December 2010
Volume 51, Issue 12
Free
Immunology and Microbiology  |   December 2010
GITR Ligand–Mediated Local Expansion of Regulatory T Cells and Immune Privilege of Corneal Allografts
Author Affiliations & Notes
  • Junko Hori
    From the Department of Ophthalmology, Nippon Medical School, Tokyo, Japan;
  • Hiroko Taniguchi
    From the Department of Ophthalmology, Nippon Medical School, Tokyo, Japan;
  • Mingcong Wang
    From the Department of Ophthalmology, Nippon Medical School, Tokyo, Japan;
  • Masamichi Oshima
    Department of Immunology, National Institute of Infectious Disease, Tokyo, Japan; and
  • Miyuki Azuma
    Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
  • Corresponding author: Junko Hori, Department of Ophthalmology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo, Tokyo, 113-8602, Japan; jhori-tky@umin.ac.jp
Investigative Ophthalmology & Visual Science December 2010, Vol.51, 6556-6565. doi:10.1167/iovs.09-4959
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Junko Hori, Hiroko Taniguchi, Mingcong Wang, Masamichi Oshima, Miyuki Azuma; GITR Ligand–Mediated Local Expansion of Regulatory T Cells and Immune Privilege of Corneal Allografts. Invest. Ophthalmol. Vis. Sci. 2010;51(12):6556-6565. doi: 10.1167/iovs.09-4959.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: The pathway between the glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and GITR ligand (GITRL) has been shown to control the function of regulatory T cells (Treg). The present study was conducted to investigate the role of this pathway and Treg in establishing immune privilege status for corneal allografts.

Methods.: Corneas of C57BL/6 mice were orthotopically transplanted into the eyes of BALB/c mice, and graft survival was assessed. A separate set of BALB/c mice received an anterior chamber injection of C57BL/6 splenocytes, and induction of allo-specific anterior chamber-associated immune deviation was assessed. Recipients were intraperitoneally administrated anti-GITRL, anti-CD25 monoclonal antibodies (mAb), or control immunoglobulin (IgG). Expressions of GITRL, GITR, and Foxp3 in the allografts were assessed. In vitro, cornea pretreated with anti-GITRL mAb or control IgG was incubated with T cells, and destruction of corneal endothelial cells and the population of Foxp3+CD25+CD4+ T cells were assessed.

Results.: GITRL was expressed constitutively in the cornea and iris-ciliary body. GITRL-expressing allografts were infiltrated with Foxp3+GITR+CD25+CD4+ T cells. Blockade of GITRL did not affect allo-specific ACAID but led to infiltration of Foxp3(−)CD4+ T cells and allograft rejection. Depletion of CD25+CD4+ Treg also accelerated allograft rejection. Destruction of corneal endothelial cells by T cells was significantly enhanced in GITRL-blocked cornea compared with control cornea. Foxp3+CD25+CD4+ T cells were increased after incubation with GITRL-expressing cornea, but not with GITRL-blocked cornea.

Conclusions.: Presence of Foxp3+CD25+CD4+ Treg in the allograft is necessary for allograft survival. GITRL-dependent expansion of Treg within the cornea is one mechanism underlying immune privilege in corneal allografts.

The eye is constructed from tissue with little or no capacity for regeneration. Specifically, corneal endothelial cells cannot proliferate in vivo. For that reason, ocular tissue damage due to excessive inflammation can lead to loss of sight. Accordingly, the eye is known to be endowed with immune privilege, 1,2 and inflammation is self-regulated to preserve organ functions. Corneal transplants are the least rejected among all organ transplants in humans, 3 5 and that characteristic is also attributable to immune privilege. 1,2,6 In the vast majority of uncomplicated cases, topical immunosuppression is sufficient to secure graft survival, with no need for systemic immunosuppression. This positive clinical experience is matched by the results obtained in experimental models where orthotopic corneal transplants have been performed in immunocompetent mice and rats. Under these conditions, considerable success has been observed for the corneal allografts. 7 9  
At present, three major lines of thought prevail regarding the molecular mechanisms of immune privilege in the eye. First are the anatomic, cellular, and molecular barriers in the eye. Normal cornea lacks blood vessels and lymphatic vessels. 10 The central part of the cornea, which is used as donor tissue, contains only a small population of major histocompatability complex (MHC) class II–expressing antigen-presenting cells (APCs). 11 Although bone marrow–derived cells recently been reported to be present within normal cornea, most of these cells show an immature phenotype lacking MHC class II expression. 12 Moreover, normal corneal cells (i.e., epithelial, stromal, and endothelial cells) express no MHC class II and only weak MHC class I antigens. 13 15 Second, anterior chamber-associated immune deviation (ACAID) is a well-known phenomenon in which antigen-specific peripheral tolerance is induced after antigen introduction into the anterior chamber. 16 The corneal graft necessarily forms the anterior surface of the anterior chamber, and donor-specific ACAID is induced after grafting. 17,18 Third, an immune-suppressive microenvironment is maintained in the eye. The anterior chamber contains biologically relevant concentrations of various immunomodulatory neuropeptides, growth factors, cytokines, and soluble cell-surface receptors, such as α-melanocyte-stimulating hormone, 19 vasoactive intestinal peptide, 20 calcitonin gene-related peptide, 21 transforming growth factor (TGF)-β, 22 thrombospondin-1, 23 macrophage migration inhibitory factor, 24 interleukin (IL)-1 receptor antagonist, 25 CD46, 26 CD55, 24 CD59, 24 and CD95L. 27 These factors suppress innate and adaptive immunity and maintain the immune suppressive microenvironment within the eye. 19 27 In addition, normal corneal endothelial cells constitutively express immune-modulating factors such as CD95L 28 and B7-H1. 29 Corneal endothelium is thus considered to play a central role in the protection of corneal allografts from immunologic rejection when transplanted orthotopically into the eye 29,30 and heterotopically beneath the kidney capsule. 31,32 The molecular mechanisms underlying corneal invulnerability are not perfectly understood. Further investigations of the mechanisms of immune privilege are necessary for the development of new therapeutic approaches to prevent blinding inflammation within the eye and ameliorate the destructive inflammation observed in other tissues and organs. 
The pathway between glucocorticoid-induced tumor necrosis factor (TNF) receptor family-related protein (GITR) and GITR ligand (GITRL) have been shown to control the function of regulatory T cells (Treg). GITR is a type I transmembrane protein of the TNF receptor superfamily. The cytoplasmic domain shares strong homology with a subgroup of the TNF receptor superfamily lacking the death domain, including CD27, CD134 (OX40), and CD137 (4–1BB). 33,34 GITR is expressed predominantly on CD25+CD4+ Treg at high levels but is also expressed constitutively at low levels on conventional CD25–CD4+ and CD8+ T cells and is rapidly upregulated after activation. 35 GITRL is a type II transmembrane protein belonging to the TNF superfamily, and cell surface expression has been observed on B cells, macrophages, and dendritic cells. 36,37  
GITR−/− mice have been shown to be resistant to splanchnic artery occlusion shock, Candida albicans infection, collagen-induced arthritis, 2,4,6-trinitrobenzenesulfonic acid–induced colitis, acute and chronic lung injury, and spinal cord injury. 38 43 Administration of soluble GITR in wild-type mice is protective against the development of these diseases. GITR−/− mice show impaired proinflammatory cytokine production, NF-κB activation, stress oxidative products, and infiltration of inflammatory cells in early responses. 38 41 Studies using an agonist anti-GITR monoclonal antibody (mAb) (DTA-1) 35,44,45 or GITRL transfectants and soluble GITRL 46,47 have revealed that the GITR-GITRL pathway positively costimulates both conventional effector CD4+ and CD8+ T cells and CD25+CD4+ Treg, despite the opposing effector functions. 
In the eye, it has been shown that GITRL is constitutively expressed in retinal pigment epithelial (RPE) cells, Müller cells, and retinal photoreceptors in vivo in human eyes. 48 Although GITRL transfection to human RPE cells has been shown to abrogate immunosuppressive function of RPE for T cells, 49 because currently available RPE cell lines do not express GITRL at protein levels on their surface, no reports have described the physiological role of the GITR/GITRL pathway in ocular immunity. 
To our knowledge, no reports have previously described the expression or role of the GITR/GITRL pathway in cornea. Although a recent report indicated that level of Foxp3 expression in Treg in draining lymph nodes (dLNs) is associated with the potential of Treg to prevent corneal allograft rejection, 50 whether Treg plays a role within the graft site after corneal grafting remains unclear. We investigated the role of the GITR-GITRL pathway in immune privilege of corneal allografts using an antagonistic anti-GITRL mAb in mouse models. Our results suggest a novel role of the GITR-GITRL pathway between T cells and corneal cells in local sites. Constitutive expression of GITRL on the corneal cells expands and maintains Foxp3+CD25+GITR+CD4+ T cells in the graft sites. This action of GITRL contributes to the immune-privileged status of corneal allografts. 
Materials and Methods
Mice and Anesthesia
Male BALB/c, C57BL/6, and C3H/He mice were purchased from Sankyo Laboratory Service Corporation (Tokyo, Japan). All mice were used at 8 to 10 weeks old and treated according to the Association for Research in Vision and Ophthalmology guidelines on the use of animals in research. All protocols in this animal study were reviewed and approved by our institutional review committee. Each mouse was anesthetized by intramuscular injection of a mixture of 3.75 mg ketamine and 0.75 mg xylazine before all surgical procedures. 
mAbs and Flow Cytometry
Anti-mouse GITRL (MIH44, rat immunoglobulin [Ig]G2a mAb) and anti-mouse CD25 (PC61.5, rat IgG1 mAb) were generated as described previously. 51,52 For flow cytometry or fluorescence immunohistohemistry, mAbs against CD3 (145–2C11, hamster IgG), CD4 (GK1.5, rat IgG2b), CD8 (53.6.72, rat IgG2a), CD25 (PC61, rat IgG1), CD11c (N418, hamster IgG), GITRL (YGL 386, rat IgG1), GITR (DTA-1, rat IgG2a), and Foxp3 (FJK16s, rat IgG2a) were used. All FITC-, PE-, allophycocyanin-, biotin-conjugated mAbs, and isotype control Ig were obtained from eBioscience (San Diego, CA) or BD Pharmingen (San Diego, CA). For biotin-conjugated mAbs, PE- or allophycocyanin-streptavidin was used. Culture supernatant from the 2.4G2 hybridoma (anti-CD16/CD32 mAb) was used to block nonspecific binding. For intracellular staining for Foxp3, a Foxp3 staining buffer set (catalog no. 00-5523; eBioscience) was used according to the manufacturer's instructions. Multicolor staining for intracellular and cell surface molecules was performed as described previously. 51 Stained cells were then analyzed (FACSCalibur system and CellQuest software; BD Biosciences). 
Orthotopic Corneal Transplantation and Treatment
Penetrating keratoplasty was performed as described previously. 29 Briefly, 2-mm diameter donor corneas were placed in a same-sized recipient bed with eight interrupted sutures (11-0 nylon; Mani, Tochigi, Japan). Sutures were removed 7 days after grafting. C57BL/6 mice were used as donors, and BALB/c mice were used as recipients. Three times a week for 8 weeks, 0.2 mg anti-mouse GITRL mAb (MIH44) or control rat IgG was administered intraperitoneally. To deplete CD25+CD4+ Treg, 0.5 mg anti-CD25 mAb was administrated intraperitoneally at 4 and 0 days before corneal transplantation. 
Evaluation of Corneal Allograft
Orthotopic grafts were observed by operative microscopy at least twice weekly. Masked assessment of orthotopic corneal grafts was performed in a masked fashion by a single observer (MW) who examined each graft for survival according to a previously reported scoring system that defines graft survival as follows: 0, clear graft; 1+, minimal superficial nonstromal opacity; 2+, minimal deep stromal opacity with pupil margin and iris vessels visible; 3+, moderate deep stromal opacity with only the pupil margin visible; 4+, intense deep stromal opacity with the anterior chamber visible; and 5+, maximum stromal opacity with total obscuration of the anterior chamber. 29 Grafts with opacity scores ≥2 after 3 weeks were considered to have been rejected. 
Reverse Transcription–Polymerase Chain Reaction
Cornea, iris-ciliary body, and neural retina were isolated from a total of 10 normal mouse eyes. Total RNA was extracted from each tissue (ISOGEN; Nippongene, Tokyo, Japan). First-strand cDNA was prepared (SuperScript First Strand Synthesis System; Invitrogen, Carlsbad, CA) from 5 μg of total RNA. Standardization of cDNA samples was based on the content of β-actin cDNA. Primers for mouse β-actin were 5′-CTACAATGAGCTGCGTGT?GG-3′ and 5′-CAACGTCACACTTCATGATGG-3′. Primers for mouse GITRL were 5′-GTCAAGTCCTCAAAGGGCAG-3′ and 5′-CAGGAATCACTTGGCCGTAG-3′. PCR was performed in a total volume of 20 μL in PCR buffer in the presence of 0.2 mM dNTP, 1 μM of each primer, and 1 U of thermostable DNA polymerase (Taq DNA polymerase; Advanced Biotechnologies, Surrey, UK). After 35 cycles of amplification, the PCR products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. 
Histology and Immunohistochemistry
Eyes bearing corneal allografts were removed for histologic assessment after transplantation, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Approximately 20 sections were prepared from each graft-bearing eye. For immunohistochemistry, normal and graft-bearing eyes were removed and frozen in optimal cutting temperature compound (Sakura Finetechnical, Tokyo, Japan) in acetone–dry ice and stored at −80°C. Cryostat sections (5 mm) were fixed in cold acetone, followed by immunofluorescent staining for the detection of mouse GITRL, GITR, CD25, Foxp3, and CD4. Briefly, after blocking with 2% bovine serum albumin, sections were incubated with FITC-, PE-, CyTM-3-, or biotin-conjugated primary antibody diluted to 4 mg/mL for 2 hours. This was followed by staining with streptavidin-allophycocyanin (eBioscience) or streptavidin-PE (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted to 4 mg/mL for 1 hour at room temperature. After washing with PBS, sections were mounted with 4′,6′-diamidino-2-phenylindole (DAPI)-containing mounting medium and observed using confocal microscopy. Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in the confocal images (magnification, ×40) were counted and compared. 
Assessment of Donor-Specific ACAID
ACAID induction was tested as described previously. 29 Briefly, recipient BALB/c mice received an anterior chamber (AC) injection of 5 × 105 donor C57BL/6 spleen cells. Two weeks after AC injection, recipients were immunized by subcutaneous injection of 1 × 107 C57BL/6 spleen cells. Seven days after immunization, 1 × 106 irradiated (2000 rad) C57BL/6 spleen cells were injected into the right ear pinna. At 24 h after the ear challenge, ear thickness was measured using a low-pressure micrometer (Mitsutoyo; MTI Corporation, Kanagawa, Japan). Ear swelling was determined as follows: specific ear swelling = (24-h measurement of right ear − 0-h measurement of right ear) − (24-h measurement of left ear − 0-h measurement of left ear) × 10−3 mm. Ear swelling responses 24 hours after injection are presented as individual values for each tested animal and as a group mean ± SD. For 3 weeks, treatments with anti-GITRL mAb or control rat IgG were performed starting from the day of AC injection and continuing until the day of ear injection. As a positive control, a similar number of irradiated spleen cells were injected into the right ear pinnae of BALB/c mice that had been immunized 1 week previously by SC injection of 10 × 106 C57BL/6 spleen cells. As a negative control, 1 × 106 irradiated C57BL/6 spleen cells were injected into the right ear pinnae of naive mice that were not previously AC-injected or immunized. 
In Vitro Assay of Corneal Endothelial Cell Destruction by Alloreactive T Cells
To examine corneal endothelial destruction by alloreactive T cells in vitro, a model of the efferent phase of corneal rejection in culture dishes was used, as described previously. 29 Fresh normal corneas from C57BL/6 eyes were incubated with 10 μg anti-GITRL mAb (MIH44) or control rat IgG for 2 h in 5% CO2 at 37°C, then washed twice with PBS. T cells were purified from the spleens of BALB/c mice that had been presensitized by subcutaneous immunization with C57BL/6 spleen cells or with third-party (C3H/He) spleen cells, or from the spleens of naive BALB/c, C57BL/6, or C3H/He mice, using the MACS magnetic cell sorting and separation system (Miltenyi Biotec) with a mouse CD4+ T-cell isolation kit (anti-CD8a [Ly-2], anti-CD45R [B220], anti-CD49b [DX5], anti-CD11b [Mac-1], and anti-Ter-119 mAbs; Miltenyi Biotec), according to the manufacturer's instructions. Purified CD4+ T cells (94%–98% pure as estimated by FACSCalibur; BD Biosciences) were suspended in RPMI1640. Corneas pretreated with anti-GITRL mAb or control rat IgG were incubated with 2.5 × 105 T cells for 6 hours in 5% CO2 at 37°C and then washed twice with PBS. Unfixed corneal samples were incubated with 50 mg/mL propidium iodide (PI) for 30 minutes to stain the nuclei of dead endothelial cells. Using confocal microscopy (magnification, ×40), PI-positive cells were counted at three randomly selected areas in the corneal endothelium of each corneal sample, as described previously. 29,53 As a positive control for corneal cell death, normal C57BL/6 cornea was incubated with Triton X-100 without antibody treatment or incubation with T cells. As negative controls, normal C57BL/6 cornea with antibody treatment and incubation without T cells and cornea without antibody treatment or incubation with T cells were used. Expression of Foxp3 in T cells after incubation with or without allogeneic cornea was also assessed by flow cytometry. 
Statistical Analyses
Corneal graft survival rates were compared using Kaplan-Meier survival curves and the Breslow-Gehan Wilcoxon test. Numbers of Foxp3+CD4+ cells and Foxp3(−)CD4+ cells, ear-swelling measurements, corneal endothelial cell death, and percentage of gated CD4+ T cells were analyzed using the two-tailed Student's t-test. Values of P < 0.05 were considered statistically significant. 
Results
Expression of GITRL in Normal Mouse Eyes
RT-PCR revealed that GITRL mRNA was expressed in freshly isolated cornea, iris-ciliary body, and the neural retina of normal mouse eyes (Fig. 1A). Immunofluorescent staining indicated that the expression of GITRL was localized to corneal endothelial cells but was not expressed on corneal stroma or epithelium of normal mouse cornea. GITRL was also expressed in the iris-ciliary body of normal mouse eyes (Fig. 1B). No expression of GITR was seen in normal cornea or the iris-ciliary body (data not shown). 
Figure 1.
 
Expression of GITRL in normal mouse eye. mRNA was extracted from freshly isolated cornea, iris-ciliary body (iris-CB), and neural retina of normal mouse eyes, and then reverse transcribed and amplified by PCR. PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining (A). Cryostat sections were stained with biotin-conjugated anti-GITRL mAb or control rat IgG, followed by streptavidin-PE (red). Nuclei were stained with DAPI (blue). CB, ciliary body; Ced, corneal endothelium; Cep, corneal epithelium; Cst, corneal stroma. Original magnification: (B) ×40).
Figure 1.
 
Expression of GITRL in normal mouse eye. mRNA was extracted from freshly isolated cornea, iris-ciliary body (iris-CB), and neural retina of normal mouse eyes, and then reverse transcribed and amplified by PCR. PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining (A). Cryostat sections were stained with biotin-conjugated anti-GITRL mAb or control rat IgG, followed by streptavidin-PE (red). Nuclei were stained with DAPI (blue). CB, ciliary body; Ced, corneal endothelium; Cep, corneal epithelium; Cst, corneal stroma. Original magnification: (B) ×40).
Accelerated Corneal Allograft Rejection after Blockade of GITRL
Normal corneas of C57BL/6 mice were transplanted orthotopically into normal eyes of BALB/c mice. In all recipients, 0.2 mg anti-GITRL mAb or control rat IgG was administered intraperitoneally 3 times/wk for 8 weeks after grafting. Graft survival was clinically assessed and compared. Approximately 50% of allografts survived >8 weeks in control IgG-treated recipients (Fig. 2A). We have previously reported that approximately 50% of corneal allografts from C57BL/6 donors survive in untreated BALB/c recipients. 29 Administration of control IgG thus does not affect corneal allograft survival. Conversely, all allografts were rejected when recipients were treated with anti-GITRL mAb. Survival of allografts was significantly shorter in anti-GITRL mAb-treated mice than in control IgG-treated mice (P < 0.01; Fig. 2A). 
Figure 2.
 
Blockade of GITRL or depletion of CD25+CD4+ Treg accelerates corneal allograft rejection. Normal corneas of C57BL/6 were transplanted orthotopically into normal eyes of BALB/c mice. After the grafting procedure, recipients were injected with 0.2 mg anti-GITRL mAb or control rat IgG intraperitoneally 3 times weekly for 8 weeks. To deplete CD25+CD4+ Treg, 0.5 mg anti-CD25 mAb was injected intraperitoneally to a different set of recipients at 4 days before and on the day of grafting. Graft survival was clinically assessed and compared. Survival of allografts treated with anti-GITRL or anti-CD25 mAbs was significantly less than that in control allografts (A, anti-GITRL, *P < 0.01; anti-CD25, **P < 0.001; n = 10 in each group). CD4+ T cells purified from spleens and dLNs of allograft recipients treated with 0.5 mg anti-CD25 mAb at 4 days before and on the day of transplantation were also assessed to confirm depletion of CD25+CD4+ Treg after transplantation (B, right). Data are displayed as dot-blot graphs (four-decade log scales), with controls comprising naive BALB/c mice. The number in the upper right corner of each graph shows the proportion of GITR+CD25+ cells among CD4+ T cells. Mean percentage of cells (±SD) of four to five mice/group.
Figure 2.
 
Blockade of GITRL or depletion of CD25+CD4+ Treg accelerates corneal allograft rejection. Normal corneas of C57BL/6 were transplanted orthotopically into normal eyes of BALB/c mice. After the grafting procedure, recipients were injected with 0.2 mg anti-GITRL mAb or control rat IgG intraperitoneally 3 times weekly for 8 weeks. To deplete CD25+CD4+ Treg, 0.5 mg anti-CD25 mAb was injected intraperitoneally to a different set of recipients at 4 days before and on the day of grafting. Graft survival was clinically assessed and compared. Survival of allografts treated with anti-GITRL or anti-CD25 mAbs was significantly less than that in control allografts (A, anti-GITRL, *P < 0.01; anti-CD25, **P < 0.001; n = 10 in each group). CD4+ T cells purified from spleens and dLNs of allograft recipients treated with 0.5 mg anti-CD25 mAb at 4 days before and on the day of transplantation were also assessed to confirm depletion of CD25+CD4+ Treg after transplantation (B, right). Data are displayed as dot-blot graphs (four-decade log scales), with controls comprising naive BALB/c mice. The number in the upper right corner of each graph shows the proportion of GITR+CD25+ cells among CD4+ T cells. Mean percentage of cells (±SD) of four to five mice/group.
Expression of GITRL and Infiltration of Foxp3+GITR+CD25+CD4+ Treg in Corneal Allografts
When normal corneas of C57BL/6 were transplanted orthotopically into normal eyes of BALB/c mice, GITRL was strongly expressed on corneal endothelial cells and stromal cells in corneal allografts in the recipients treated with control rat IgG (Fig. 3A). A small number of GITR-expressing cells were observed in surviving allografts in these recipients at 3 weeks after grafting. Those GITR-positive cells were Foxp3+GITR+CD25+CD4+ T cells (Figs. 3B–D). Infiltration of Foxp3+CD25+CD4+ Treg into surviving corneal allografts was thus demonstrated for the first time. When the recipients were treated with 0.2 mg anti-GITRL mAb intraperitoneally 3 times weekly, the majority of CD4+ T cells that had infiltrated allografts were Foxp3-negative (Figs. 3E, 3F). Few CD25+ cells were seen among CD4+ cells in allografts with GITRL-blockade (Fig. 3G). 
Figure 3.
 
Expression of GITRL and infiltration of Foxp3+GITR+CD25+ CD4+ Treg in corneal allografts. Normal corneas of C57BL/6 were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes from recipients treated with control rat IgG (A, BD) or anti-GITRL mAb (EG) were isolated at 3 weeks. Cryostat sections of graft-bearing eyes were examined by hematoxylin and eosin staining and multicolor immunofluorescence staining with PE-conjugated anti-GITRL (A), anti-GITR (B, E), or anti-CD4 (D, G), and FITC-conjugated anti-CD4 (B, C, E, F) or anti-CD25 (D, G), and biotin-conjugated anti-Foxp3 mAbs followed by streptavidin-allophycocyanin (B, C, E, F). Nuclei were stained with DAPI. Merged color images are also shown. High-magnification images (C, F) show intracellular staining of Foxp3 and cell-surface staining of CD4. Graft center (A) and the graft junction (BG) are shown, as are representative sections. Ced, corneal endothelium; Cep, corneal epithelium; Cst, corneal stroma (original magnification, ×40).
Figure 3.
 
Expression of GITRL and infiltration of Foxp3+GITR+CD25+ CD4+ Treg in corneal allografts. Normal corneas of C57BL/6 were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes from recipients treated with control rat IgG (A, BD) or anti-GITRL mAb (EG) were isolated at 3 weeks. Cryostat sections of graft-bearing eyes were examined by hematoxylin and eosin staining and multicolor immunofluorescence staining with PE-conjugated anti-GITRL (A), anti-GITR (B, E), or anti-CD4 (D, G), and FITC-conjugated anti-CD4 (B, C, E, F) or anti-CD25 (D, G), and biotin-conjugated anti-Foxp3 mAbs followed by streptavidin-allophycocyanin (B, C, E, F). Nuclei were stained with DAPI. Merged color images are also shown. High-magnification images (C, F) show intracellular staining of Foxp3 and cell-surface staining of CD4. Graft center (A) and the graft junction (BG) are shown, as are representative sections. Ced, corneal endothelium; Cep, corneal epithelium; Cst, corneal stroma (original magnification, ×40).
Numbers of Foxp3+CD4+ cells in GITRL-expressing control allografts were significantly higher than the numbers in allografts treated with anti-GITRL mAb. Conversely, Foxp3(−)CD4+ cells in allografts with GITRL-blockade were significantly higher than the number in controls. Moreover, the numbers of Foxp3+CD4+ cells were significantly lower than the numbers of Foxp3(−)CD4+ cells in allografts with GITRL-blockade (Fig. 4). Blockade of GITRL thus let Foxp3(−)CD4+ T cells dominate in the corneal allograft. 
Figure 4.
 
Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in corneal allografts. Normal corneas of C57BL/6 were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes from recipients treated with isotype control rat IgG or anti-GITRL mAb were isolated at 3 weeks. Cryostat sections of graft-bearing eyes were examined by immunofluorescence staining with FITC-conjugated anti-CD4 and biotin-conjugated anti-Foxp3 mAbs followed by streptavidin-allophycocyanin. Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in the confocal images (magnification, ×40) were counted and compared. *P < 0.05, **P < 0.01, ***P < 0.001; n = 4–5 grafts in each group.
Figure 4.
 
Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in corneal allografts. Normal corneas of C57BL/6 were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes from recipients treated with isotype control rat IgG or anti-GITRL mAb were isolated at 3 weeks. Cryostat sections of graft-bearing eyes were examined by immunofluorescence staining with FITC-conjugated anti-CD4 and biotin-conjugated anti-Foxp3 mAbs followed by streptavidin-allophycocyanin. Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in the confocal images (magnification, ×40) were counted and compared. *P < 0.05, **P < 0.01, ***P < 0.001; n = 4–5 grafts in each group.
Accelerated Corneal Allograft Rejection after Deletion of CD25+CD4+ T Cells
The above results led us to hypothesize that CD25+CD4+ Treg is necessary for corneal allograft survival. To test this, before corneal transplantation from normal C57BL/6 donors to normal BALB/c recipients, 0.5 mg anti-CD25 mAb was administered intraperitoneally at 4 days before and on the day of transplantation, to deplete CD25+CD4+ Treg. Depletion of CD25+CD4+ Treg in PC61-treated mice was confirmed to be <1% of CD4+ cells in dLNs and spleen at 4 weeks after transplantation in a different set of recipients (Fig. 2B). All allografts were swiftly rejected when recipients were treated with anti-CD25 mAb (Fig. 2A). Survival of allografts was significantly shorter in anti-CD25 mAb-treated mice than in control IgG-treated mice (P < 0.001). These results indicate that CD25+CD4+ Treg are indeed necessary for corneal allograft survival. Rejection in these recipients occurred in a similar fashion to allograft with GITRL blockade. Survival of allografts in anti-CD25 mAb-treated mice was statistically indistinguishable from that seen in anti-GITRL mAb-treated mice (Fig. 2A). 
Induction of ACAID after Blockade of GITRL
Eye-associated tolerance, known as ACAID, is one of the major mechanisms for immune privilege of the eyes and maintains acceptance of corneal allografts. 1,2 We hypothesized that the vulnerability of corneal allografts noted after blockade of GITRL might result from failure of ACAID-inducing Treg. To examine this, we tested the effects of GITRL blockade on alloantigen-specific ACAID induction using a simple model. B6 spleen cells were used as alloantigens and injected into the right anterior chamber of normal BALB/c eyes. After 2 weeks, B6 spleen cells were injected SC to sensitize the mice. After one more week, B6 spleen cells were challenged into the ear pinnae to determine the delayed hypersensitivity (DH) response 24 hours later. For 3 weeks, treatments with anti-GITRL mAb were applied starting from the day of AC injection and until the day of ear challenge. DH response was induced in sensitized mice without prior AC injection (positive controls) compared with unsensitized naive mice (negative controls) (Fig. 5). Prior AC injection significantly suppressed the DH response in control IgG-treated mice, indicating induction of ACAID. Treatments with anti-GITRL mAb did not significantly affect induction of ACAID (Fig. 5). These results indicate that GITR/GITRL interactions are not involved in the induction of ACAID. 
Figure 5.
 
Blockade of GITRL does not abolish ACAID. B6 spleen cells were used as alloantigens and injected into the right AC of BALB/c normal eyes. Two weeks later, B6 spleen cells were injected SC to sensitize the mice. After one more week, a challenge was performed by injecting B6 spleen cells into the right ear pinna of each mouse. After 24 hours, specific ear swelling was measured as an indication of DH. Treatments with anti-GITRL mAb were performed starting from the day of AC injection and continuing until the day of the ear challenge. DH responses were similarly suppressed in anti-GITRL mAb or control rat IgG-treated groups, and no significant differences were observed between these groups. Positive control mice (Posi.C) received SC immunization and the ear challenge without previous AC injection. Negative control mice (Nega.C) received only the ear challenge without AC injection or immunization.
Figure 5.
 
Blockade of GITRL does not abolish ACAID. B6 spleen cells were used as alloantigens and injected into the right AC of BALB/c normal eyes. Two weeks later, B6 spleen cells were injected SC to sensitize the mice. After one more week, a challenge was performed by injecting B6 spleen cells into the right ear pinna of each mouse. After 24 hours, specific ear swelling was measured as an indication of DH. Treatments with anti-GITRL mAb were performed starting from the day of AC injection and continuing until the day of the ear challenge. DH responses were similarly suppressed in anti-GITRL mAb or control rat IgG-treated groups, and no significant differences were observed between these groups. Positive control mice (Posi.C) received SC immunization and the ear challenge without previous AC injection. Negative control mice (Nega.C) received only the ear challenge without AC injection or immunization.
GITRL-Mediated Protection of Corneal Endothelial Cells from Killing by CD4+ T Cells In Vitro
The above results led us to hypothesize that constitutive expression of GITRL in the cornea has the capacity to protect corneal allografts from effector T cells in the cornea. To further substantiate this possibility, we used a model of corneal endothelial cell destruction by alloreactive T cells in vitro, as described previously. 29 In previous studies by others and the present authors, corneal endothelial cells have been documented as the target of alloreactive T cells in both human and rodent corneal transplantations. 29,53 As a model of the effector phase of corneal rejection, normal C57BL/6 corneas were incubated with purified CD4+ T cells from the spleens of BALB/c mice presensitized against C57BL/6 antigens. Purified CD4+ T cells from the spleens of BALB/c mice presensitized against third-party C3H/He antigens were used as nonallospecific activated CD4+ T cells. Splenic CD4+ T cells from naive BALB/c, C57BL/6, or C3H/He mice were used as allogeneic, syngeneic, or third-party naive CD4+ T cells, respectively. The number of dead corneal endothelial cells was significantly larger in anti-GITRL mAb-treated corneas than in control IgG-treated corneas after incubation with alloreactive CD4+ T cells (P < 0.01). Interestingly, the number of dead corneal endothelial cells was also significantly larger in anti-GITRL mAb-treated corneas than in control IgG-treated corneas after incubation with CD4+ T cells activated against third-party alloantigens (P < 0.05). Moreover, the number of dead corneal endothelial cells was significantly larger in anti-GITRL mAb-treated corneas than in control IgG-treated corneas after incubation with CD4+ T cells from naive BALB/c and C3H/He mice (P < 0.05, P < 0.01, respectively) (Fig.6). These results suggest that GITRL protects corneal endothelial cells from being killed by CD4+ T cells, irrespective of prior sensitization. 
Figure 6.
 
GITRL protects corneal endothelial cells from being killed by T cells in vitro. C57BL/6 corneas pretreated with anti-GITRL mAb or control IgG were incubated with purified T cells from the spleens of BALB/c mice presensitized against C57BL/6 antigens (α-B6/BALB-T cells) or third-party C3H/He antigens (α-C3H/BALB-T cells), or from the spleens of naive BALB/c (naive BALB-T cells), C57BL/6 (naive B6-T cells), or C3H/He mice (naive C3H-T cells). After a 6-hour incubation, corneal endothelial cell death was detected by staining unfixed tissue with PI followed by confocal microscopic examination. Positive control corneas were incubated with Triton X-100, without antibody treatment or incubation with T cells. As negative controls, corneas with antibody treatment and incubation without T cells, and corneas without antibody treatment or incubation with T cells were used. Data are presented as the mean (±SD) number of PI+ corneal endothelial cells from five to six corneas in each group (*P < 0.05, **P < 0.01).
Figure 6.
 
GITRL protects corneal endothelial cells from being killed by T cells in vitro. C57BL/6 corneas pretreated with anti-GITRL mAb or control IgG were incubated with purified T cells from the spleens of BALB/c mice presensitized against C57BL/6 antigens (α-B6/BALB-T cells) or third-party C3H/He antigens (α-C3H/BALB-T cells), or from the spleens of naive BALB/c (naive BALB-T cells), C57BL/6 (naive B6-T cells), or C3H/He mice (naive C3H-T cells). After a 6-hour incubation, corneal endothelial cell death was detected by staining unfixed tissue with PI followed by confocal microscopic examination. Positive control corneas were incubated with Triton X-100, without antibody treatment or incubation with T cells. As negative controls, corneas with antibody treatment and incubation without T cells, and corneas without antibody treatment or incubation with T cells were used. Data are presented as the mean (±SD) number of PI+ corneal endothelial cells from five to six corneas in each group (*P < 0.05, **P < 0.01).
GITRL-Expressing Cornea-Dependent Expansion of Foxp3+CD4+ T Cells In Vitro
We next wanted to know the mechanisms of GITRL-dependent corneal protection from T cells. To test the possibility that GITRL expressed in the cornea induces and/or maintains Foxp3+GITR+CD25+CD4+ Treg, we studied expression of Foxp3 and CD25 on CD4+ T cells after incubation with allogeneic cornea in vitro. Results revealed that when purified splenic CD4+ T cells from normal BALB/c mice were incubated for 6 hours with normal GITRL-expressing B6 cornea treated with control IgG, the population of Foxp3+CD25+CD4+ T cells was significantly increased than those of pre-incubation or post–6-hour incubation without cornea (P < 0.05). However, the population of Foxp3+CD25+CD4+ T cells was unchanged after incubation with B6 cornea treated with anti-GITRL mAb (Fig.7). 
Figure 7.
 
GITRL-mediated expansion of Foxp3+CD25+CD4+ T cells in the cornea. Purified CD4+ T cells from the spleens of BALB/c mice were incubated with C57BL/6 corneas pretreated with anti-GITRL mAb or control IgG. After a 6-hour incubation, expression of Foxp3 and CD25 in CD4+ T cells was assessed by flow cytometry. Data are displayed the proportion (±SD) of Foxp3+CD25+ cells among CD4+ T cells, with those in controls comprising purified CD4+ T cells from the spleens of BALB/c mice incubated without cornea for 0 hours (pre-incubation) and 6 hours. Data are representative of two independent experiments (n = 4 mice per group). *P < 0.05.
Figure 7.
 
GITRL-mediated expansion of Foxp3+CD25+CD4+ T cells in the cornea. Purified CD4+ T cells from the spleens of BALB/c mice were incubated with C57BL/6 corneas pretreated with anti-GITRL mAb or control IgG. After a 6-hour incubation, expression of Foxp3 and CD25 in CD4+ T cells was assessed by flow cytometry. Data are displayed the proportion (±SD) of Foxp3+CD25+ cells among CD4+ T cells, with those in controls comprising purified CD4+ T cells from the spleens of BALB/c mice incubated without cornea for 0 hours (pre-incubation) and 6 hours. Data are representative of two independent experiments (n = 4 mice per group). *P < 0.05.
Discussion
The present study was designed to investigate whether the interaction between GITRL and GITR is involved in the immune-privileged status of corneal allo-transplantation and to explore the underlying mechanisms. 
We demonstrated that normal cornea constitutively expresses GITRL. GITRL was localized on corneal endothelium in normal cornea. GITR was expressed on CD4+ T cells infiltrating into corneal allografts or adhering to corneal endothelium. If GITRL was blocked by peritoneal injection of antagonistic mAbs in recipients of corneal allografts, the allografts became more vulnerable to rejection. These results indicate that GITRL/GITR interactions play an important role in protecting corneal allografts from rejection. 
We also demonstrated for the first time that Foxp3+ CD25+CD4+ Treg are present in surviving corneal allografts. The presence of these Treg in corneal allografts was dependent on GITRL expression in the cornea. When GITRL was blocked by mAb treatment, Foxp3-negative CD4+ T cells dominantly infiltrated into corneal allografts, and graft destruction occurred, leading to accelerated rejection. These results suggest that GITRL expressed in the cornea functions as a Foxp3+CD25+CD4+ Treg-inducing or -maintaining molecule in the cornea. Constitutive expression of GITRL in cornea is thus at least partially responsible for the immune-privileged status of corneal allografts. 
We explored two possible mechanisms for GITRL-mediated corneal allograft protection from rejection via Treg. One possibility was that GITRL/GITR interactions might be involved in the induction of antigen-specific systemic immune tolerance to eye-derived antigens, known as ACAID. It has been suggested that antigens placed in the anterior chamber are captured by resident APCs, which then migrate through the trabecular meshwork out of the eye and into the blood. Once these cells reach the marginal zone of the spleen, active TGF-β, IL-10, and CCL5 attract and activate antigen-specific CD4+ and CD8+ T cells, which differentiate into antigen-specific regulatory T cells, as so-called ACAID-inducing Treg, inhibiting induction and expression of delayed hypersensitivity. 1,2 Induction of donor-specific ACAID has been confirmed to be associated with long-term graft acceptance and promotes the survival of corneal allografts. 1,2 Our results demonstrate that ACAID was induced in recipients treated with control IgG, because antigen-specific DH was suppressed. DH was also similarly suppressed in recipients treated with anti-GITRL mAb. These results indicate that induction of ACAID is independent of the GITRL/GITR interaction and that acceptance of corneal grafts can be abrogated even if ACAID remains intact. GITRL-mediated protection of corneal allografts from immune rejection via Treg is due to a mechanism other than ACAID-inducing Treg. 
The other possible mechanism is that corneal GITRL supports the immunosuppressive intraocular microenvironment via Treg, in which effector T cells are suppressed within the eye. 
To further substantiate GITRL-mediated protection of corneal allografts from effector T cells, we evaluated corneal endothelial cell destruction by CD4+ T cells in vitro. Killing of corneal endothelial cells by alloreactive T cells in vitro was significantly enhanced in corneas pretreated with anti-GITRL mAb compared with those pretreated with control IgG. This finding demonstrates that GITRL expressed on corneal endothelial cells plays a substantial role in the protection of corneal endothelium from destruction by effector T cells. Interestingly, GITRL-mediated protection was also observed after incubation with third-party-reactive T cells, indicating that GITRL protects cornea from bystander injury by activated T cells. Surprisingly, GITRL-mediated protection was still observed even after incubation with allogeneic T cells from naive mice. This suggests that naive T cells were sensitized with allo-antigens by corneal endothelial cells to promote their injury. These results indicate that constitutive expression of GITRL on corneal endothelial cells inhibits peripheral sensitization. Because corneas were pretreated with anti-GITRL mAb and then washed before incubation with CD4+T cells, anti-GITRL mAb exerts its blocking effect on the corneal cells, but not on CD4+T cells in this assay. The most surprising finding was that the population of Foxp3+CD25+CD4+ T cells from naive BALB/c was increased after incubation with GITRL-expressing B6 cornea in vitro, but not with GITRL-blocked cornea. This is evidence of GITRL-expressing corneal cell-mediated local expansion of CD25+CD4+ Treg. Although our data for flow cytometry show an increased proportion of Treg after incubation of T cells and cornea, this is not a cell-proliferation assay. The possibility thus remains that conversion of conventional T cells to Treg occurs. Our hypothesis is that GITRL signals from corneal cells via GITR expressed on T cells induce local proliferation of CD25+CD4+ Treg, as previously shown in the interaction between GITRL-transfected tumor cells and CD4+T cells. 51 The underlying molecular mechanisms for how CD25+CD4+ Treg protect corneal cells from effector T cells within the cornea remain to be established. 
The role of the GITR/GITRL pathway remains controversial. GITR costimulation reportedly attenuates the suppressive functions of Treg, 47,54 and GITR-engagement using agonistic mAb enhances effector T cells (Teff). 47 GITR/GITRL thus costimulates both Teff and Treg by in vitro engagement of GITR using agonistic mAb or via binding of soluble or cell surface GITRL expressed on GITRL-transfectants. 55,56 In contract, in vivo GITR engagement using agonistic mAb on Treg reportedly does not directly abrogate the suppressive functions, but instead increases expansion of Treg and promotes IL-10 production. 57 A very recent report has found that GITRL-costimulation activates proliferation of Treg, maintaining suppressive function in a coculture system using GITRL-transfected tumor cells without any APCs. 51 Most of these studies have used an agonistic anti-GITR mAb, GITRL transfectants, or soluble GITRL, none of which represents the physiological involvement of the native GITR-GITRL pathway in T-cell immune responses. 
The present study demonstrated the physiological function of constitutively expressed GITRL in immune privileged tissue. We propose that GITRL-mediated protection of the cornea is mediated by local expansion and/or maintenance of Foxp3+GITR+CD25+CD4+ Treg in the cornea. We have previously shown that expression of an immune-regulating molecule such as B7-H1 is upregulated, and localization of expression was expanded not only to the corneal endothelium, but also to stromal cells after grafting or in an inflammatory environment. 58 The possibility remains that GITRL is also upregulated on stromal cells after grafting. Further studies are needed to clarify the mechanisms underlying GITRL upregulation. Of note is the finding that GITRL-induced Treg functions have only been observed in immune-privileged tissues or sites such as tumors 51 and cornea so far. This suggests that GITRL can induce Treg functions only when GITRL-expressing tissues also express other immune-regulating factors. The cornea constitutively expresses various immune-regulating factors, such as CD95L, 30 B7-H1, 29 MHC class Ib, 59 and CD59, 60 in distinct localization patterns within the tissue. Moreover, the posterior surface of the cornea faces the anterior chamber, which is rich in TGF-β2. 1,2 The fact that the immune-privileged status of the cornea can be abolished by dysfunction of even just one of these molecules suggests that each of these molecules plays a nonredundant or cooperative role in maintaining immune privilege. Further studies are needed to address this possibility. 
In summary, the present results indicate that GITRL plays a critical role in maintaining the immune-privileged status of corneal allografts. GITRL constitutively expressed on corneal endothelial cells induces expansion of Foxp3+GITR+CD25+CD4+ Treg within the cornea. Our findings also indicate that acceptance of the corneal allograft depends on the presence of Foxp3+CD25+CD4+ Treg within the cornea. The cornea thus exists as an immune-privileged tissue in part due to constitutive expression of GITRL. 
Footnotes
 Supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JH).
Footnotes
 Disclosure: J. Hori, None; H. Taniguchi, None; M. Wang, None; M. Oshima, None; M. Azuma, None
References
Streilein JW . Ocular immune privilege: therapeutic opportunities from an experiment of nature [Review]. Nat Rev Immunol. 2003;3:879–889. [CrossRef] [PubMed]
Niederkorn JY . See no evil, hear no evil, do no evil: the lessons of immune privilege [Review]. Nat Rev Immunol. 2006;7:354–359. [CrossRef]
Coster DJ Williams KA . The impact of corneal allograft rejection of the long-term outcome of corneal transplantation. Am J Ophthalmol. 2005;140:1112–1122. [CrossRef] [PubMed]
Al-Yousuf N Mavrikakis I Mavrikakis M Daya SM . Penetrating keratoplasty: indications over a 10 year-period. Br J Ophthalmol. 2004;88:998–1001. [CrossRef] [PubMed]
Randleman JB Stulting RD . Prevention and treatment of corneal graft rejection: current practice patterns (2004). Cornea. 2006;25:286–290. [CrossRef] [PubMed]
Hori J . Mechanisms of immune privilege in the anterior segment of the eye: what we learn from corneal transplantation. J Ocul Biol Dis Inform. 2008;1:94–100. [CrossRef]
Sonoda Y Streilein JW . Orthotopic corneal transplantation in mice—evidence that the immunogeneic rules of rejection do not apply. Transplantation. 1992;54:694–704. [CrossRef] [PubMed]
Williams KA Coster DJ . Penetrating corneal transplantation in the inbred rat: a new model. Invest Ophthalmol Vis Sci. 1985;26:23–30. [PubMed]
Sonoda Y Streilein JW . Impaired cell-mediated immunity in mice bearing healthy orthotopic corneal allograft. J Immunol. 1993;150:1727–1734. [PubMed]
Streilein JW Toews GB Bergstresser PR . Corneal allografts fail to express Ia antigens. Nature. 1979;282:326–327. [CrossRef] [PubMed]
Sosnova M Bradl M Forrester JV . CD34+ corneal stromal cells are bone marrow-derived and express hemopoietic stem cells markers. Stem Cells. 2005;23:507–515. [CrossRef] [PubMed]
Hamrah P Zhang Q Liu Y Dana MR . Novel characterization of MHC class II-negative population of resident corneal Langerhans cell-type dendritic cells. Invest Ophthalmol Vis Sci. 2002;43:639–646. [PubMed]
Whitsett CF Stulting RD . The distribution of HLA antigens on human corneal tissue. Invest Ophthalmol Vis Sci. 1984;25:519–524. [PubMed]
Wang HM Kaplan HJ Chan WC Johnson M . The distribution and ontogeny of MHC antigens in murine ocular tissue. Invest Ophthalmol Vis Sci. 1987;28:1383–1389. [PubMed]
Treseler PA Sanfilippo F . The expression of major histocompatibility complex and leukocyte antigens by cells in the rat cornea. Transplantation. 1986;41:248–252. [CrossRef] [PubMed]
Kaplan HJ Streilein JW . Immune response to immunization via the anterior chamber of the eye, I: F1 lymphocyte-induced immune deviation. J Immunol. 1977;118:809–814. [PubMed]
Niederkorn JY . Anterior chamber-associated immune deviation and its impact on corneal allograft survival. Curr Opin Organ Transplant. 2006;11:360–365. [CrossRef]
Sonoda Y Ksander B Streilein JW . Evidence that active suppression contributes to the success of H-2-incompatible orthotopic corneal allografts in mice. Transplant Proc. 1993;25(1 pt 2):1374–1376. [PubMed]
Namba K Kitaichi N Nishida T Taylor AW . Identification a-melanocyte stimulating hormone and transforming growth factor-β2. J Leukoc Biol. 2002;72:946–952. [PubMed]
Kaiser CJ Ksander BR Streilein JW . Inhibition of lymphocyte proliferation by aqueous humor. Reg Immunol. 1989;2:42–49. [PubMed]
Taylor AW Yee DG Streilein JW . Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor. Invest Ophthalmol Vis Sci. 1998;39:1372–1378. [PubMed]
Willbanks GA Mammolenti MM Streilein JW . Studies on the induction of anterior chamber-associated immune deviation (ACAID), III: induction of ACAID depends upon intraocular transforming growth factor-b. Eur J Immunol. 1992;22:165–173. [CrossRef] [PubMed]
Sheibani N Sorenson CM Cornelius LA Frazier WA . Thrombospondin-1, a natural inhibitor of angiogenesis, is present in vitreous and aqueous humor and is modulated by hyperglycemia. Biochem Biophys Res Commun. 2000;267:257–268. [CrossRef] [PubMed]
Apte RS Sinha D Mayhew E Wistow GL Niederkorn JY . Role of macrophage migration inhibitory factor in inhibition NK cell activity. J Immunol. 1998;160:5693–5696. [PubMed]
Kennedy MC Rosenbaum JT Brown J . Novel production of interleukin-1 receptor antagonist peptides in normal human cornea. J Clin Invest. 1995;95:82–88. [CrossRef] [PubMed]
Shon JH Kaplan HJ Suk HJ Bora PS Bora NS . Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59. Invest Ophthalmol Vis Sci. 2000;41:4195–4202. [PubMed]
Sugita S Taguchi C Takase H . Soluble Fas ligand and soluble Fas in ocular fluid of patients with uveitis. Br J Ophthalmol. 2000;84:1130–1134. [CrossRef] [PubMed]
Griffith TS Brunner T Fletcher SM Green DR Ferguson TA . FAS ligand-induced apoptosis as a mechanism of immune privilege. Science. 1995;270:1189–1192. [CrossRef] [PubMed]
Hori J Wang MC Miyashita M . B7–H1-induced apoptosis as a mechanism of immune privilege of corneal allografts. J Immunol. 2006;177:5928–5935. [CrossRef] [PubMed]
Stuart PM Griffith TS Usui N Pepose J Yu X Ferguson X . CD95 ligand (FasL)-induced apoptosis is necessary for corneal allograft survival. J Clin Invest. 1997;99:396–402. [CrossRef] [PubMed]
Hori J Joyce N Streilein JW . Epithelium-deficient corneal allografts display immune privilege beneath the kidney capsule. Invest Ophthalmol Vis Sci. 2000;41:443–452. [PubMed]
Hori J Joyce NC Streilein JW . Immune privilege and immunogenicity reside among layers of the mouse cornea. Invest Ophthalmol Vis Sci. 2000;41:3032–3042. [PubMed]
Nocentini G Giunchi L Ronchetti S . A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis. Proc Natl Acad Sci U S A. 1997;94:6216–6221. [CrossRef] [PubMed]
Gurney AL Marsters SA Huang RM . Identification of a new member of the tumor necrosis factor family and its receptor, a human ortholog of mouse GITR. Curr Biol. 1999;9:215–218. [CrossRef] [PubMed]
Kanamaru F Youngnak P Hashiguchi M . Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. J Immunol. 2004;172:7306–7314. [CrossRef] [PubMed]
Yu KY Kim HS Song SY Min SS Jeong JJ Youn BS . Identification of a ligand for glucocorticoid-induced tumor necrosis factor receptor constitutively expressed in dendritic cells. Biochem Biophys Res Commun. 2003;310:433–438. [CrossRef] [PubMed]
Tone M Tone Y Adams E . Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc Natl Acad Sci U S A. 2003;100:15059–15064. [CrossRef] [PubMed]
Cuzzocrea S Nocentini G Di Paola R . Glucocorticoid-induced TNF receptor family gene (GITR) knockout mice exhibit a resistance to splanchnic artery occlusion (SAO) shock. J Leukocyte Biol. 2004;76:933–940. [CrossRef] [PubMed]
Agostini M Cenci E Pericolini E . The glucocorticoid-induced tumor necrosis factor receptor-related gene modulates the response to Candida albicans infection. Infect Immun. 2005;73:7502–7508. [CrossRef] [PubMed]
Cuzzocrea S Ayroldi E Di Paola R . Role of glucocorticoid-induced TNF receptor family gene (GITR) in collagen-induced arthritis. FASEB J. 2005;19:1253–1265. [CrossRef] [PubMed]
Cuzzocrea S Nocentini G Di Paola R . Proinflammatory role of glucocorticoid-induced TNF receptor-related gene in acute lung inflammation. J Immunol. 2006;177:631–641. [CrossRef] [PubMed]
Santucci L Agostini M Bruscoli S . GITR modulates innate and adaptive mucosal immunity during the development of experimental colitis in mice. Gut. 2007;56:52–60. [CrossRef] [PubMed]
Cuzzocrea S Ronchetti S Genovese T . Genetic and pharmacological inhibition of GITR-GITRL interaction reduces chronic lung injury induced by bleomycin instillation. FASEB J. 2007;21:117–129. [CrossRef] [PubMed]
Ronchetti S Zollo O Bruscoli S . GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur J Immunol. 2004;34:613–622. [CrossRef] [PubMed]
Esparza EM Arch RH . Glucocorticoid-induced TNF receptor, a costimulatory receptor on naive and activated T cells, uses TNF receptor-associated factor 2 in a novel fashion as an inhibitor of NF-B activation. J Immunol. 2005;174:7875–7882. [CrossRef] [PubMed]
Stephens GL McHugh RS Whitters MJ . Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J Immunol. 2004;173:5008–5020. [CrossRef] [PubMed]
Ji HB Liao G Faubion WA . Cutting edge: the natural ligand for glucocorticoid-induced TNF receptor-related protein abrogates regulatory T cell suppression. J Immunol. 2004;172:5823–5827. [CrossRef] [PubMed]
Kim BJ Li Z Fariss RN . Constitutive and cytokine-induced GITR ligand expression on human retinal pigment epithelium and photoreceptors. Invest Ophthalmol Vis Sci. 2004;45:3170–3176. [CrossRef] [PubMed]
Mahesh SP Li Z Liu B Fariss RN Nussenblatt RB . Expression of GITR ligand abrogates immunosuppressive function of ocular tissue and differentially modulates inflammatory cytokines and chemokines. Eur J Immunol. 2006;36:2128–2138. [CrossRef] [PubMed]
Chauhans SK Saban DR Lee HK Dana R . Levels of Foxp3 in regulatory T cells reflect their functional status in transplantation. J Immunol. 2009;182:148–153. [CrossRef] [PubMed]
Igarashi H Cao Y Iwai H . GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells. Biochem Biophys Res Commun. 2008;369:1134–1138. [CrossRef] [PubMed]
Kamimura Y Iwai H Piao J Hashiguchi M Azuma M . The glucocorticoid-induced TNF receptor-related protein (GITR) ligand pathway acts as a mediator of cutaneous dendritic cell migration and promotes T cell-mediated acquired immunity. J Immunol. 2009;182:2708–2716. [CrossRef] [PubMed]
Hori J Streilein JW . Dynamics of donor cell persistence and recipient cell replacement in orthotopic corneal allografts in mice. Invest Ophthalmol Vis Sci. 2001;42:1820–1828. [PubMed]
Shimizu J Yamazaki S Takahashi T Ishida Y Sakaguchi S . Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol. 2002;3:135–142. [CrossRef] [PubMed]
Ko K Yamazaki S Nakamura K . Treatment of advanced tumors with agonistic anti-GITR mAn and its effects on tumor-infiltrating Foxp3+ CD25+CD4+ regulatory T cells. J Ext Med. 2005;202:885–891. [CrossRef]
La S Kim E Kwon B . In vivo ligation of glucocorticoid-induced TNF receptor enhances the T-cell immunity to herpes simplex virus type 1. Exp Mol Med. 2005;37:193–198. [CrossRef] [PubMed]
Zhou P Litalien L Hodges D Schebye XM . Pivotal roles of CD4+ effector T cells in mediating agonistic anti-GITR mAb-induced-immune activation and tumor immunity in CT26 tumors. J Immunol. 2007;179:7365–7375. [CrossRef] [PubMed]
Hori J . Molecular mechanisms of immune-suppressive microenvironment in the cornea. Cornea. 2009;28:s58–s64. [CrossRef]
Niederkorn JY Chiang EY Ungchusri T Stroynowski I . Expression of a nonclassical MHC class 1b molecule in the eye. Transplantation. 1999;68:1790–1799. [CrossRef] [PubMed]
Bora NS Gobleman CI Atkinson JP Pepose JS Kaplan HJ . Differential expression of the complement regulatory proteins in the human eye. Invest Ophthalmol Vis Sci. 1993;34:3579–3584. [PubMed]
Figure 1.
 
Expression of GITRL in normal mouse eye. mRNA was extracted from freshly isolated cornea, iris-ciliary body (iris-CB), and neural retina of normal mouse eyes, and then reverse transcribed and amplified by PCR. PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining (A). Cryostat sections were stained with biotin-conjugated anti-GITRL mAb or control rat IgG, followed by streptavidin-PE (red). Nuclei were stained with DAPI (blue). CB, ciliary body; Ced, corneal endothelium; Cep, corneal epithelium; Cst, corneal stroma. Original magnification: (B) ×40).
Figure 1.
 
Expression of GITRL in normal mouse eye. mRNA was extracted from freshly isolated cornea, iris-ciliary body (iris-CB), and neural retina of normal mouse eyes, and then reverse transcribed and amplified by PCR. PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining (A). Cryostat sections were stained with biotin-conjugated anti-GITRL mAb or control rat IgG, followed by streptavidin-PE (red). Nuclei were stained with DAPI (blue). CB, ciliary body; Ced, corneal endothelium; Cep, corneal epithelium; Cst, corneal stroma. Original magnification: (B) ×40).
Figure 2.
 
Blockade of GITRL or depletion of CD25+CD4+ Treg accelerates corneal allograft rejection. Normal corneas of C57BL/6 were transplanted orthotopically into normal eyes of BALB/c mice. After the grafting procedure, recipients were injected with 0.2 mg anti-GITRL mAb or control rat IgG intraperitoneally 3 times weekly for 8 weeks. To deplete CD25+CD4+ Treg, 0.5 mg anti-CD25 mAb was injected intraperitoneally to a different set of recipients at 4 days before and on the day of grafting. Graft survival was clinically assessed and compared. Survival of allografts treated with anti-GITRL or anti-CD25 mAbs was significantly less than that in control allografts (A, anti-GITRL, *P < 0.01; anti-CD25, **P < 0.001; n = 10 in each group). CD4+ T cells purified from spleens and dLNs of allograft recipients treated with 0.5 mg anti-CD25 mAb at 4 days before and on the day of transplantation were also assessed to confirm depletion of CD25+CD4+ Treg after transplantation (B, right). Data are displayed as dot-blot graphs (four-decade log scales), with controls comprising naive BALB/c mice. The number in the upper right corner of each graph shows the proportion of GITR+CD25+ cells among CD4+ T cells. Mean percentage of cells (±SD) of four to five mice/group.
Figure 2.
 
Blockade of GITRL or depletion of CD25+CD4+ Treg accelerates corneal allograft rejection. Normal corneas of C57BL/6 were transplanted orthotopically into normal eyes of BALB/c mice. After the grafting procedure, recipients were injected with 0.2 mg anti-GITRL mAb or control rat IgG intraperitoneally 3 times weekly for 8 weeks. To deplete CD25+CD4+ Treg, 0.5 mg anti-CD25 mAb was injected intraperitoneally to a different set of recipients at 4 days before and on the day of grafting. Graft survival was clinically assessed and compared. Survival of allografts treated with anti-GITRL or anti-CD25 mAbs was significantly less than that in control allografts (A, anti-GITRL, *P < 0.01; anti-CD25, **P < 0.001; n = 10 in each group). CD4+ T cells purified from spleens and dLNs of allograft recipients treated with 0.5 mg anti-CD25 mAb at 4 days before and on the day of transplantation were also assessed to confirm depletion of CD25+CD4+ Treg after transplantation (B, right). Data are displayed as dot-blot graphs (four-decade log scales), with controls comprising naive BALB/c mice. The number in the upper right corner of each graph shows the proportion of GITR+CD25+ cells among CD4+ T cells. Mean percentage of cells (±SD) of four to five mice/group.
Figure 3.
 
Expression of GITRL and infiltration of Foxp3+GITR+CD25+ CD4+ Treg in corneal allografts. Normal corneas of C57BL/6 were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes from recipients treated with control rat IgG (A, BD) or anti-GITRL mAb (EG) were isolated at 3 weeks. Cryostat sections of graft-bearing eyes were examined by hematoxylin and eosin staining and multicolor immunofluorescence staining with PE-conjugated anti-GITRL (A), anti-GITR (B, E), or anti-CD4 (D, G), and FITC-conjugated anti-CD4 (B, C, E, F) or anti-CD25 (D, G), and biotin-conjugated anti-Foxp3 mAbs followed by streptavidin-allophycocyanin (B, C, E, F). Nuclei were stained with DAPI. Merged color images are also shown. High-magnification images (C, F) show intracellular staining of Foxp3 and cell-surface staining of CD4. Graft center (A) and the graft junction (BG) are shown, as are representative sections. Ced, corneal endothelium; Cep, corneal epithelium; Cst, corneal stroma (original magnification, ×40).
Figure 3.
 
Expression of GITRL and infiltration of Foxp3+GITR+CD25+ CD4+ Treg in corneal allografts. Normal corneas of C57BL/6 were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes from recipients treated with control rat IgG (A, BD) or anti-GITRL mAb (EG) were isolated at 3 weeks. Cryostat sections of graft-bearing eyes were examined by hematoxylin and eosin staining and multicolor immunofluorescence staining with PE-conjugated anti-GITRL (A), anti-GITR (B, E), or anti-CD4 (D, G), and FITC-conjugated anti-CD4 (B, C, E, F) or anti-CD25 (D, G), and biotin-conjugated anti-Foxp3 mAbs followed by streptavidin-allophycocyanin (B, C, E, F). Nuclei were stained with DAPI. Merged color images are also shown. High-magnification images (C, F) show intracellular staining of Foxp3 and cell-surface staining of CD4. Graft center (A) and the graft junction (BG) are shown, as are representative sections. Ced, corneal endothelium; Cep, corneal epithelium; Cst, corneal stroma (original magnification, ×40).
Figure 4.
 
Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in corneal allografts. Normal corneas of C57BL/6 were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes from recipients treated with isotype control rat IgG or anti-GITRL mAb were isolated at 3 weeks. Cryostat sections of graft-bearing eyes were examined by immunofluorescence staining with FITC-conjugated anti-CD4 and biotin-conjugated anti-Foxp3 mAbs followed by streptavidin-allophycocyanin. Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in the confocal images (magnification, ×40) were counted and compared. *P < 0.05, **P < 0.01, ***P < 0.001; n = 4–5 grafts in each group.
Figure 4.
 
Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in corneal allografts. Normal corneas of C57BL/6 were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes from recipients treated with isotype control rat IgG or anti-GITRL mAb were isolated at 3 weeks. Cryostat sections of graft-bearing eyes were examined by immunofluorescence staining with FITC-conjugated anti-CD4 and biotin-conjugated anti-Foxp3 mAbs followed by streptavidin-allophycocyanin. Numbers of Foxp3+CD4+cells and Foxp3(−)CD4+ cells in the confocal images (magnification, ×40) were counted and compared. *P < 0.05, **P < 0.01, ***P < 0.001; n = 4–5 grafts in each group.
Figure 5.
 
Blockade of GITRL does not abolish ACAID. B6 spleen cells were used as alloantigens and injected into the right AC of BALB/c normal eyes. Two weeks later, B6 spleen cells were injected SC to sensitize the mice. After one more week, a challenge was performed by injecting B6 spleen cells into the right ear pinna of each mouse. After 24 hours, specific ear swelling was measured as an indication of DH. Treatments with anti-GITRL mAb were performed starting from the day of AC injection and continuing until the day of the ear challenge. DH responses were similarly suppressed in anti-GITRL mAb or control rat IgG-treated groups, and no significant differences were observed between these groups. Positive control mice (Posi.C) received SC immunization and the ear challenge without previous AC injection. Negative control mice (Nega.C) received only the ear challenge without AC injection or immunization.
Figure 5.
 
Blockade of GITRL does not abolish ACAID. B6 spleen cells were used as alloantigens and injected into the right AC of BALB/c normal eyes. Two weeks later, B6 spleen cells were injected SC to sensitize the mice. After one more week, a challenge was performed by injecting B6 spleen cells into the right ear pinna of each mouse. After 24 hours, specific ear swelling was measured as an indication of DH. Treatments with anti-GITRL mAb were performed starting from the day of AC injection and continuing until the day of the ear challenge. DH responses were similarly suppressed in anti-GITRL mAb or control rat IgG-treated groups, and no significant differences were observed between these groups. Positive control mice (Posi.C) received SC immunization and the ear challenge without previous AC injection. Negative control mice (Nega.C) received only the ear challenge without AC injection or immunization.
Figure 6.
 
GITRL protects corneal endothelial cells from being killed by T cells in vitro. C57BL/6 corneas pretreated with anti-GITRL mAb or control IgG were incubated with purified T cells from the spleens of BALB/c mice presensitized against C57BL/6 antigens (α-B6/BALB-T cells) or third-party C3H/He antigens (α-C3H/BALB-T cells), or from the spleens of naive BALB/c (naive BALB-T cells), C57BL/6 (naive B6-T cells), or C3H/He mice (naive C3H-T cells). After a 6-hour incubation, corneal endothelial cell death was detected by staining unfixed tissue with PI followed by confocal microscopic examination. Positive control corneas were incubated with Triton X-100, without antibody treatment or incubation with T cells. As negative controls, corneas with antibody treatment and incubation without T cells, and corneas without antibody treatment or incubation with T cells were used. Data are presented as the mean (±SD) number of PI+ corneal endothelial cells from five to six corneas in each group (*P < 0.05, **P < 0.01).
Figure 6.
 
GITRL protects corneal endothelial cells from being killed by T cells in vitro. C57BL/6 corneas pretreated with anti-GITRL mAb or control IgG were incubated with purified T cells from the spleens of BALB/c mice presensitized against C57BL/6 antigens (α-B6/BALB-T cells) or third-party C3H/He antigens (α-C3H/BALB-T cells), or from the spleens of naive BALB/c (naive BALB-T cells), C57BL/6 (naive B6-T cells), or C3H/He mice (naive C3H-T cells). After a 6-hour incubation, corneal endothelial cell death was detected by staining unfixed tissue with PI followed by confocal microscopic examination. Positive control corneas were incubated with Triton X-100, without antibody treatment or incubation with T cells. As negative controls, corneas with antibody treatment and incubation without T cells, and corneas without antibody treatment or incubation with T cells were used. Data are presented as the mean (±SD) number of PI+ corneal endothelial cells from five to six corneas in each group (*P < 0.05, **P < 0.01).
Figure 7.
 
GITRL-mediated expansion of Foxp3+CD25+CD4+ T cells in the cornea. Purified CD4+ T cells from the spleens of BALB/c mice were incubated with C57BL/6 corneas pretreated with anti-GITRL mAb or control IgG. After a 6-hour incubation, expression of Foxp3 and CD25 in CD4+ T cells was assessed by flow cytometry. Data are displayed the proportion (±SD) of Foxp3+CD25+ cells among CD4+ T cells, with those in controls comprising purified CD4+ T cells from the spleens of BALB/c mice incubated without cornea for 0 hours (pre-incubation) and 6 hours. Data are representative of two independent experiments (n = 4 mice per group). *P < 0.05.
Figure 7.
 
GITRL-mediated expansion of Foxp3+CD25+CD4+ T cells in the cornea. Purified CD4+ T cells from the spleens of BALB/c mice were incubated with C57BL/6 corneas pretreated with anti-GITRL mAb or control IgG. After a 6-hour incubation, expression of Foxp3 and CD25 in CD4+ T cells was assessed by flow cytometry. Data are displayed the proportion (±SD) of Foxp3+CD25+ cells among CD4+ T cells, with those in controls comprising purified CD4+ T cells from the spleens of BALB/c mice incubated without cornea for 0 hours (pre-incubation) and 6 hours. Data are representative of two independent experiments (n = 4 mice per group). *P < 0.05.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×