+ cells during the early (3d) post-graft (pg) stage but lymphatic density was similar to WT grafted mice by 7d pg. CONCLUSIONS: Our results indicate that the chemokine scavenger receptor, ACKR2, has no role to play in the survival of allogeneic grafts. A minor role in regulation of lymphangiogenesis in the early stage of wound healing in syngeneic grafts is suggested, but this effect is probably masked by the more pronounced lymphangiogenic inflammatory response in allogeneic grafts. No additional effect was observed with the deletion of the resident macrophage gene, F4/80.

Autoimmunity, Autoinflammation, and Infection in Uveitis.

PURPOSE: To review the pathogenesis of uveitis in light of recent advances in our understanding of innate and adaptive immune responses and their regulation. DESIGN: Perspective. METHODS: Methods included a review of prevailing views on the pathogenesis of uveitis and an analysis of developments in immunology that impact on its conceptual basis, particularly the concept of immunologic tolerance and its loss in autoimmunity. Importantly, the role of infection in the pathogenesis of uveitis is evaluated. RESULTS: The results comprise a reappraisal of the pathogenesis of anterior vs posterior uveitis in the context of the blood-retinal barrier and its relation to autoimmune, autoinflammatory, and infectious uveitis. Autoimmunity is seen as a possible cause of certain forms of uveitis but definitive proof is lacking. Autoinflammatory disease, involving activated innate immune mechanisms, is considered causative in a second set of uveitis conditions. A place for infection in uveitis generally is proposed within a unifying concept for the pathogenesis of uveitis. CONCLUSION: Infection may be implicated directly or indirectly in many forms of noninfectious or undifferentiated uveitis. In addition to the growing recognition that foreign antigen, including reactivatable infectious agents, might hide within ocular tissues, the possibility that a dysregulated microbiome might generate T cells that cause immune-mediated ocular inflammation has now been demonstrated experimentally. An uncontrolled, overexuberant host immune response may cause continuing irreversible tissue damage even after the infection has been cleared.

Partial retinal photoreceptor loss in a transgenic mouse model associated with reduced levels of interphotoreceptor retinol binding protein (IRBP, RBP3).

Organ-specific transgenic membrane expression of hen egg lysozyme (HEL) as a "neo-self antigen" has been used in several models to study immunological tolerance. In this study we report the changes which occur in the B10.BR mouse retina when membrane-bound HEL is expressed in photoreceptors under the control of the promoter for interphotoreceptor retinoid binding protein (IRBP, RBP3). On direct clinical examination of the single transgenic (sTg-IRBP:HEL) mouse fundus, a low-level increase in retinal degeneration compared to non-transgenic controls was observed, presenting as drusenoid deposits and occasional small patches of atrophy. On histological examination, there was an overall shortening of outer segments and loss of photoreceptor nuclei in sTg-IRBP:HEL mice, which was more pronounced in the retinal periphery, particularly inferiorly. The fundoscopically observed lesions did not correlate with the photoreceptor shortening/loss but appeared to be located at the level of the retinal pigment epithelium/choriocapillaris layer and were an exaggeration in size and number of similar age-related changes found in wild type (WT) mice. In addition, neither the atrophic lesions nor the photoreceptor shortening were associated with common retinal degeneration genes, nor were they caused by exposure to light damage since mice housed at both high and low ambient light levels had similar degrees of retinal degeneration. Instead, sTg-IRBP:HEL mice expressed reduced levels of soluble retinal IRBP compared to WT mice which were present from postnatal day16 (P16) and preceded development of photoreceptor shortening (onset P21). We propose that insertion of the HEL transgene in the photoreceptor membrane disrupted normal photoreceptor function and led to reduced levels of soluble IRBP and retinal thinning. A similar phenotype has been observed in IRBP deficient mice. Despite the retinal thinning, the amount of HEL expressed in the retina was sufficient to act as an autoantigenic target when the mice were crossed to the HEL T cell receptor Tg mouse, since double transgenic (dTg-IRBP:HEL) mice spontaneously developed a severe uveoretinitis with onset at weaning. We suggest that, although membrane expression of foreign transgene products is likely to modify the structure and function of tissues and cells, the technology provides useful models to investigate mechanisms of antigen-specific immunological tolerance.

Mesenchymal stem cell therapy for retro-corneal membrane - A clinical challenge in full-thickness transplantation of biosynthetic corneal equivalents.

Artificial corneas (keratoprostheses) and biosynthetic collagen-based corneal equivalents are surgical implants designed to ease the global burden of corneal blindness. However, keratoprostheses in many cases fail due to development of fibrous retro-corneal membranes (RCM). Fibrous membranes which develop in the anterior chamber after prosthesis implantation do so on a matrix of fibrin. This study investigated fibrin deposition and RCM formation after full-thickness collagen-based hydrogel implants and compared them with syngeneic and allogeneic corneal grafts in mice. Fibrin cleared from the anterior chamber within 14 days in both allo- and syn-grafts but, persisted in hydrogel implants and developed into dense retro-corneal membrane (RCM) which were heavily infiltrated by activated myofibroblasts. In contrast, the number of CD11b+ macrophages infiltrating the initial deposition of fibrin in the anterior chamber (AC) after hydrogel implantation was markedly reduced compared to syn- and allo-grafts. Inoculation of mesenchymal stem cells prior to collagen gel implant promoted clearance of gel-associated fibrin from the anterior chamber. We propose that a failure of macrophage-mediated clearance of fibrin may be the cause of RCM formation after collagen-based hydrogel implants and that mesenchymal stem cell therapy promotes clearance of fibrin and prevents RCM formation. STATEMENT OF SIGNIFICANCE: The manuscript addresses the potential value of bone marrow-derived mesenchymal stem cell therapy for retro-corneal membrane (RCM) formation in full-thickness transplantation of biosynthetic corneal equivalents. This work reports the pathophysiological changes in the anterior chamber of the mouse eye following full-thickness recombinant human cross-linked collagen-based hydrogel implants in which persistent fibrin promotes the development of dense RCM. Furthermore, pre-treatment with mesenchymal stem cells reduces RCM formation and enhances corneal transparency.

High-Risk Corneal Graft Rejection in the Setting of Previous Corneal Herpes Simplex Virus (HSV)-1 Infection.

PURPOSE: The "high-risk phenotype" of corneal graft recipients is considered to be related to preexisting vascularization such as that associated with herpes simplex virus-1 (HSV-1) keratitis (HSK). The purpose of this study was to investigate the immunologic mechanisms underlying accelerated corneal graft rejection using a mouse model of HSK. METHODS: Herpes simplex virus type 1 keratitis was induced in BALB/c mice. Syngeneic and allogeneic (C57BL/6 mice) corneal grafts were performed in mice with HSK at different times after infection. Some grafts were performed on HSV-infected CD4 T cell-deficient BALB/c mice. Clinical, histologic, immunologic, and virus detection studies were performed on samples of cornea, draining lymph node (LN), and trigeminal ganglion (TG) cells. RESULTS: Corneal grafts in mice with HSK rejected with higher frequency and more rapid tempo compared with grafts in uninfected mice. In corneas with HSK and vascularization at the time of grafting, both syngeneic and allogeneic corneal grafts failed with similar frequency and tempo. However, in the absence of preexisting inflammation and vascularization, syngeneic grafts were accepted when the grafts were performed at a late time point after HSV infection (42 days), whereas allografts were rejected at this time. In contrast, syngeneic grafts in nonvascularized HSV-infected recipients failed if they were performed within 10 days of HSV infection, an effect that was dependent on CD4 T cells, as demonstrated using CD4 deficient mice. Importantly, a variably sustained but strongly positive anti-HSV T-cell response was detected in allografted HSK recipients with a similar but lesser response in syngeneic hosts. CONCLUSIONS: A previous HSV-1 corneal infection predisposes donor grafts to a high risk of failure by both innate and adaptive immune mechanisms in which an anti-HSV CD4 T-cell response plays a prominent role.

High-risk corneal allografts: A therapeutic challenge.

Corneal transplantation is the most common surgical procedure amongst solid organ transplants with a high survival rate of 86% at 1-year post-grafting. This high success rate has been attributed to the immune privilege of the eye. However, mechanisms originally thought to promote immune privilege, such as the lack of antigen presenting cells and vessels in the cornea, are challenged by recent studies. Nevertheless, the immunological and physiological features of the cornea promoting a relatively weak alloimmune response is likely responsible for the high survival rate in "low-risk" settings. Furthermore, although corneal graft survival in "low-risk" recipients is favourable, the prognosis in "high-risk" recipients for corneal graft is poor. In "high-risk" grafts, the process of indirect allorecognition is accelerated by the enhanced innate and adaptive immune responses due to pre-existing inflammation and neovascularization of the host bed. This leads to the irreversible rejection of the allograft and ultimately graft failure. Many therapeutic measures are being tested in pre-clinical and clinical studies to counter the immunological challenge of "high-risk" recipients. Despite the prevailing dogma, recent data suggest that tissue matching together with use of systemic immunosuppression may increase the likelihood of graft acceptance in "high-risk" recipients. However, immunosuppressive drugs are accompanied with intolerance/side effects and toxicity, and therefore, novel cell-based therapies are in development which target host immune cells and restore immune homeostasis without significant side effect of treatment. In addition, developments in regenerative medicine may be able to solve both important short comings of allotransplantation: (1) graft rejection and ultimate graft failure; and (2) the lack of suitable donor corneas. The advances in technology and research indicate that wider therapeutic choices for patients may be available to address the worldwide problem of corneal blindness in both "low-risk" and "high-risk" hosts.

Local targeting of the CD200-CD200R axis does not promote corneal graft survival.

Corneal graft rejection is primarily a CD4(+) T cell-mediated mechanism in which macrophages may play an important inflammatory role. CD200Fc fusion protein is an artificial agonist of CD200R1, a receptor expressed predominantly on myeloid cells, engagement of which is known to down-regulate macrophage function. We therefore wished to test whether CD200Fc could be used as a therapeutic agent to prolong corneal graft survival. The distribution of CD200R1 and CD200, its natural ligand, was examined by immunohistology in the cornea and conjunctiva of unoperated rats and rats that had received corneal allografts. Mouse CD200Fc was injected subconjunctivally into transplanted rats on six occasions from the day of surgery until day 10 after transplantation. Control groups received injections of mouse IgG or diluent PBS. Allo-transplants were also performed in CD200(-/-) and control mice. The ability of CD200Fc to bind rat macrophages in vitro and to inhibit nitric oxide production was tested. Mean day of rejection in CD200Fc, IgG and PBS-treated rats was 12, 10 and 9 respectively (p=0.24). Mean day of rejection in CD200(-/-) and wild type mice was 17.5 and 16.0 respectively (p=0.07). Mouse CD200Fc bound to rat macrophages in a dose-dependent manner, but was unable to inhibit nitric oxide production. The fact that treatment with CD200Fc did not inhibit graft rejection and the failure of CD200 deficiency to affect graft survival suggests that local targeting of the CD200-CD200R axis to suppress macrophage activation is not a useful therapeutic strategy in corneal graft rejection.