Molecular Vision 2007; 13:1902-1911 <http://www.molvis.org/molvis/v13/a214/>
Received 24 July 2007 | Accepted 2 October 2007 | Published 5 October 2007
Download
Reprint


Ovalbumin serves as a neo-transplantation antigen in retinal pigment epithelial cells

Janine R. Wajchman,1 James C. Zimring,2 Jeffrey P. Lake,3 Jing Wen,4 Judith A. Kapp5
 
 

1Department of Ophthalmology, 2Department of Pathology, Emory University, Atlanta, GA, 3The Jackson Laboratory, Bar Harbor ME, 4Department of Surgery, Emory University, Atlanta, GA, 5Departments of Ophthalmology and Pathology, University of Alabama at Birmingham, Birmingham, AL

Correspondence to: Judith A. Kapp, PhD, Room W287 Spain Wallace Building, 619 South 19th Street, University of Alabama at Birmingham, Birmingham, AL 35294-2170; Phone (205)-975-7081; FAX (205)-996-2435; email: jkapp@uab.edu


Abstract

Purpose: Our long-term goal is to determine the optimal methods for inducing allograft tolerance to facilitate transplantation of retinal pigment epithelial cells or stem cells for the treatment of retinal degenerative diseases. These goals have been hampered by the extreme complexity of allograft rejection and the heterogeneity of responding T cells. The current studies were undertaken to develop a simplified transplant model for studying rejection and tolerance in the unique environment of the eye.

Methods: To provide a defined transplantation antigen, transgenic C57BL/6 (B6) mice were produced, which express the exogenous chicken egg ovalbumin (OVA) gene under the regulation of the mouse tyrosinase related protein-1 (TRP-1) promoter that is transcriptionally active in retinal pigmented epithelial (RPE) cells. To determine whether the transgene was expressed as a neo-transplantation antigen, RPE from TRP-1-OVA mice were injected into the subretinal space of B6 mice or B6 mice expressing the OVA-specific (OT1) TCR transgenes and examined for inflammatory cell infiltration.

Results: The TRP-1-OVA transgenic mice expressed OVA mRNA in the brain and eye but not the heart or kidney. RPE cells from TRP-1-OVA transgenic mice expressed mRNA and protein encoded by the OVA gene and RPE expressing TRP-1-OVA induced an inflammatory response within the subretinal space of OT1 mice but not in B6 mice.

Conclusions: OVA serves as a defined, neo-transplantation antigen in RPE that is recognized by mice whose CD8+ T cells recognize OVA peptide. These observations provide new tools for future studies of the mechanisms of rejection and prolongation of RPE transplants in the eye.


Introduction

Age-related macular degeneration (AMD) is a leading cause of blindness in the United States. Although the cause of AMD is unknown, replacement of dead or damaged cells with healthy retinal cells is thought to be a promising, albeit challenging, approach to the treatment of this and other retinal diseases [1-3]. Because the eye is a relatively privileged site, in terms of immunological responses, it was thought that ocular grafts could be transplanted with impunity. Indeed, corneal transplants between unrelated individuals are the singular most successful transplants performed on humans and neither histocompatibility matching nor prolonged administration of immunosuppressive drugs is necessary for most recipients. Although the subretinal space (SRS) is an immunologically privileged site and neonatal RPE are immunologically privileged cells [4], suspensions of adult RPE are rejected when transplanted into the subretinal space albeit with prolonged kinetics [5,6]. However, transplantation of normal allogeneic RPE cells into the SRS of Royal College of Surgeons (RCS) rats, which have a mutation in the Mertk gene that causes a defect in phagocytosis of photoreceptor outer segments by RPE cells leading to blindness [7], significantly prolongs retinal function [8] if recipients were treated with immunosuppressive drugs. This observation provides proof of the concept that transplantation of RPE may protect against photoreceptor death in retinal degeneration. However, survival of allogeneic RPE in RCS rats requires continuous treatment with immunosuppressive drugs to prevent allograft rejection. Prolonged treatment with immunosuppressive drugs is less than ideal clinically because chronic graft rejection can still occur. Moreover, complications and side effects of the drugs are a problem and life-long drug therapy is expensive and onerous. Thus, transplant rejection will have to be overcome before this approach could be applied to humans for the treatment of AMD.

Our long-term goal is to optimize methods for the treatment of retinal degenerative diseases by inducing allograft tolerance to facilitate transplantation of RPE or stem cells, which also will be subject to immune rejection upon differentiation [9,10]. A fundamental problem in the assessment of various tolerance-inducing strategies in allograft rejection is the extreme complexity of the alloantigens and the heterogeneity of the responding T cells. To reduce this complexity, we set out to develop a transplant model in which the nominal antigen is a defined exogenous protein driven by a tissue specific promoter, an approach used by many investigators, including some who have used promoters that are expressed primarily in ocular tissues [11-13]. To reduce the heterogeneity of responding T cells, we have used TCR transgenic mice specific for the transgenic antigen. Here we report the production of transgenic B6 mice that express the chicken OVA transgene under control of the mouseTRP-1 promoter, which limits expression primarily to pigmented cells [14,15]. The TRP-1-OVA gene is expressed in RPE of these mice and RPE from TRP-1-OVA transgenic mice induced inflammatory responses when transplanted into the SRS of transgenic OT-1 mice, whose T cells express a receptor for OVA presented in the context of major histocompatibility complex (MHC) class I protein, H-2Kb, but not in non-transgenic B6 mice.


Methods

Experimental Animals

B6 mice and B6 mice expressing the enhanced green fluorescent protein (GFP) under the control of the chicken β-actin promoter and cytomegalovirus enhancer (C57BL/6-Tg(ACTB-EGFP)1Osb/J) referred to as B6.GFP, which expresses GFP in all tissues except erythrocytes and hair, were purchased from the Jackson Laboratory (Bar Harbor, ME). T cell receptor (TCR) transgenic B6 mice [16], which are referred to as OT-1 mice, and TRP-1-OVA (described below) transgenic mice were bred in the animal facilities at Emory University and the University of Alabama at Birmingham. The OT-I mice, have an MHC class I restricted TCR that is specific for the OVA257-264 peptide and the Kb MHC class I molecule. Mice used in these experiments were 8 weeks or older. All procedures on animals were conducted according to the principals in the guidelines of the Committee on Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council and approved by the institutional animal care and use committee (IACUC).

Isolation and culturing retinal pigment epithelium from adult mice

Mouse RPE cells were isolated from TRP-1-OVA mice using a modification of the methods for human RPE [17,18]. Eyes were removed aseptically and incubated at 37 °C with collagenase and hyaluronidase for 40 min followed by an additional 50 min with trypsin. The sclera and choroid were dissected from the eye and discarded; the RPE were gently rubbed from the neurosensory retina, which remained attached to the vitreous. Approximately 105 RPE cells/eye were routinely obtained. RPE were cultured as described by Sheedlo et al. [19] to expand the number of RPE so multiple mice could be injected with the same preparation of RPE and so they could be incubated with microbeads for tracking purposes. RPE were transplanted between passages 2 and 5.

Cell lines

The EL4 thymoma (H-2b) was purchased from American Type Culture Collection (ATCC, Rockville, MD). E.G7-OVA, generated by transduction of EL4 with the OVA cDNA [20], was provided by Dr. Michael J. Bevan (University of Washington, Seattle, WA). Cell lines were grown in standard growth media (SGM), which consisted of RPMI 1640 (Mediatech, Washington, DC) supplemented with 10% FCS, 1 mM L-glutamine, 1 mM sodium pyruvate, 50 μM 2-ME (Sigma Chemical, St. Louis, MO) and antibiotics. They were incubated at 37 °C in a humidified 5% CO2 atmosphere. The cell lines were maintained mycoplasma-free.

Generation of TRP-1-OVA transgenic mice

cDNA encoding OVA was obtained by performing RT-PCR on RNA extracted from E.G7-OVA using the following primers: 5' primer cgg aat tcg cca gcc atg ggc tcc atc cgg cga agc aag ca; 3' primer gct cta gat taa ggg gaa aca cat ctg cca aag. A single band of approximately 1.2 kb was obtained, which contained the intact OVA open reading frame. This fragment was digested with EcoR1 and Xba 1 and ligated into the commercially available pCI mammalian expression vector (Promega, Madison, WI) that had been digested with the same enzymes. The CMV promoter was excised by cutting TRP-1-OVA with Nco I, blunt ending with Klenow and then cutting with BglII. The TRP-1 promoter was amplified by PCR from B6 mouse spleen DNA using the following published primers: 5' primer gga aga tct tct aga ctt ttc tgt tta atg ttt t, 3' primer aac tgc agc tgt taa ttg ccc gaa gag att ttc [21]. The product was cut with BglII and ligated into the digested pCI-OVA. The ligation mixture was then transformed into DH5α E.coli and recombinant clones were isolated. The DNA was purified from E.coli using a Qiagen plasmid prep kit (Qiagen, Valencia, CA). The final construct was sequenced to verify that no mutations had been introduced during PCR. The TRP-1-OVA construct was injected into pronuclei from B6 mice following standard techniques by the Transgenic Mouse Facility at Emory University. A founder mouse was identified and then crossed to B6 mice.

Identification of TRP-1-OVA transgenic mice

Transgenic mice were identified by PCR of genomic tail DNA. Genomic DNA was purified using a Purgene DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN) according to the manufacturer's instructions. The PCR analysis was performed using 500 ng of genomic DNA in the presence of 2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.5), 0.1% Triton X-100, 5% DMSO, 0.2 mM each dNTP, 0.1 μM each primer and 1 unit of Taq DNA polymerase (Fisher Scientific, Springfield, NJ) in a final volume of 20 μl. The primers for OVA were: forward primer: tca gct cta gcc atg gta tac c, reverse primer: ggc att gct tgt gtg tct tc which generated a 580 bp fragment. After the initial denaturation at 94 °C for 3 min, amplification was performed during 34 cycles of 93 °C for 1 min, 58 °C for 2 min and 72 °C for 2 min followed by a final extension at 72 °C for 4 min in a thermal cycler (Ericomp, San Diego, CA). Amplification of β-actin was used as a control to rule out the presence of PCR inhibitors in negative samples. The mastermix for β-actin was the same as described above except that β-actin specific primers were used, the reaction volume was 50 μl, and 1.5 mM MgCl2 and 2 units of Taq DNA polymerase were used. The primers for β-actin amplification were: forward primer: tgg aat cct gtg gca tcc atg aaa c, reverse primer: taa aac gca gct cag taa cag tcc g resulting in a 324 bp fragment. The cycling conditions were the following: initial denaturation 94 °C for 3 min followed by amplification with 34 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The final extention time was for 4 min at 72 °C. Amplified DNA from tails was loaded onto agarose gels along with positive and negative controls. Transgenic mice, confirmed to have the OVA gene in their genomic DNA, were used in subsequent experiments.

Reverse transcriptase-polymerase chain reaction for tissue expression of ovalbumin mRNA

RPE cells from B6 and TRP-1-OVA mice were harvested and cultured, as previously described [22], to obtain sufficient RNA for the analysis. Various tissues were harvested from B6 TRP-1-OVA mice, which were digested with collagenase and RNA extracted using RNAzol (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. After extraction, the samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI) according to the manufacturer's instructions. The samples were read on a spectrophotometer to determine quantity and purity of RNA. RT-PCR was performed with a set of outside primers for OVA: forward primer: cat tgc cat cat gtc agc tc, reverse primers: aga agc cat tga tgc cac tc, an Access RT-PCR kit (Promega) and 200 ng of RNA in the presence of AMV/Tfl buffer, 25 mM MgSO4, 10 mM each dNTP, 5U Tfl DNA polymerase and 5U AMV reverse transcriptase in a final volume of 50 μl. The cycling conditions were first strand cDNA synthesis at 48 °C for 45 min then AMV RT inactivation and RNA/cDNA primer denaturation at 94 °C for 2 min followed by 40 cycles of second strand cDNA synthesis and PCR amplification at 94 °C for 30 s, 60 °C for 1 min and 68 °C for 2 min then a final extension at 68 °C for 7 min and finishing with 4 °C until removal from the thermal cycler. Samples were also tested for the GAPDH housekeeping gene as an internal control with the following primers: forward primer: acc aca gtc cat gcc atc ac, reverse primer: tcc acc acc ctg ttg ctg ta in the presence of 25 mM MgCl2, 10 mM dNTPs, Taq DNA polymerase and PCR buffer in a final volume of 50 ul. The cycling conditions were denaturation at 94 °C for 2 min followed by amplification for 40 cycles of 94 °C for 30 s, 60 °C for 1 min and 68 °C for 2 min with a final extension at 68 °C for 7 min and finishing with 4 °C until removal from the thermal cycler. The cDNA was analyzed by Southern blotting; the OVA product is a 484 bp fragment whereas the GAPDH product is a 452 bp fragment.

Immunofluorescence staining

Nonadherent EL4 and E.G7-OVA cells were harvested from cultures, washed and affixed to slides using a cytocentrifuge to serve as negative and positive controls for staining for intracellular OVA. RPE cells were cultured in chambered slides to allow them to adhere to the surface. Slides were air dried, fixed with acetone for 10 min, and frozen at -80 °C until use. The slides with cells or tissue sections were thawed at room temperature and rehydrated in phosphate buffered saline (PBS) for 5 min. Nonspecific binding sites were blocked by incubating the slides for 20 min in a moist chamber with PBS + 1% BSA. After washing in PBS, unconjugated rabbit anti-OVA antibody (Crystal Chem, Chicago, IL) diluted in PBS + 1% BSA was added and the slides were incubated for 1 h. The slides were washed and incubated with the FITC goat anti-rabbit 2 °C antibody (Vector Laboratories, Burlingame, CA) for 1 h. Following another wash with PBS, Evan's blue (Sigma Chemical, St. Louis, MO) was added to the tissue sections, as a counterstain, and incubated for 5 min. After a final wash with PBS, Vectashield mounting media (Vector Laboratories, Burlingame, CA) was added to the slides to maintain fluorescence and coverslips applied. The slides were examined using fluorescent microscopy and images were made with a digital camera interfaced to a computer using MagnaFire software (Optronics, Goleta, CA).

Immunohistochemistry

RPE cells were harvested from cultures, washed, and affixed to slides by cytocentrifugation. The slides were stained with an isotype control or a primary mouse monoclonal anti-human cytokeratin 8 (Dako Corp., Carpinteria, CA) to verify that they were epithelial cells [23]. The slides were then treated with secondary biotinylated goat anti-mouse IgG, Horseradish Peroxidase labeled Avidin D, and 3,3'-diaminobenzidine Substrate Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's directions. The slides were counter-stained with Evan's Blue and examined by light microscopy.

Transplantation of ovalbumin transgenic retinal pigment epithelium into the subretinal space

Superparamagnetic microbeads (4.5 μm, DynabeadsTM, Dynal Inc., Lake Success, NY; referred to as microbeads) were used to track injections into the SRS. The microbeads were blocked with BSA, washed, and mixed with equal numbers of cultured RPE cells from TRP-1-OVA mice and injected into the SRS of either normal B6 or OT-1 transgenic mice as previously described [22]. Microbeads were added to the cells to verify that the transplanted cells were delivered to the SRS and not the choroid, which is a more immunogenic site, in each recipient. The microbeads do not induce an inflammatory response in the SRS and are retained in the eye for long periods, if not indefinitely [22]. Eyes were removed at various intervals after injection, fixed with acetone, and embedded in paraffin. Six micron sections were cut and stained with hematoxylin and eosin (H&E) to determine if the donor RPE cells induced an inflammatory cell infiltrate and/or disorganization of the retina.

Slides were examined bright-field, phase contrast or fluorescent microscopy, as appropriate. Digital images were prepared at 200X magnification, unless otherwise noted.


Results

Characterization of the TRP-1-OVA transgenic mice

The TRP-1 promoter was chosen for production of OVA transgenic mice because it preferentially targets expression to the developing RPE during embryogenesis and expression of TRP-1 persists throughout the life of the RPE cells [14]. Various tissues from B6 TRP-1-OVA and non-transgenic B6 mice were examined for expression of OVA mRNA by RT-PCR (Figure 1). OVA mRNA was detected in the brain and eye but not the heart or kidney of B6 TRP-1-OVA mice. RNA from E.G7-OVA was used as a positive control for OVA amplification. No signal was detected in any tissues of the normal B6 mice (Figure 1). The absence of amplification was not an artifact of mRNA degradation or PCR inhibitors, as RT-PCR detected GAPDH mRNA in all samples of cells and tissues (not shown).

Freshly isolated RPE from various strains of normal and transgenic mice showed typical pigmentation and a hexagonal shape, as illustrated in Figure 2A. For tracking purposes, RPE were isolated from transgenic B6 mice expressing the GFP transgene; consequently, these RPE also exhibited typical green fluorescence (Figure 2B). RPE used for transplantation were expanded by culturing in vitro for 2 to 5 passages, but under these conditions they rapidly loose pigmentation and the typical hexagonal shape. To verify that the cells that had been cultured for two passages were epithelial cells, they were stained with an isotype control (Figure 2C) or mouse anti-human cytokeratin 8 [23] (Figure 2D), which cross reacts with mouse cytokeratin 8 because of the extremely conserved sequences of cytokeratins. Virtually all of the cultured RPE showed cytoplasmic staining with the anti-cytokeratin antibody.

To verify that RPE from B6 TRP-1-OVA mice expressed the OVA gene, mRNA isolated from cultured B6 and B6 TRP-1-OVA RPE was examined by RT-PCR. RPE from B6 TRP-1-OVA, but not B6 mice, showed expression of OVA-specific RNA (Figure 3). Amplification of GAPDH RNA from all tissues confirmed the integrity of the isolated RNA.

Immunofluorescent staining with anti-OVA antiserum was used to determine whether RPE from B6 TRP-1-OVA mice expressed the OVA protein. To verify the specificity of this antibody, it was used to stain the OVA transduced E.G7-OVA cells, giving a bright cytoplasmic staining pattern (Figure 4A), whereas it did not stain the parental EL4 cells (Figure 4B). RPE cells from B6 TRP-1-OVA mice expressed lower levels of reactivity with this antibody (Figure 4C) than seen with E.G7-OVA, whereas B6 RPE gave no detectable signal (Figure 4D). The reason for differences in staining between E.G7-OVA and the RPE from B6 TRP-1-OVA has not been identified but expression of OVA is regulated by the actin promoter in E.G7-OVA, whereas it is regulated by the TRP-1 promoter in B6 TRP-1-OVA RPE, which may be expressed at different levels. Alternatively, the RNA and protein levels may be the same but the volume of the RPE is much greater than E.G7-OVA, which could dilute the signal. In addition, expression of OVA may be low in the cultured B6 TRP-1-OVA RPE because they do not produce high levels of pigment in vitro.

Transplantation of retinal pigment epithelium into the subretinal space of the eye

The OVA protein could not be detected in frozen sections after injection of cultured B6 TRP-1-OVA RPE into the SRS of B6 mice (not shown). Thus, we set out to examine transplant rejection of cultured RPE, first using RPE from B6.GFP mice to verify that we were able to inject them into the SRS. One day after injection, the transferred cells were clearly identifiable in the space between the host RPE and the photoreceptor cells as shown by H&E staining (Figure 5A,C) and by fluorescent microscopy (Figure 5B,D). Note the presence of the beads, seen as small orange spheres, among the RPE in the subretinal space but not in the vitreous, neural retina or the choroid. The separation of the injected cells from the RPE is thought to be caused by the detachment induced by the injected solution that had not been fully resorbed at 24 h after injection.

To test whether OVA serves as a transplantation antigen, RPE cells from TRP-1-OVA mice were transplanted into the SRS of the eyes of B6 and OT-1 TCR transgenic mice. Normal retinal architecture is observed by H&E staining of eyes from B6 and OT-1 transgenic mice that were not transplanted (Figure 6). RPE from TRP-1-OVA mice were mixed with microbeads and injected into the sub-retinal space and the recipient mice euthanized one or two weeks later. Representative images show that there was no disruption of the normal architecture of the retina and no inflammatory cell infiltration in B6 recipients at either one or two weeks, although transplanted cells and microbeads were detectable (see arrows in the SRS of Figure 6). In contrast, TRP-1-OVA RPE cells induced a fulminate cellular infiltrate that was detected at two weeks, but not at one week (Figure 6). While retinal detachments were noted in all of the transplanted mice, the retinal structures of the B6 eyes appear to be normal even in the area over the collection of transplanted cells and beads. The retinal layers of the OT-1 mice, however, were very disorganized by the second week due to the vigorous inflammatory response consisting of a large proportion of lymphocytes (small round cells consisting of a large nucleus and a small rim of cytoplasm). Because of the severe infiltration seen at two weeks, it was not possible to determine if there were cell types other than lymphocytes by morphology. However, in a different experiment, the beginning stages of inflammation were observed in OT1 recipient mice at one week after transplantation (Figure 7). A low power image of most of the globe shows that RPE and microbeads were distributed over almost half of the SRS (Figure 7A). Higher magnifications illustrate that the microbeads have been taken up by the RPE (Figure 7B) but it is not clear whether these are host or donor RPE. In addition to the RPE and microbeads, small numbers of macrophages (large cells with peri-centric nuclei and abundant cytoplasm) and lymphocytes were morphologically detectable in the SRS (Figure 7C).


Discussion

Transgenic B6 mice, produced with a construct containing the OVA gene under control of the TRP-1 promoter, express OVA mRNA in the brain and eyes but not the heart or kidney. Expression in the brain and the eye is expected, given that pigmented cells are found in both tissues. By fluorescence microscopy, the OVA protein is also expressed by RPE isolated from TRP-1-OVA transgenic mice, albeit at lower levels than the OVA gene driven by the actin promoter in the transduced E.G7-OVA cell line. Whether expression of the OVA gene as a neo-self antigen induces tolerance in these transgenic mice, as reported for other exogenous antigens regulated by ocular promoters [11-13], is an interesting question that is currently under investigation.

Despite the fact that OVA and the TRP-1 proteins are cytoplasmic proteins, they serve as transplantation antigens because cytosolic proteins are digested by proteasomes and the resulting peptides are loaded onto the MHC class I proteins that are subsequently transported to the surface of the cells [24,25]. CD8+ T cells expressing TCR that bind to the complex formed by the peptides and the class I protein are activated to proliferate and differentiate into effector T cells within draining lymph nodes [26]. The resulting effector T cells immigrate from the lymph nodes to circulate through the body in search of cells expressing the peptide loaded MHC class I molecules (reviewed by Weninger et al. [27]). CD8+ T cells that bind to the peptide MHC class I complex on the cell surface deliver cytolytic molecules inducing their death [28]. In addition, CD8+ T cells that are activated by the peptide MHC class I complex may secrete cytokines that recruit and activate macrophages, which can induce apoptosis of the RPE directly or indirectly (reviewed in [29]).

RPE from TRP-1-OVA transgenic mice to induced a fulminate leukocytic infiltrate in the subretinal space of OT-1 mice but not in B6 mice. We believe that this results from the frequency of the OVA peptide specific T cells in the TCR transgenic mice, which is very high, and because OT1 T cells are known to be of very high avidity [30]. The massive infiltrate of lymphocytes and macrophages observed in the eyes of OT1 mice after transplantation with TRP-1-OVA-RPE represents a full blown immune response as observed in the livers OT1 mice injected with the OVA peptide [31] but not the small accumulation of unactivated (tolerant OT1 T cells) seen in OVA expressing tumors [32]. The failure of TRP-1-OVA to induce rejection in normal, nontrangenic B6 mice is not unexpected since other OVA expressing cells, such as E.G7-OVA tumors, do not elicit rejection in B6 mice whether injected subcutaneously [33] or into the anterior chamber of the eye [34], unless the animals are primed with an immunogenic form of OVA.

These experiments provide proof of the principal that OVA peptides expressed specifically in the RPE can serve as neotransplantation antigens. Under these circumstances, the OVA peptides are equivalent to minor histocompatibility antigens, which are known to be potent inducers of corneal graft rejection in the eye [35-37]. The observation that OVA-specific CD8+ T cells in OT1 mice mediate inflammation in the retina when transplanted with OVA expressing RPE seems to be at odds with the current dogma that allograft rejection is mediated by CD4+ T cells. However, there is mounting evidence that CD8+ T cells can mediate rejection in CD4 knockout mice [38-40] suggesting that there are both CD4 dependent and independent mechanisms for organ rejection in general and for rejection in the eye specifically. Moreover, McPherson et al. have shown that activated TCR transgenic CD8+ T cells specific for β-galactosidase can trigger autoimmune elimination of photoreceptors in mice expressing β-galactosidase in photoreceptor cells [13]. The TCR expressed by the OT1 T cells are known to be of very high avidity and purified, Rag1 deficient OT1 T cells are known to be activated by OVA bearing APC in the absence of CD4+ T cells [41]. Thus, the T cells in the OT1 TCR transgenic mice may be activated by antigens derived from the TRP-1-OVA RPE in the absence of significant numbers of CD4+ T cells, as occurs in vitro [41]. The observation that both lymphocytes and macrophages infiltrate the SRS in transplanted OT1 mice suggests that rejection could be mediated directly by killing the TRP-1-OVA RPE [28] and/or indirectly by activation of cells of the innate system [29].

Induction of inflammation by the transplantation of RPE from TRP-1-OVA mice into CD8+ OT-1 TCR transgenic T cells provides evidence that OVA can serve as a neotransplantation antigen that can be recognized by specific CD8+ effector T cells. Future studies to test the cellular requirements for rejection and prevention of rejection by induction of specific tolerance will be performed in B6 recipients that will be adoptively transferred with OT1 T cells plus or minus CD4+ T cells before transplantation of TRP-1-OVA-RPE.


Acknowledgements

We thank Dr. Hans Grossniklaus (Emory University, Atlanta, GA) for help with identifying the inflammatory cell infiltrates in the ocular sections of the transplant recipients. We thank Jing Ming Liu and Brent Barron for excellent technical assistance and Dr. Michael J. Bevan (University of Washington, Seattle, WA) for providing E.G7-OVA.

This work was supported by a grant from the Foundation for Fighting Blindness, an NEI core grant (P30EY006360), a grant from Research to Prevent Blindness, research grants (EY13459 and EY14877) from NIH, and a gift from Malcolm and Musette Powell. J.R. Wajchman is the recipient of an NRSA grant F32 EY06985 from the National Eye Institute. J.A. Kapp is the recipient of the Jules and Doris Stein Professorship in Ophthalmology awarded by Research to Prevent Blindness.


References

1. Lund RD, Ono SJ, Keegan DJ, Lawrence JM. Retinal transplantation: progress and problems in clinical application. J Leukoc Biol 2003; 74:151-60.

2. Ng TF, Klassen HJ, Hori J, Young MJ. Retinal transplantation. Chem Immunol Allergy 2007; 92:300-16.

3. Thomas BB, Aramant RB, Sadda SR, Seiler MJ. Retinal transplantation. A treatment strategy for retinal degenerative diseases. Adv Exp Med Biol 2006; 572:367-76.

4. Streilein JW, Ma N, Wenkel H, Ng TF, Zamiri P. Immunobiology and privilege of neuronal retina and pigment epithelium transplants. Vision Res 2002; 42:487-95.

5. Kohen L, Enzmann V, Faude F, Wiedemann P. Mechanisms of graft rejection in the transplantation of retinal pigment epithelial cells. Ophthalmic Res 1997; 29:298-304.

6. Zhang X, Bok D. Transplantation of retinal pigment epithelial cells and immune response in the subretinal space. Invest Ophthalmol Vis Sci 1998; 39:1021-7.

7. Vollrath D, Feng W, Duncan JL, Yasumura D, D'Cruz PM, Chappelow A, Matthes MT, Kay MA, LaVail MM. Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proc Natl Acad Sci U S A 2001; 98:12584-9.

8. Lund RD, Kwan AS, Keegan DJ, Sauve Y, Coffey PJ, Lawrence JM. Cell transplantation as a treatment for retinal disease. Prog Retin Eye Res 2001; 20:415-49.

9. Priddle H, Jones DR, Burridge PW, Patient R. Hematopoiesis from human embryonic stem cells: overcoming the immune barrier in stem cell therapies. Stem Cells 2006; 24:815-24.

10. Boyd AS, Higashi Y, Wood KJ. Transplanting stem cells: potential targets for immune attack. Modulating the immune response against embryonic stem cell transplantation. Adv Drug Deliv Rev 2005; 57:1944-69.

11. Lai JC, Fukushima A, Wawrousek EF, Lobanoff MC, Charukamnoetkanok P, Smith-Gill SJ, Vistica BP, Lee RS, Egwuagu CE, Whitcup SM, Gery I. Immunotolerance against a foreign antigen transgenically expressed in the lens. Invest Ophthalmol Vis Sci 1998; 39:2049-57.

12. Gregerson DS, Torseth JW, McPherson SW, Roberts JP, Shinohara T, Zack DJ. Retinal expression of a neo-self antigen, beta-galactosidase, is not tolerogenic and creates a target for autoimmune uveoretinitis. J Immunol 1999; 163:1073-80.

13. McPherson SW, Yang J, Chan CC, Dou C, Gregerson DS. Resting CD8 T cells recognize beta-galactosidase expressed in the immune-privileged retina and mediate autoimmune disease when activated. Immunology 2003; 110:386-96.

14. Beermann F, Hunziker A, Foletti A. Transgenic mouse models for tumors of melanocytes and retinal pigment epithelium. Pigment Cell Res 1999; 12:71-80.

15. Galibert MD, Yavuzer U, Dexter TJ, Goding CR. Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter. J Biol Chem 1999; 274:26894-900.

16. Hogquist KA, Jameson SC, Heath WR, Howard JL, Bevan MJ, Carbone FR. T cell receptor antagonist peptides induce positive selection. Cell 1994; 76:17-27.

17. Castillo BV Jr, del Cerro M, White RM, Cox C, Wyatt J, Nadiga G, del Cerro C. Efficacy of nonfetal human RPE for photoreceptor rescue: a study in dystrophic RCS rats. Exp Neurol 1997; 146:1-9.

18. Tezel TH, Del Priore LV, Kaplan HJ. Harvest and storage of adult human retinal pigment epithelial sheets. Curr Eye Res 1997; 16:802-9.

19. Sheedlo HJ, Li L, Turner JE. Effects of RPE age and culture conditions on support of photoreceptor cell survival in transplanted RCS dystrophic rats. Exp Eye Res 1993; 57:753-61.

20. Moore MW, Carbone FR, Bevan MJ. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell 1988; 54:777-85.

21. Lowings P, Yavuzer U, Goding CR. Positive and negative elements regulate a melanocyte-specific promoter. Mol Cell Biol 1992; 12:3653-62.

22. Wen J, McKenna KC, Barron BC, Langston HP, Kapp JA. Use of superparamagnetic microbeads in tracking subretinal injections. Mol Vis 2005; 11:256-62 <http://www.molvis.org/molvis/v11/a30/>.

23. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 1982; 31:11-24.

24. Rock KL, York IA, Saric T, Goldberg AL. Protein degradation and the generation of MHC class I-presented peptides. Adv Immunol 2002; 80:1-70.

25. Osterloh P, Linkemann K, Tenzer S, Rammensee HG, Radsak MP, Busch DH, Schild H. Proteasomes shape the repertoire of T cells participating in antigen-specific immune responses. Proc Natl Acad Sci U S A 2006; 103:5042-7.

26. Lakkis FG, Arakelov A, Konieczny BT, Inoue Y. Immunologic 'ignorance' of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat Med 2000; 6:686-8.

27. Weninger W, Manjunath N, von Andrian UH. Migration and differentiation of CD8+ T cells. Immunol Rev 2002; 186:221-33.

28. Gregerson DS, Lew KL, McPherson SW, Heuss ND, Ferrington DA. RPE cells resist bystander killing by CTLs, but are highly susceptible to antigen-dependent CTL killing. Invest Ophthalmol Vis Sci 2006; 47:5385-94.

29. Wyburn KR, Jose MD, Wu H, Atkins RC, Chadban SJ. The role of macrophages in allograft rejection. Transplantation 2005; 80:1641-7.

30. Jameson SC, Bevan MJ. T cell receptor antagonists and partial agonists. Immunity 1995; 2:1-11.

31. Kennedy NJ, Russell JQ, Michail N, Budd RC. Liver damage by infiltrating CD8+ T cells is Fas dependent. J Immunol 2001; 167:6654-62.

32. Shrikant P, Mescher MF. Control of syngeneic tumor growth by activation of CD8+ T cells: efficacy is limited by migration away from the site and induction of nonresponsiveness. J Immunol 1999; 162:2858-66.

33. Ke Y, Kapp LM, Kapp JA. Inhibition of tumor rejection by gammadelta T cells and IL-10. Cell Immunol 2003; 221:107-14.

34. McKenna KC, Kapp JA. Accumulation of immunosuppressive CD11b+ myeloid cells correlates with the failure to prevent tumor growth in the anterior chamber of the eye. J Immunol 2006; 177:1599-608. Erratum in: J Immunol. 2006 Oct 15; 177(8):5748.

35. Osawa H, Streilein JW. MHC class I and II antigens as targets of rejection in penetrating keratoplasty in low- and high-risk mouse eyes. Cornea 2005; 24:312-8.

36. Sano Y, Ksander BR, Streilein JW. Murine orthotopic corneal transplantation in high-risk eyes. Rejection is dictated primarily by weak rather than strong alloantigens. Invest Ophthalmol Vis Sci 1997; 38:1130-8.

37. Sano Y, Streilein JW, Ksander BR. Detection of minor alloantigen-specific cytotoxic T cells after rejection of murine orthotopic corneal allografts: evidence that graft antigens are recognized exclusively via the "indirect pathway". Transplantation 1999; 68:963-70.

38. Niederkorn JY, Stevens C, Mellon J, Mayhew E. CD4+ T-cell-independent rejection of corneal allografts. Transplantation 2006; 81:1171-8.

39. Zhai Y, Meng L, Busuttil RW, Sayegh MH, Kupiec-Weglinski JW. Activation of alloreactive CD8+ T cells operates via CD4-dependent and CD4-independent mechanisms and is CD154 blockade sensitive. J Immunol 2003; 170:3024-8.

40. Zhan Y, Corbett AJ, Brady JL, Sutherland RM, Lew AM. CD4 help-independent induction of cytotoxic CD8 cells to allogeneic P815 tumor cells is absolutely dependent on costimulation. J Immunol 2000; 165:3612-9.

41. Kapp JA, Honjo K, Kapp LM, Xu X, Cozier A, Bucy RP. TCR transgenic CD8+ T cells activated in the presence of TGFbeta express FoxP3 and mediate linked suppression of primary immune responses and cardiac allograft rejection. Int Immunol 2006; 18:1549-62.


Wajchman, Mol Vis 2007; 13:1902-1911 <http://www.molvis.org/molvis/v13/a214/>
©2007 Molecular Vision <http://www.molvis.org/molvis/>
ISSN 1090-0535