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Subretinal Space and Vitreous Cavity as Immunologically Privileged Sites for Retinal Allografts Luke Qi Jiang, Marianela Jorquera, and J. Wayne Streilein Purpose. Because immune rejection is likely to be a major barrier to successful retinal transplantation, it is important to determine whether immune privilege for allogeneic retinal grafts is a feature of the subretinal space and vitreous cavity. Methods. Newborn neural retinas of C57BL/6 mice were implanted into the subretinal space, vitreous cavity, or subconjunctival space of eyes of adult BALB/c (disparate from C57BL/6 at major and minor histocompatibility loci). At postimplantation day 12, the recipients were evaluated for donor-specific delayed hypersensitivity and examined clinically and histologically for evidence of rejection. Results. Newborn neural retina allografts in the subconjunctival space were destroyed by postimplantation day 12 and these recipients displayed intense donor-specific delayed hypersensitivity. By contrast, grafts in the subretinal space and vitreous cavity at postimplantation day 12 were found to be well differentiated and with no evidence of inflammation; these recipients failed to display donor-specific delayed hypersensitivity. Moreover, their spleens contained regulatory T cells that suppressed donor-specific delayed hypersensitivity in naive syngeneic recipients. Conclusions. Allogeneic newborn neural retinal grafts implanted in the subretinal space and vitreous cavity experience immune privilege and induce deviant immune responses resembling anterior chamber associated immune deviation. Invest Ophthalmol Vis Sci. 1993; 34:33473354. Urthotopic retinal transplantation holds promise as a means of restoring vision to eyes blinded by destructive retinal disease. Experimental studies have demonstrated that neural retina or retinal pigment epithelium implanted into the subretinal space can display function to a limited extent, and can survive for a variable period of time.1 Clinical use of retinal grafts, however, must be associated with full function and longterm graft survival. To achieve these objectives, researchers must overcome two major obstacles. The aggregate limits of mammalian neural regeneration, plasticity, and maintenance constitute the first obstacle. These limits must be stretched if retinal grafts are to be fully functional. Even if grafts are able to survive, immune rejection represents the second obstacle, and, it is this issue that is addressed here. From the Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida. This ivork was supported by USPHS Grant EY 09595 and a grant from the National Relinilis Pigmentosa Foundation. Submitted for publication March 12, 1993; accepted April 30, 1993. Property interest category: N. Reprint requests: Luke Qi Jiang, Schepens Eye Research Institute, Harvard School of Medicine, 20 Stamford Street, Boston, MA 02114. Two factors appear to influence the potential of immune rejection of retinal grafts placed in the eye: the inherent immunogenicity of the retinal grafts, and the anatomic site of implantation. Although histocompatibility antigens (known to be potent inducers of systemic immunity) are normally expressed sparsely in the neural retina and other neural tissue, the expression of such molecules is upregulated after transplantation.2 We have demonstrated in mice that neural retinal grafts are immunogenic, and that the immunity induced by neural retinal cells is directed at both transplantation antigens and retinal restricted autoantigens.3 However, the type of immunity elicited depends on the sites at which the retina is implanted. Implantation of neural retinal grafts into the subconjunctival space (SCon) has been found to induce antigen-specific delayed hypersensitivity (DH), whereas implantation of similar retinal grafts into the anterior chamber (AC) induce a deviant form of immunity (anterior chamber associated immune deviation, ACAID).45 In this deviant response, antigen-specific suppressor T cells are generated and DH reactivity is selectively impaired. Thus, grafts placed in the AC are protected by ACAID, that is, they enjoy immune privilege. Investigative Ophthalmology & Visual Science, November 1993, Vol. 34, No. 12 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933171/ on 05/06/2017 3347 3348 Investigative Ophthalmology & Visual Science, November 1993, Vol. 34, No. 12 From a therapeutic point of view, the subretinal space (SR) and vitreous cavity (VC)—both of which are adjacent to the retina—are the most likely potential sites for therapeutic retinal grafts. Therefore, determining whether these posterior compartments of the eye are also immunologically privileged sites is essential. In this study, we implanted allogeneic newborn neural retinal (NNR) tissue into the eyes of adult mice, either into the SR or VC. We subsequently conducted clinical, histologic, and immunologic examinations to determine the characteristics of the systemic immune responses evoked by these implants. MATERIALS AND METHODS Animals Donor retinal tissue was obtained from newborn C57BL/6 mice (aged < 24 hours). Adult male BALB/c mice (aged 7 to 12 weeks) served as recipients. For retinal transplantation into the VC, we repeated two experiments switching the donor and recipient strains. All experimental mice were obtained from our breeding colony at the University of Miami School of Medicine, Miami, Florida. Mice were maintained in a common room of the vivarium where an overhead fluorescent light provided 12-hour cycles of light and dark. Inoculations, clinical examinations, and enucleations of grafted eyes were performed under anesthesia induced by intramuscular injections of ketamine (Ketalar, Parke Davis, Shawnee, KS) 0.075 mg/g body weight, and xylazine (Rompun, Haver-Lockhart, Morris Plains, NJ), 0.006 mg/g body weight. All experimental procedures conformed to the ARVO Resolution on the Use of Animals in Research. Preparation of Donor Neural Retina Donor C57BL/6 newborn mice were decapitated. Their eyes were immediately enucleated and placed in ice-cold calcium-magnesium-free Hanks balanced salt solution (HBSS). Each eye was cut open along the edge of the cornea using microsurgical scissors. After the lens and iris were removed, the neural retina was gently separated from the eyecup and transferred to a new petri dish containing cold calcium-magnesiumfree HBSS. After two changes to fresh medium, the entire neural retina was halved and each half was used for transplantation. Intraocular or Subconjunctival Implantation of Newborn Neural Retina Tissue Recipient mice received general anesthesia. For implantation of retina tissue into the SR or VC, the eyelids were kept open and the eyeballs were held steady with forceps. A 0.3-mm penetrating wound was made at the posterior portion of the wall of the eye using a microsurgical knife with a 15° angle (Edward Week and Company, Inc., Research Triangle Park, NC). For retinal implantations into the SCon, a penetrating wound was made in the fornix portion of the conjunctiva. The retinal tissue was then drawn into a glass needle made from a glass bore of a 1O-jul micropipetter (diameter of 200 /mi). The retinal tissue, along with about 1 /il of HBSS, was slowly injected via the wound into the SR, VC, or SCon of the eye. Clinical Examination The mice were anesthetized and their eyes examined with a dissecting microscope on designated days. The pupils were dilated with 0.5% Mydriacyl (Alcon Inc., Humacao, PR) and 2.5% phenylephrine hydrochloride and the fundus was visualized through a contact lens so the posterior segments of the eyes could be viewed. Histologic Examination Sectioning Procedure. The graft was localized by clinical examinations and sections were cut through the portion of the eye containing the graft. For hematoxylin and eosin staining, the eyes were fixed with 10% buffered formalin embedded with paraffin and cut 5 /xm thick. For immunohistochemical staining, the eyes were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffer, pH 7.2. After fixation for 24 hours, the eyes were immersed in 30% phosphate-buffered sucrose and embedded with Tissue-Tek II O.C.T. compound (Lab-Tek Products, Elkhart, IN). Cryostat sections of 7 ixm were cut transversely, mounted on gelatin-coated slides, and stored at 4°C. Immunocytochemical Staining for S-Antigen. Cryostat sections were washed in phosphate-buffered saline (PBS) pH 7.2 for 1 minute for immunofluorescent staining, then incubated with primary antibody for 30 minutes. Guinea pig anti-S antigen antiserum (donated by Dr. Carolyn Kalsow, University of Rochester, Rochester, NY), which cross reacts with mouse S-antigen, was diluted with PBS at 1:10 and used as the primary antibody. For negative controls, sections were incubated with PBS only. The sections were washed in PBS and incubated for 30 minutes with the secondary antibody, fluorescein-conjugated F(ab')2 fragments of goat anti-guinea pig immunoglobulin G (Rockland, Gilbertsville, PA) diluted with PBS at 1:50. After three washes in PBS, sections were mounted with polyvinyl alcohol in PBS and glycerol, and examined under an Olympus research microscope (Olympus Corp., Lake Success, NY) using epifluorescence and a Blue Filter block (excitation light wavelength 380 to 490 nm). Photographs Photographs of the fundus were taken with a 35-mm camera system attached to an Olympus dissection mi- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933171/ on 05/06/2017 3349 Immune Privilege and Retinal Allografts croscope (using Kodak Plus-X pan 125 black-andwhite print film; Eastman Kodak Co., Rochester, NY). Micrographs of sections stained with hematoxylin and eosin technique and immunocytochemical technique were taken with a 35-mm camera system attached to an Olympus research microscope (using Kodak Plus-X pan 125 black-and-white print film). For immunofluorescent staining, micrographs were taken with a 35mm camera system attached to an Olympus research microscope with epifluorescence and a Blue Filter block (excitation light wavelength 380 to 490 nm). Kodak Tri-X pan 400 black-and-white printfilmwas used. DH Response to Retinal Grafts DH was measured based as ear swelling, as previously described.6 Briefly, half of the neural retina of newborn donor mice was implanted into the SR of recipient mice. For positive controls, mice received similar retinal grafts into the SCon. Normal BALB/c mice without transplants of neural retina served as negative controls. On day 12 after transplantation, both ear pinnae of each mouse were challenged and measured for DH responses. A Mitutoyo engineer's micrometer (Mitutoyo Corp., Tokyo, Japan) was used to measure the thickness of both ears immediately before challenge. For challenge, 2 X 106 (BALB/c X C57BL/6) Fl spleen cells (irradiated) were suspended in 10 ^1 of HBSS and injected into the subcutaneous tissue of the left ear pinnae. The right ear served as an untreated control. The difference in measured ear thickness after 24 hours was used as a measurement of DH intensity. Results were expressed as specific ear swelling = (24 hour measurement — 0 hour measurement) experimental ear — (24 hour measurement — 0 hour measurement) negative control ear X 10~3 mm. A twotailed Student's t test was performed on the data presented and significance was assumed to exist if P<0.05. Adoptive Transfer of Capacity to Induce Suppression of Alloantigen-Specific DH Spleens from BALB/c mice bearing C57BL/6 retinal grafts in the SR were collected aseptically. Single cell suspensions were prepared by pressing whole spleens through stainless steel screens (60-mesh) as described elsewhere.6 Spleen cells were then washed and resuspended in HBSS. Each naive BALB/c recipient received 5 X 107 spleen cells in 100 v\ of HBSS via the tail vein. For positive control, a similar number of naive BALB/c spleen cells was infused intravenously into naive BALB/c recipients. Negative controls received no infusion. Within 2 hours, all experimental mice received implants in SCon of neural retina grafts from immature C57BL/6 mice (aged 8 to 14 days). Twelve days later, DH reactivity, as described earlier, was assayed by ear challenge. RESULTS Clinical Appearance and Course of Retinal Graft Implanted into the Subretinal Space or the Vitreous Cavity Entire neural retinas isolated from the eyes of newborn C57BL/6 mice were halved and each half was implanted into the SR or the VC of adult BALB/c mice. As the medium containing the retinal grafts was injected into the SR, detachment of the retina could be observed as a white reflection through the dilated pupil. On examination of the fundus with a contact lens, the detached retina with a retinal implant underneath appeared as a white drapelike projection, smooth surfaced with a well-defined edge. Within 3 days, the projection gradually flattened (possibly because of reabsorption of the medium), and the retinal graft became recognizable through the translucent ret- Graft- Retina] Vessel -V B FIGURE 1. (A) Fundus appearance of the eye of adult BALB/ c mouse 12 days after receiving allogeneic C57BL/6 newborn neural retina graft into SR. The graft (arrow) appears white with no sharp limit. The arrowhead indicates the vessels of the host retina, which is superior to the graft. (B) A schematic diagram showing the relationship between the vessel and the SR graft. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933171/ on 05/06/2017 Investigative Ophthalmology & Visual Science, November 1993, Vol. 34, No. 12 3350 ina. Subretinal grafts had a white color with an irregular shape and no clearly denned edge. Since the portion of the host retina that covered the graft was virtually detached, a climbing pattern of vessels was often observed at the edge of the graft (Fig. 1, A and B). Retinal folds usually surrounded the grafts. No hemorrhage or inflammatory exudate was in the AC, VC, SR or the retina itself during the time course of these experiments. When retinal tissues were implanted into the VC, the implant could be observed directly under the microscope. Through the dilated pupil, the white reflection of the retinal graft appeared immediately behind the lens. Within a few days, the cloudlike graft tissue gradually aggregated and formed a white mass with an irregular shape and a clearly defined edge. The graft typically attached to the retinal surface superior to the retinal vessels (Fig. 2, A and B). Because no neovessels FIGURE 3. (A) Histologic appearance of an allogeneic C57BL/6 newborn neural retinal graft implanted into SR of the eye of an adult BALB/c mouse. The graft (G) consists mainly of photoreceptor cells, which form rosettes (Rs), and it is adjacent to the host retina (H). (B) Inimunofluorescent staining for S antigen reveals a majority of cells in the SR graft to be positive. Photoreceptor cells (Ph) of the host retina (H) are positive with S antigen. Graft Retinal Vessel— B 2. (A) Fundus appearance of the eye of an adult C57BL/6 mouse 12 days after receiving allogeneic BALB/c newborn neural retina graft into VC. The graft (arrow) has a white translucent appearance with the edge clearly denned. The graft is attached to the surface of the retina and is superior to the retinal vessels (arrowhead). (B) A schematic diagram showing the relationship between the vessels and the VC graft. FIGURE developed on the VC surface of the graft, the relationship of the graft to the retinal vessels (i.e., beneath or superior; Figs. IB, 2B) became an important clinical sign for differentiating VC grafts from SR grafts, particularly the flat-type graft. No inflammatory exudate or hemorrhage was detected in media of the eye, retinal grafts, or host retina. On deep illumination, grafts appeared translucent and they retained this appearance for the remainder of the experiment. Histologic Examination SR Retinal Crafts. The eyes of five animals that received retinal grafts into the SR 12 days earlier were enucleated and processed for hematoxylin and eosin staining and for inimunofluorescent staining for S an- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933171/ on 05/06/2017 3351 Immune Privilege and Retinal Allografts tigen. Examination of the hematoxylin and eosin sections revealed that the grafts were well developed and differentiated, and located between the host neural retina and retinal pigment epithelium. Retinal grafts contained a dominant population of photoreceptor cells that formed rosettes (Fig. 3A). On immunofluorescent staining, the photoreceptor cells associated with rosettes showed S antigen-positive staining (Fig. 3B). The rosettes, which resembled the outer nuclear layer of the retina, were surrounded by cells of the inner nuclear layer. Ganglion cells were not usually recognizable, probably because they were scant and did not form a discrete layer. No inflammatory infiltration was detected in either the retinal graft or in the host retina; however, a few macrophages occasionally appeared around the graft margins. VC Retinal Grafts. Retinal grafts were implanted into the VC offiveanimals and their eyes were enucleated 12 days later and processed for histologic examination in a similar fashion to that described earlier for SR grafts. Examination of both hematoxylin and eosin and immunofluorescent sections revealed results resembling those found in the SR grafts (Fig. 4). By clinical and histologic criteria, our evidence indicates that grafts implanted into either SR or VC survive and proceed to differentiate toward the morphology associated with the mature retina. Moreover, this process occurs without evidence of immunologic rejection. In contrast, retinal grafts implanted into the SCon soon lost their identity, and histologic examination revealed that the SCon implants contained an obvious inflammatory infiltrate, but little unidentifiable retinal structures (results published previously).7 24 Hours After Challenge 80 _ 0 24 Hours After Challenge FIGURE 5. (A) SR graft; (B) VC graft. Capacity of allogeneic retinal graft to induce DH in recipient mice. Allogeneic NNR was implanted into SR or VC of recipient mice on day 0; ears were challenged on day 12 and ear swelling was assessed 24 hours later. Positive control mice received allogeneic NNR in SCon. Negative controls received ear challenge only. Bar represents mean ±SEM. Responses of the SCon group are significantly higher than those of the SR, VC, or negative control groups. (P < 0.01) Aspects of Systemic Immunity Induced by Allogeneic Neural Retinal Grafts Implanted into the SR and VC FIGURE 4. Histologic appearance of an allogeneic C57BL/6 newborn neural retinal graft implanted into the VC of an adult BALB/C mouse eye 12 days previously. The graft (G) contains mostly photoreceptor cells which form rosettes (Rs). Host retina (H). Our previous studies indicated that allogeneic NNR implanted into the AC induced a deviant form of immunity (ACATD) in recipient mice, and that ACAID played a role in protecting these grafts from rejection.7 Because the clinical and histologic studies of SR or VC retinal grafts yielded findings similar to those of AC grafts, we next examined the respective fates of SR, VC, and SCon retinal grafts and their impact on systemic immunity. Impaired DH Induced by Allogeneic SR or VC NNR Grafts. Allogeneic C57BL/6 NNR were implanted into the SR and VC of adult BALB/c mice. Five mice were used as recipients for each experimental group and each recipient received one half a donor Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933171/ on 05/06/2017 3352 Investigative Ophthalmology 8c Visual Science, November 1993, Vol. 34, No. 12 retina. Mice receiving similar NNR grafts into the SCon served as positive controls, while negative control mice received a sham operation or no treatment at all. Twelve days later, the ears of all mice received subcutaneous injections of irradiated (1500 R) suspended spleen cells (106 cells/10 /x\) isolated from adult (BALB/c X C57BL/6)F1 mice. Ear swelling was measured by micrometer 24 and 48 hours later. Figure 5 shows the results of a representative experiment. Vigorous DH reactivity directed at the alloantigens of C57BL/6 donors was generated in BALB/c recipients of C57BL/6 NNR grafts in SCon, whereas recipients of allogeneic NNR grafts in the SR and VC displayed no significant alloantigen-specific DH reactivity. These results indicate that allogeneic NNR grafts implanted into intraocular compartments (SR or VC) as opposed to an extraocular site (SCon) evoked different forms of systemic immunity. In contrast to conventional DH reactivity, which was induced by SCon NNR grafts, impaired DH was generated in recipients of SR or VC NNR grafts. The experiment was repeated twice with similar results. In addition, two additional experiments were performed in which the donor and recipient strains were switched. The pattern of immune responses was unchanged in these experiments. Generation of Suppressor Spleen Cells in Recipients of SR or VC Grafts. We demonstrated previously that failure to develop conventional DH in recipients of allogeneic NNR grafts in the AC is associated with the generation of antigen-specific suppressor splenic cells, a key feature in expression of ACAID.4'5 To determine whether C57BL/6-specific suppressor splenic cells were induced in the BALB/c recipients by grafts in the SR or VC, we conducted adoptive transfer assays. Spleens were removed from five adult BALB/c mice that received C57BL/6 NNR grafts into the SR or VC 12 days previously. As positive controls, spleens were harvested from five naive adult BALB/c mice. Two groups of spleens were minced into single cell suspensions and used for intravenous infusion into naive BALB/c mice (5 per group) at a dose of 50 X 106 per mouse. Within 2 hours, each recipient received an NNR graft from C57BL/6 mice in the SCon space. Twelve days later, the ears of all mice were challenged by injection of irradiated spleen cells of (BALB/c X C57BL/6)F1 mice. Ear swelling was measured 24 and 48 hours after challenge. Figure 6 gives the results of representative experiments. Naive BALB/c mice that received spleen cells from donors bearing C57BL/6 NNR grafts in the SR or VC failed to acquire alloantigen-specific DH reactivity. However, BALB/c mice of the control group, which received intravenous infusions of naive spleen cells, developed a strong alloantigen-specific DH after receiving a C57BL/6 NNR graft in the SCon space. Thus, the DH impair- •Control Test -Control 24 Hours After Challenge 80 n +Control Test 0 -Control 24 Hours After Challenge FIGURE 6. (A) SR graft; (B) VC graft. Adoptive transfer of impaired DH reactivity induced by allogeneic NNR in recipient mice. For test groups, naive mice received spleen cells (50 X 106) intravenously from mice bearing SR or VC allogeneic NNR grafts. As positive control, a similar number of normal spleen cells were infused intravenously into naive syngeneic recipients. One hour later recipients received implants of allogeneic NNR grafts in SCon. Twelve days later their ears were challenged; ear swelling responses were measured 24 hours later. Negative controls were as described in Figure 5. Responses of positive control group are significantly greater than those of either test group or negative control groups (p < 0.01). ment displayed in BALB/c mice bearing C57BL/6 NNR grafts in SR or VC was associated with generation of alloantigen-specific suppressor splenic T cells, consistent with the existence of ACAID. DISCUSSION During the past two decades, understanding of immune privilege in the eye and central nervous system has advanced greatly. It has been learned that immune privilege is a dynamic physiological process achieved through active downregulation of systemic cell-me- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933171/ on 05/06/2017 Immune Privilege and Retinal Allografts diated immunity.45 For example, after antigens such as allogeneic tumor cells,8 soluble antigens,9 or neural retinal cells4 are injected into the AC of the eye, recipients display a deviant form of antigen- specific systemic immunity (e.g., AC AID). This phenomenon has also been described in response to antigens injected into the VC.8'10 However, the nature of immune privilege in the SR, an important compartment of the eye related to retinal transplantation and retinal disease, has not been determined previously. Our data indicate that allogeneic NNR grafts implanted into the SR and VC induce an antigen-specific deficit in DH associated with suppressor splenic T cells, characteristic features of ACAID. Moreover, NNR grafts implanted in the SR or VC developed and differentiated into recognizable retinal structures within 12 days of engraftment. In contrast, NNR grafts in the SCon induced conventional DH immunity and were rejected by postimplantation day 12. These results imply that immune privilege is extended to NNR grafts implanted into the SR and VC, and that ACAID plays a protective role in the success of these retinal grafts. Immune privilege in intraocular compartments can be considered to be the consequence of an interplay between the immune system and the intraocular microenvironment. It is known that under physiological conditions, the AC possesses an immunosuppressive microenvironment.11 This microenvironment, in turn, plays a regulatory role in modulating systemic immunity directed at antigens introduced into the AC.12 On one hand, the suppressive microenvironment sustained in the AC inhibits the local expression of preexisting systemic immunity. On the other hand, the intraocular microenvironment participates in modifying the primary immune responses to ocular antigen. The AC constitutively contains suppressive molecules such as transforming growth factor /3, and other as-yet-unknown cytokines.7 Although it is not clear which immunosuppressive molecules are represented in the SR or VC, this study implies that both the SR and VC also contain immunosuppressive microenvironments that are sustained for at least 12 days after implantation of NNR grafts. Because both the SR and VC are adjacent to the retina, it would be reasonable to suggest that retinal cells may significantly contribute to the immune suppressive microenvironment. In support of this idea are the following: production of transforming growth factor /5 by astrocytes is upregulated under pathological conditions,12 retinal pigment epithelium is an intraocular producer of transforming growth factor /3,13 and Muller's cells (glia) of the retina suppress T cell proliferation by a direct contact mechanism.14 In addition, the endothelium of retinal vessels, Bruch's membrane, and the pigment epithelium to- 3353 gether form the so-called ocular-blood barrier. It is interesting that the SR, although only a potential space, has features of an immunologically privileged site even after retinal detachment and the disruption of the retinal-blood barrier by implantation. It would appear that the SR's immune privilege is able to withstand passive disruption of the ocular-blood barrier, implying that local factors may actively downregulate systemic immunity. Because neural retinal grafts express both transplantation antigens and retinal autoantigens,3 this study's results do not exclude the possibility that NNR grafts in the SR may induce ACAID directed at retinarestricted antigens. We have previously reported that both allogeneic and syngeneic NNR grafts implanted into the AC or VC induced ACAID directed at retinarestricted autoantigens.10 It would be interesting to determine whether retinal autoantigen-specific ACAID can be induced by syngeneic or allogeneic NNR grafts, or by soluble retinal autoantigens (such as S antigen and interphotoreceptor retinoid binding protein) injected into the SR. Such information will be vital in determining the potential mechanisms involved in retinal graft rejection. It will also provide insight into the immunopathogenesis of autoimmune retinitis. Retinal autoantigens, which are expressed on retinal grafts, can evoke conventional DH reactivity when the grafts were implanted in the immunologically nonprivileged SCon space. Such DH reactivity presents a potential risk both to retinal grafts and to the host retina.3 Therefore, knowing whether the SR is an immunologically privileged site for both alloantigens or autoantigens, is essential for predicting the fate of retinal grafts in this space. The SR is surrounded by retinal pigment epithelium and photoreceptor cells, both of which constitutively express immunogenic autoantigens.1516 It therefore is reasonable to speculate that these autoantigens may be released into the SR under pathological (perhaps even physiological) conditions and that ACAID may constantly exist under such conditions. Thus, the immunologic features of the normal SR (e.g., immune suppressive or nonsuppressive) may play a critical role in preventing autoimmune retinitis. Both the SR and VC are immunologically privileged sites for NNR grafts, implying that the posterior compartments are favorable sites for retinal grafts. However, the immune privilege induced by NNR grafts in the SR or VC may not be absolute or permanent. It must be noted that allogeneic P815 tumor cells implanted into the AC of C57BL/6 mice induced only transient ACAID.17 We have preliminary results that reveal that allogeneic retinal grafts placed in the AC are eventually destroyed,18 suggesting that conventional immunity can emerge and overcome ACAID. A Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933171/ on 05/06/2017 3354 Investigative Ophthalmology & Visual Science, November 1993, Vol. 34, No. 12 study is underway to determine whether long-standing retinal grafts in the SR and VC are similarly affected. Key Words immune privilege, ACAID, allogeneic retinal graft, subretinal space, vitreous cavity 10. Acknowledgments The authors thank Ms. Debra Bunch Ghosh for her editorial assistance and Ms. Barbara French for photographic assistance. 11. References 12. 1. LaVail MM, Li L, Turner JE, Yasumura, D. Retinal pigment epithelial cell transplantation in RCS rats: normal metabolism in rescued photoreceptors. Exp Eye Res. 1992;55:555-562. 2. Rao K, Lund RD, Kunz HW, Gill TJ III. The role of MHC and non-MHC antigens in the rejection ofintracerebral allogeneic neural grafts. Transplantation. 1989;48:1018-1021. 3. Jiang LQ, Streilein JW. Immune responses elicited by transplantation and tissue-restricted antigens expressed on retinal tissues implanted subconjunctivally. Transplantation. 1991;529:513-513. 4. Streilein JW. Anterior chamber associated immune deviation: the privilege of immunity in the eye. Surv Ophthalmol. 1990; 35:67-73. 5. Niederkorn JY. Immune privilege and immune regulation in the eye. Adv Immunol. 1990;48:191-226. 6. Streilein JW, Niederkorn JY, Shadduck JA. Systemic immune unresponsiveness induced in adult mice by anterior chamber presentation of minor histocompatibility antigens. / Exp Med. 1980; 152:1121-1125. 7. Streilein JW, Wilbanks GA, Cousins SW. Immunoregulatory mechanisms of the eye. J Neuroimmunol. 1992; 39:185-200. 8. Jiang LQ, Streilein JW. Immune privilege extended to allogenic tumor cells in the vitreous cavity. Invest Ophthalmol VisSci. 1991;32:224-228. 9. Hara Y, Caspi RR, Wiggert B, Chan C-C, Wilbanks 13. 14. 15. 16. 17. 18. GA, Streilein JW. Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber associated immune deviation with interphotoreceptor retinoid binding protein. J Immunol. 1992;148:1685-1692. Jiang LQ, Streilein JW. Immunity and immune privilege elicited by autoantigens expressed on syngeneic neonatal neural retinal grafts. Curr Eye Res. 1992; 11:697-709. Cousins SW, McCabe MM, Danielpour D, Streilein JW. Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor. Invest Ophthalmol VisSci. 1991;32:2201-2211. Wahl SM, Allen JB, McCartney-Francies N, et al. Macrophage- and astrocyte-derived transforming growth factor j8 as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome. J Exp Med. 1991; 173:981-991. Tanihara H, Yoshida M, Matsumoto M, Yoshimura N. Identification of transforming growth factor-/? expressed in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1993; 34:413-419. Caspi RR, Roberge FG, Nussenblatt RB. Organ-resident, non-lymphoid cells suppress proliferation of autoimmune T lymphocytes. Science. 1987; 237:10291032. Donoso LA, Marryman CF, Edelberg KE, Naids R, Kalsow C. S-antigen in the developing retina and pineal gland. A monoclonal antibody study. Invest Ophthalmol VisSci. 1985;26:561-567. Broekhuyse RM, Kuhlmann ED, Winkens HJ. Experimental autoimmune anterior uveitis (EAAU). II. Dose-dependent induction and adoptive transfer using a melanin-bound antigen of the retinal pigment epithelium. Exp Eye Res. 1992;55:401-411. Ksander BR, Bando Y, Acevedo J, Streilein JW. The infiltration and accumulation of precursor cytotoxic T cells increases with time in progressively growing ocular tumors. Cancer Res. 1991;51:3153-3158. Jiang LQ, Streilein JW. Survival of intraocular neural retina grafts is influenced by microenvironmental factors as well as immunogenetic disparity. Soc Neurosci Abs. 1991;17:1137. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933171/ on 05/06/2017