Special Reviews

Juntendo Medical Journal 2017. 63(1), 2-7 A New Immunotherapy Using Regulatory T-Cells for High-Risk

TAKENORI INOMATA＊

＊Department of Ophthalmology, Juntendo University Faculty of , 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 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 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

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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 (1:2,000, Jackson ImmunoRe- humans 15) 16). While these and other studies have search Laboratories, West Grove, PA, USA) was shed light on the antigen-specificity of Tregs on then added as a secondary antibody and incubated graft survival, Chauhan SK, et al. showed that the for 2 hours. Stained whole mount corneas were adoptive transfer of Tregs to corneal transplant mounted in Vectashield with DAPI. The area of recipients can prolong graft survival 17). This has blood vessels and lymphatic vessels was then raised important questions especially regarding the calculated using ImageJ (Figure-1 for the represen- differential contributions of Treg subsets to the tative image of CD31, LYVE-1 and calculated vessel natural allotolerance that is developed in vivo.

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Understanding the natural involvement and precise Tregs on corneal transplantation at Juntendo function of Treg subsets in allotolerance is essential University. for the effective development of Treg-based strat- Corneal allografts normally enjoy very high egies in transplantation. Recently, independent survival rates due to tolerogenic mechanisms that animal studies have shown that Foxp3 + natural account for ocular immune privilege 26); however, in Tregs (nTregs; thymic-derived Tregs [tTregs]), settings of graft site inflammation (ʻhigh-riskʼ which are generated in the thymus, are primarily transplantation), the risk of a prompt rejection neuropillin-1 +(Nrp-1 +) while Foxp3 + peripherally becomes a near absolute certainty 27)-29). In accord- induced Tregs (pTregs), which are generated upon ance with previous studies reporting reduced Treg antigen exposure in the periphery 18), are Nrp-1 − 19) 20). frequencies in solid organ allograft rejection 30)-32), This differentiation has permitted studies evaluat- we revealed a reduction in Treg frequencies at the ing these distinct Treg subsets in a variety of graft site of high-risk grafted hosts. Chauhan SK, et immunopathologies. In the current study, we used a al. showed decreased Foxp3 expression in Tregs of mouse model of corneal transplantation 17) 21) 22) to rejected recipients at the mRNA and protein levels. delineate the differential function and susceptibility Because Foxp3 is the key transcription factor for of tTregs and pTregs from allograft recipients, Tregs and its expression level correlates with Treg including healthy (low-risk) hosts with normal suppressive function 17) 33), Tregs from rejected immune homeostatic mechanisms who develop recipients with less Foxp3 expression display allotolerance naturally and ʻhigh-riskʼ hosts with decreased inhibitory functions ex vivo. inflamed graft beds who are prone to swift rejection Tregs constitute an attractive therapeutic target of their transplants 22). Our study had clearly given their essential role in controlling autoimmu- showed impaired function of pTregs, but not nity. However, recent animal studies provide tTregs, mediates the loss of immune tolerance and evidence for functional heterogeneity and lineage promotes allograft rejection 22). plasticity within the Treg compartment. Although optimized sorting strategies have dramatically Discussion improved the yield and purity of sorted Tregs, the current use of surrogate surface markers for Tregs have attracted broad interest because of selection cannot identify a 100% pure population of their role in supporting allograft survival. However, stable Foxp3-expressing T-cells. Furthermore, the we have yet to develop effective amplification need for in vitro expansion opens up the possibility methods of Tregs for humans. Strategies to induce for outgrowth of non-Tregs during culture or the and expand Ag-specific Tregs are being developed, potential loss of a regulatory phenotype by and whether various types of Tregs are suppres- expanded nTregs. This raises a significant safety sive in inflammatory conditions needs to be deter- concern that expanded Tregs with some inherent mined. Tregs can be induced and expanded in vitro autoreactivity may precipitate tissue damage after activating CD4+ T-cells in the presence of rather than prevent it 16). Given the importance of TGF-β and IL-2 23). Currently, Tahvildari M, et al. these issues, further studies are needed. showed that administration of low dose IL-2 Amazingly, Juntendo University, Tokyo, Japan expands Treg function and corneal allograft and Hokkaido University, Hokkaido, Japan has survival 24). The other potential key cytokine is succeeded in breaking away from immunosuppres- IL-33, which is an atypical IL-1 family member that sive agents for human liver transplantation using ex facilitates type 2 responses typified by enhanced vivo-generated regulatory T-cell-enriched cells 34). Th2 cell activity. IL-33 has been found to have the After liver transplantations, 10 adult patients were capacity to expand functional CD4+Foxp3+ Tregs, given a novel regulatory T-cell-based cell therapy which mediate the capacity of IL-33 to promote and have been drug free for 16-33 months since. cardiac allograft survival 25). Therefore, the effects This surprising immunosuppressive function of of IL-33 on corneal transplantation outcomes has Treg therapy can be extended to other organ been noted, and we have currently started transplantations including high-risk corneal trans- researching the relationship between IL-33 and plantation.

5 Inomata: Treg therapy to corneal transplantation

Conclusion 10) Inomata T, Mashaghi A, Di Zazzo A, Lee SM, Chiang H, Dana R: Kinetics of angiogenic responses in corneal Here we showed that Tregs constitute an transplantation. Cornea, 2017; 36: 491-496. attractive therapeutic target given their essential 11) Sakaguchi S, Sakaguchi N: Regulatory T cells in immunologic self-tolerance and autoimmune disease. Int role in regulating autoimmunity. Strategies to Revimmunol, 2005; 24: 211-226. induce and expand Ag-specific Tregs are being 12) Gupta S: Immune homeostasis: regulatory T cells developed, but future studies are needed to (Treg) and molecules. J Clin Immunol, 2008; 28: determine whether various types of Tregs are 617-618. suppressive in inflammatory conditions. 13) Monteiro M, Almeida CF, Caridade M, et al: Identifica- tion of regulatory Foxp3 + invariant NKT cells induced Acknowledgments by TGF-beta. J Immunol, 2010; 185: 2157-2163. 14) Tsang JY, Tanriver Y, Jiang S, et al: Indefinite mouse The author thanks Tina Shiang for editing the heart allograft survival in recipient treated with CD4(+) CD25(+) regulatory T cells with indirect allospecificity manuscript. and short term immunosuppression. Transpl Immunol, 2009; 21: 203-209. Disclosure 15) Sagoo P, Lombardi G, Lechler RI: Relevance of regula- tory T cell promotion of donor-specific tolerance in solid The author has no financial conflicts of interest. organ transplantation. Front Immunol, 2012; 3: 184. 16) Haribhai D, Williams JB, Jia S, et al: A requisite role for References induced regulatory T cells in tolerance based on expand- ing antigen receptor diversity. Immunity, 2011; 35: 1) Zirm EK: Eine erfolgreiche totale Keratoplastik (a 109-122. successful total keratoplasty). 1906. Refract Corneal 17) Chauhan SK, Saban DR, Lee HK, Dana R: Levels of Surg, 1989; 5: 258-261. Foxp3 in regulatory T cells reflect their functional status 2) Williams KA, Brereton HM, Coster DJ: Prospects for in transplantation. J Immunol, 2009; 182: 148-153. genetic modulation of corneal graft survival. Eye 18) Abbas AK, Benoist C, Bluestone JA, et al: Regulatory T (Lond), 2009; 23: 1904-1909. cells: recommendations to simplify the nomenclature. 3) Eye Bank Association of America. Medical Standards Nat Immunol, 2013; 14: 307-308. 2016 2014 [Available from: http://restoresight.org/wp- 19) Yadav M, Louvet C, Davini D, et al: Neuropilin-1 content/uploads/2016/10/Med-Standards-October-2016. distinguishes natural and inducible regulatory T cells pdf]. among regulatory T cell subsets in vivo. J Exp Med, 4) Streilein JW, Yamada J, Dana MR, Ksander BR: 2012; 209: 1713-1722, S1-19. Anterior chamber-associated immune deviation, ocular 20) Weiss JM, Bilate AM, Gobert M, et al: Neuropilin 1 is immune privilege, and orthotopic corneal allografts. expressed on thymus-derived natural regulatory T Transplant Proc, 1999; 31: 1472-1475. cells, but not mucosa-generated induced Foxp3+ T reg 5) Qazi Y, Hamrah P: Corneal allograft rejection: immuno- cells. J Exp Med, 2012; 209: 1723-1742, S1. pathogenesis to therapeutics. J Clin Cell Immunol, 2013; 21) Inomata T, Mashaghi A, Di Zazzo A, Dana R: Ocular 2013 Suppl 9: 006. surgical models for immune and angiogenic responses. J 6) Streilein JW, Masli S, Takeuchi M, Kezuka T: The eyeʼs Biol Methods, 2015; 2: e27. view of antigen presentation. Hum Immunol, 2002; 63: 22) Inomata T, Hua J, Di Zazzo A, Dana R: Impaired func- 435-443. tion of peripherally induced regulatory T cells in hosts at 7) Inomata T, Mashaghi A, Di Zazzo A, Dana R: Ocular high risk of graft rejection. Sci Rep, 2016; 6: 39924. surgical models for immune and angiogenic responses. J 23) Nguyen TL, Sullivan NL, Ebel M, Teague RM, DiPaolo Biol Methods, 2015; 2: e27. RJ: Antigen-specific TGF-beta-induced regulatory T 8) Sonoda Y, Streilein JW: Orthotopic corneal transplanta- cells secrete chemokines, regulate T cell trafficking, and tion in mice--evidence that the immunogenetic rules of suppress ongoing autoimmunity. J Immunol, 2011; 187: rejection do not apply. Transplantation, 1992; 54: 1745-1753. 694-704. 24) Tahvildari M, Omoto M, Chen Y, et al: In vivo expansion 9) Hua J, Jin Y, Chen Y, et al: The resolvin D1 analogue of regulatory T cells by low-dose interleukin-2 treat- controls maturation of dendritic cells and suppresses ment increases allograft survival in corneal transplanta- alloimmunity in corneal transplantation. Invest Ophthal- tion. Transplantation, 2016; 100: 525-532. mol Vis Sci, 2014; 55: 5944-5951. 25) Turnquist HR, Zhao Z, Rosborough BR, et al: IL-33

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