Special Reviews Juntendo Medical Journal 2017. 63(1), 2-7 A New Immunotherapy Using Regulatory T-Cells for High-Risk Corneal Transplantation TAKENORI INOMATA＊ ＊Department of Ophthalmology, Juntendo University Faculty of Medicine, Tokyo, Japan Corneal transplantation is the most commonly performed tissue transplantation worldwide. Despite very high (above 90%) survival rates in recipients with nonvascularized and noninflamed graft beds, survival rates significantly decline (under 50%) when grafts are placed onto vascularized or inflamed host beds associated with conditions such as previous graft rejection, infection, or trauma. These results are seen despite treatment with high doses of nonspecific immunosuppressive medications, which often do not promote long-term survival. Therefore, new strategies are required to modulate the immune system without conventional immunosuppressive agents and improve transplant survival in“high-risk”patients with inflamed host beds. Regulatory T-cells (Treg) are key modulators of the immune response and may play a crucial role in a new therapy for high-risk corneal transplantation. Here we introduce the murine high-risk corneal transplantation model and review the implications of Tregs for corneal transplantation. Key words: corneal transplantation, regulatory T-cell, high-risk corneal transplantation, neovascularization Introduction eyeʼs refractive power. The cornea has three layers, including the epithelium, stroma and endothelium, 1. Outline of corneal transplantation divided by Bowmanʼs membrane and Descemetʼs The first corneal transplantation was successfully membrane respectively. Corneal transplantation is performed by Dr. Eduard Zirm in 1905 1). Along with a surgical procedure where a damaged or diseased the growing demand for corneal transplantation, the cornea is replaced by donated corneal tissue. first institutional eye bank was born in New York in Corneal transplantation has evolved into an array of 1944. In Japan, the first eye bank was established by techniques focused on the selective replacement of Juntendo University School of Medicine and Keio diseased layers of the cornea such as lamellar University in 1963. Nowadays, cornea transplanta- corneal transplantation, Descemet stripping auto- tion is one of the most frequently performed solid mated endothelial keratoplasty (DSAEK) and organ transplantation worldwide with 65,000 cases penetrating keratoplasty. These procedures are annually. There are more than 40,000 cases in the tailored specifically to the underlying pathologic US and 1,500 cases in Japan annually 2). condition causing the corneal dysfunction. How- The cornea is the transparent, dome shaped layer ever, the majority of corneal transplantations are that covers the anterior eye and has the dual still penetrating keratoplasties 3). function of protecting the inner contents of the eye In uninflamed low-risk host beds (e. g. simple as well as providing approximately two thirds of the corneal scars), corneal transplants enjoy very high Takenori Inomata Department of Ophthalmology, Juntendo University Faculty of Medicine 3-1-3 Hongo, Bunkyo-ku, Tokyo 113-8431, Japan TEL: +81-3-5802-1228 FAX: +81-3-5689-0394 E-mail: [email protected] 339th Triannual Meeting of the Juntendo Medical Society: Medical Research Update〔Held on May 21, 2016〕 〔Received Nov. 25, 2016〕 Copyright © 2017 The Juntendo Medical Society. This is an open access article distributed under the terms of Creative Commons Attribution Li- cense (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original source is properly credited. doi: 10.14789/jmj.63.2 2 Juntendo Medical Journal 63(1), 2017 rates of success (above 90%). However, host factors corneal transplantation is able to track the behavior such as graft bed inflammation and neovasculariza- and contribution of donor and host-derived cells tion lead to high rejection rates (over 50%) even during an immune response with different allograft with the use of topical steroids. These results are types from different strains of mice as well as in seen despite treatment with high doses of nonspe- mice that lack (knockout) or overexpress (trans- cific immunosuppressive medications, which often genic) specific genes. do not promote long-term survival and may have We have developed a murine corneal transplanta- severe side effects such as glaucoma, cataracts, and tion model 7). Briefly, the central cornea (2-mm opportunistic infections 4) 5). diameter) was excised from a donor C57BL/6 In all forms of transplantation, certain hosts are mouse using scissors (Vannas; Storz Instruments, known to be at particularly high risk for graft rejec- San Dimas, CA). The graft bed was prepared by tion. High-risk transplant recipients often abruptly excising a 1.5-mm site in the central cornea of a and irreversibly reject their grafts regardless of the BALB/c mouse. The donor button was then placed magnitude of immunosuppression. Thus, under- onto the recipient bed and secured with eight standing the underlying mechanisms of high interrupted 11-0 nylon sutures. Corneal sutures rejection rates in high-risk corneal transportation is were removed 7 days after surgery. Graft survival essential for controlling the transplant immune was evaluated for 8 weeks using a slit-lamp response. biomicroscope. We used a standard opacity-grad- ing (range, 0-5+) scheme to define rejection 8); 2. Immune privilege of cornea corneas with an opacity score of 2+ for two consec- Immune privilege is well developed in the eye, utive examinations were considered rejected. brain, pregnant uterus, testis, ovary adrenal cortex and certain solid tumors 6). It protects against Analysis of neovascularization post high-risk collateral damage from immune-mediated inflam- corneal transplantation mation. In the eye, the cornea, anterior chamber, vitreous cavity and subretinal space are considered 1. Murine high-risk corneal transplantation model to be immune-privileged sites. Immune privilege is For understanding the underlying mechanisms of defined as the reduced incidence and activity of high rejection rate in high-risk corneal transplanta- immune rejection in corneal allografts compared to tion, we also developed a murine high-risk corneal other categories of organ allografts performed transplantation model 7). Inflamed, neovascularized under the same conditions. Skin allografts trans- (ʻhigh-riskʼ) host beds were created using three planted across various MHC or minor histocompati- intrastromal sutures placement into the central bility barriers undergo rejection in approximately cornea using 11-0 nylon sutures 14 days before 100% of the hosts. By contrast, orthotopic corneal corneal transplantation to induce host beds prone to allografts experience longer-term survival in 50% graft rejection. For confirming the neovasculariza- to 90% of the hosts, depending on the histocompati- tion and lymphoangiogenessis in the cornea, we bility barriers that confront the host. The capacity developed original software using ImageJ 9) 10). Our of corneal allografts to evade immune rejection is software calculates the areas of neovascularization attributable to multiple anatomical, physiological (Figure-1A; CD31) and lymphoangiogenessis and immunoregulatory conditions that act to pre- (Figure-1B; LYVE-1) after immunofluorescent vent the induction and expression of alloimmunity. staining as the percentage of vessels (as outlined in white, Figure-1C and D) growing into the cornea. Murine model of corneal transplantation We have confirmed significantly high neovasculari- zation and lymphoangiogenessis in high-risk host Corneal transplantation in the murine model is an beds 14 days after suture placement 10). Then, we excellent model for researching cornea and ocular excised a 1.5 mm corneal button from the high-risk surface immunology. The accessibility of the cornea host beds and performed the corneal transplanta- enables ease of manipulation and observation of tion with 2.0 mm donor corneas via 8 sutures using graft survival. In addition, the murine model of 11-0 nylon suture. 3 Inomata: Treg therapy to corneal transplantation CD31 LYVE－1 AB IHC CD Result Figure-1 Representative image showing (A) CD31 (blood vessels), (B) LYVE-1 (lymphatic vessels) staining of the cornea and (C & D) the processed image (calculated area) using ImageJ. (magnification, ×4). 2. Corneal whole mount and immunofluorescent area (%) by ImageJ). staining Freshly excised corneas were washed in phos- Regulatory T-cell therapy for corneal phate-buffered saline. Corneal epithelium was transplantation removed after incubation with 20 mM EDTA for 60 minutes in 37℃; fixed in acetone for 15 minutes at Regulatory T-cells (Tregs) were discovered by room temperature, and blocked in 2% bovine serum Shimon Sakauguchi in 1995. Tregs maintain albumin for 60 minutes. Then, the corneas were immune homeostasis by dampening inflammatory double stained with CD31 (Santa Cruz Biotechnol- responses toward self- and alloantigens 11) 12), ogy, Dallas, TX, USA) and lymphatic vessel thereby playing a crucial role in allograft survival. endothelial hyaluronan receptor (LYVE)-1 (R&D Studies in solid organ transplantation have shown AF2125) as previously described using goat anti- that Treg-based therapies can be effective in mouse CD31 FITC (1:100) and LYVE-1 purified promoting long-term tolerance to skin and heart goat-anti mouse (1:400) overnight. Cy3 conjugated grafts in experimental animals 13) 14) as well as donkey anti-goat