Report. Immune Privilege Extended to Allogeneic Tumor Cells in the Vitreous Covity

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1 Investigative Ophthalmology & Visual Science, Vol. 32, No. 1, January 1991 Copyright Association for Research in Vision and Ophthalmology Report Immune Privilege Extended to Allogeneic Tumor Cells in the Vitreous Covity Luke Qi Jiang and J. Wayne Srreilein Inoculation of alloantigenic P815 tumor cells into the vitreous cavity of eyes of BALB/c mice resulted in the intraocular development of progressively growing tumors. We observed by clinical and histologic examination that the tumors acquired a blood supply, lacked significant necrosis or degeneration, and gradually penetrated the globe into the orbit. Mice bearing these intraocular tumors did not develop tumor-specific delayed hypersensitivity (DH), and their spleens contained lymphocytes capable of suppressing tumor-specific DH when transferred adoptively into naive, syngeneic recipients. The authors conclude that the vitreous cavity (VC) is an immunologically privileged site for histoincompatible tumor cells and that the privilege is mediated by active suppression of DH, similar to anterior chamber (AC)-associated, immune deviation. Invest Ophthalmol Vis Sci 32: ,1991 The anterior chamber (AC) of the eye functions as an immunologically privileged site, 1 and antigenic material placed in the AC elicits anterior chamber associated immune deviation (ACAID). ACAID is a unique and selective, antigen-specific immune deficiency characterized by impaired delayed hypersensitivity (DH), T-cells that suppress delayed hypersensitivity, and inability to produce complement-fixing antibodies.' The vitreous cavity (VC) and the AC are similar in that both sites lack significant lymphatic drainage pathways, possess a blood:tissue barrier, and are surrounded by tissues deficient in antigen-presenting cells. There is no agreement about the existence of immune privilege in the VC. 2 " 4 Because our long-term goal is to develop strategies for placing functioning grafts of retinal tissues into the posterior compartment of the eye, and because the presence of immune privilege in the VC could be beneficial for the survival of allogeneic retinal tissues, we conducted experiments designed to confirm, or refute, the immune-privileged status of the VC of eyes of mice. Materials and Methods. Animals: Adult male BALB/c mice, aged 9-12 weeks, were obtained from the animal facilities at the University of Miami School of Medicine. For inoculations, clinical examinations, and enucleation of the eye, mice were anesthetized with intramuscular injections of ketamine (Ketalar, Parke Davis, Shawnee, KS) mg/g body weight, and xylazine (Rompun, Haver-Lockhart, Morris Plains, NJ), mg/g body weight. Tumor Cells and Injections: P815 mastocytoma cells (DBA/2 origin) were grown as previously described. 5 For VC, AC, and subconjunctival (SC) injections, an 0.3-mm penetrating wound was made in the posterior portion of the wall of the eye, in the fornix of the conjunctiva, and in the peripheral portion of the cornea, respectively, with a microsurgical knife of 15 angle (Edward Week and Co., Inc., Research Triangle Park, NC). From a glass micropipette connected to a 20-/il syringe, a 3 /A volume containing 2 X 10 5 P815 cells was slowly injected into the wound. Ocular Examinations: The anterior segment of eyes was examined clinically with a Topcon slitlamp (Tokyo Optical Co., Ltd., Tokyo, Japan), and the posterior segment was examined with a dissecting microscope, through a contact lens. For histologic evaluation, tumor-containing eyes were enucleated, immersion-fixed with 10% phosphate-buffered formalin for 24 hr, immersed in 30% phosphate-buffered sucrose, embedded with Tissue-Tek IIOCT Compound (Miles, Inc., Elkhart, IN); 10 /xm cryostat sections were cut and stained with H&E. Assay for DH and Suppression: Ear swelling was measured as described previously 5 : 5 X 10 5 irradiated (20,000 R) P815 cells lo/ul were injected into the ear pinnae and were measured immediately before and 24 hr later with an engineers' micrometer. Results were expressed as specific ear swelling = [(24 hr - hr) measurement of experimental ear (24 hr -0 hr) measurement of negative control ear] X 10~ 3 mm. A two-tailed student's t-test was performed on the results, and significance was assumed at P < For adoptive transfer studies, splenocytes obtained from 99/1

No. 1 Reporr ocular tumor-bearing mice were injected intravenously (6 X 107 spleen cells) into naive BALB/c mice. Within 2 hr, each mouse received a subcutaneous injection of 2 X 105 P815 cells.

2 No. 1 Reporr ocular tumor-bearing mice were injected intravenously (6 X 107 spleen cells) into naive BALB/c mice. Within 2 hr, each mouse received a subcutaneous injection of 2 X 105 P815 cells. DH was assayed 10 days after transfer of spleen cells. 225 Results. In the following experiments} P815 tumor cells were inoculated into the intravitreal space of eyes of adult BALB/c mice. A clinical and histopathologic examination of inoculated eyes was performed during the 3 week interval postinoculation, and the 1C Fig. 1A. Fundus examination of the eye inoculated with VC P815 tumor cells 5 days previously. The multiple mass-like tumors (arrow) associated with the vessels are seen in the vitreous cavity. Fig. IB. Examination on day 7 p.i. of the anterior segment of the eye shown in Figure 1 A. P815 tumor tissues has extended into the anterior chamber. The arrows indicate two tumor nodules which are attached to the iris near the pupil edge. The cornea and the lens have a cloudy appearance. Fig. 1C. Histological section of an eye bearing a P815 tumor on day 10 p.i. Tumor cells fill the VC (v), the AC (a), and the subretina space (sr). Tumor cells invade the orbit via the posterior wall (arrow) of the eye. Tumor cells also infiltrate the retina (r), choroid, ciliary body and iris, but the cornea is relatively free from tumor cells. Fig. ID. A high magnification of the section shown in figure 1C. The tumor is highly vascularized, necrosis is rare.

226 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1991 Vol. 32 capacity of recipient mice to develop and display P815-specific DH was assessed.

3 226 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1991 Vol. 32 capacity of recipient mice to develop and display P815-specific DH was assessed. Clinical Evaluation of Intravitreal Growth ofp815 Tumor Cells: Tumor tissue was first detected 3 days post-inoculation (pi) as translucent nodules within the VC and near the retina. By day 5 pi, tumor nodules were visible as multiple massive nodules within the VC, and tumor-associated vessels were clearly evident (Fig. 1 A). At 7 days pi, tumor tissue was apparent within the AC (Fig. 1B), the aqueous humor appeared to be cloudy, and the chamber had become shallow. The lens was cloudy, iris vessels were engorged, and the pupil was fixed and dilated. The cornea was swollen and mildly cloudy. By day 10 pi, the anterior segment of the eye was heavily infiltrated with tumor tissue, the cornea was cloudy, and the eyes were enlarged and proptotic. Since subconjunctival injections of P815 cells into BALB/c mice did not produce significant tumor masses and were rejected, the VC appeared to function as an immunologically privileged site for minor histo-incompatible tumor cells. Histopathologic Evaluation of Intravitreally Growing P815 Cells: Tumor-bearing eyes were enucleated at day 10 pi. By this time, tumor tissue occupied the VC and subretinal space and was vascularized without evidence of necrosis. Tumor cells had invaded the ocular orbit through the posterior wall of the eye, and malignant cells were present throughout the retina, ciliary body, and iris (Fig. 1C). The distribution of tumor cells suggested that local spread and extension was dictated by the original needle tract and by the physiologic pathways by which intraocular fluids communicate. Absence of necrosis implies that the tumor had established a nourishing vascular supply (Fig. ID). Delayed Hypersensitivity Responses of Mice with Intravitreal P815 Tumors: To determine whether antigen-specific DH developed in AC tumor-bearing animals, mice received intravitreal injections of P815 cells. Tumor-bearing eyes were enucleated on day 10 pi, and the ears of these mice received intrapinna injections of irradiated P815 cells on day 14 pi. Positive control mice were immunized with P815 cells injected SC, and negative control mice were ear-challenged. As displayed in Figure 2, mice that received P815 cells SC mounted vigorous DH responses, whereas mice that received P815 cells intravitreally made ineffectual responses. To determine whether the spleens of mice with VC tumors contained suppressor cells, a panel of BALB/c mice received P815 cells intravitreally. Seven days later, their spleens were removed and injected intravenously into naive BALB/c mice (50 X 10 6 spleen cells per recipient). One hour later, recipient mice received P815 cells (2 X 10 5 ) subcutaneously. Seven days later, the ears were challenged with irradiated P815 cells. The swelling response was assayed 24 and 48 hr later. Figure 3 shows that the spleens of mice with VC tumors, but not the spleens of normal mice, contain suppressor cells that prevent the induction of DH in normal BALB/c mice. Discussion. The VC appears to function as an immunologically privileged site. Allogeneic tumor cells (P815) injected into the vitreal space are accepted by BALB/c mice, grow, and induce a deviant form of systemic immunity characterized by impaired expression of DH and the presence of suppressor cells in the spleens of recipient mice. Since similar numbers of P815 cells injected subcutaneously into BALB/c mice, do not form progressively growing tumors, are rejected, and elicit vigorous DBA/2-specific DH, 1 ' 5 we conclude that immune privilege is extended to tumor allografts within the VC. The systemic immune responses of mice with VC tumors resemble the responses of mice with AC tumors, where the phenomenon of ACAID has been described. 1 The current findings are consistent with the reports of Atherton et al 2 and of Pepose and Whittum-Hudson 3 that intravitreal injections of HSV-1 (KOS strain) elicit a deviant form of systemic immunity. Francois et al 6 have shown that allogeneic and xenogeneic hours after challenge Fig. 2. Capacity of intravitreal P815 cells to induce delayed hypersensitivity in BALB/c mice. P815 cells (2 X 10 5 ) were inoculated VC on day 0; tumor-containing eye was enucleated on day 10. Ears were challenged with irradiated P815 cells on day 14; ear swelling was assessed 24 hours later. Positive controls received P815 cells SC on day 0. Negative controls were challenged only with irradiated tumor cells. Bar represents mean ± SEM. Response of SC group was significantly higher than those of either AC group or negative control group (P < 0.01).

No. 1 Reporr 227 100- ' 60- M 40-1 0-24 hours after challenge Test + Control Control Fig. 3. Adoptive transfer of impaired DH reactivity induced by intravitreal P815 cells in BALB/c mice.

4 No. 1 Reporr ' 60- M hours after challenge Test + Control Control Fig. 3. Adoptive transfer of impaired DH reactivity induced by intravitreal P815 cells in BALB/c mice. Spleen cells (50 X 10 6 ) from donor BALB/c mice that received P815 cells VC on day 0, (their eyes were enucleated on day 7 and they were sacrificed on day 10) were infused i.v. into naive BALB/c recipients. One hour later, recipients received 10 6 P815 cells SC. Ten days later, ears were challenged with irradiated P815 cells; ear swelling response measured 24 hours later. Positive and negative controls as described in legend to Figure 2. hyalocytes can be successfully transplanted into the vitreous body of normal rabbit eyes, eliciting no detectable immune response. Foster et al 4 have reported that azobenzene arsonate (ABA)-derivatized syngeneic spleen cells, which induce immune deviation when injected into the AC of the eye, induce conventional immunity when injected into the vitreous body, although VC injection of cross-reactive, idiotypic antibody induced suppressed ABA-specific cell- mediated immunity. It is not obvious why disparate results are obtained with ABA-SC, on one hand, and P815 tumor cells, HSV-1, and allogeneic hyalocytes on the other. The murine VC is a small space (<5 ix\ total volume). In the experiments of Foster et al, 4 the intravitreal injection volume was 101, whereas we injected 3 fi\. We suspect that the larger the injected volume, the greater the extent of leakage. Injection of soluble antigens into the VC of rabbits is a model system used to study immunogenic uveitis, mediated chiefly by antibodies. 7 It seems paradoxical that the same intraocular site should support the induction of both immune deviation and immunogenic uveitis. There are several possible explanations for this paradox. First, mouse and rabbit eyes differ remarkably in their capacities to display inflammatory responses. Rabbit eyes respond with vigorous inflammation to intraocular instillation of bacterial lipopolysaccharide (LPS), 8 whereas mouse eyes are much less responsive (personal communication, Dr. Scott Cousins). Similarly, it is possible to create local graft versus host reactions in the eyes of rabbits, 7 but not in mouse eyes. 8 Second, the amount of antigen injected may be crucial. In general, greater than 1 mg of soluble protein antigen has been injected intravitreally to induce immunogenic uveitis in rabbit eyes, 7 whereas ACAID has been elicited with much smaller amounts (50 /Lig) of soluble antigens in mouse eyes. 9 The rapidity with which intravitreal P815 tumors acquired a blood supply deserves comment. Since the vitreous body is known to contain anti-angiogenic factors, 10 rapid emergence of new vessels to support intravitreal tumor growth implies that the tumor cells are capable of overcoming this natural inhibition against new vessel formation. Moreover, intravitreal tumors were found to be free of necrosis, emphasizing the failure of the hosts to mount an anti-tumor immune response. Rapid acquisition of a blood supply by intravitreal allogeneic tumors did not prejudice the development of a deviant systemic immune response. In induction of ACAID via the AC, the blood serves as the medium through which antigen leaves the immune-privileged site and reaches the spleen. We suspect that by quickly establishing a blood supply, intravitreal tumor allografts help to arrange their own protection from eventual immune attack. Key words: ACAID, alloantigen, vitreous cavity, immune privilege, intravitreal tumor Acknowledgments. The authors thank Dr. Norman H. Altman and Dr. Douglas Anderson for use of their equipment for sectioning. We appreciate the advice and counsel of Dr. Bruce Ksander and the technical assistance of Ms. Debra A. Bradley and Ms. Michele M. Mammolenti. From the Departments of Microbiology and Immunology, and of Ophthalmology, School of Medicine, University of Miami, Miami, FL. Submitted for publication: May 23, 1990; accepted August 7, Supported by NEI Grant EY All experimental procedures conformed to the ARVO Resolution on the Use of Animals in research. Reprint requests: J. Wayne Streilein, MD, University of Miami School of Medicine, Department of Microbiology and Immunology, P.O. Box (R-138), Miami, FL References 1. Streilein JW: Immune regulation and the eye: a dangerous compromise. FASEB J 1:199, Atherton SS, Pesicka GA, and Streilein JW: Retinitis and deviant immune responses following intravitreal inoculation of HSV-1. Invest Ophthalmol Vis Sci 28:859, Pepose JS and Whittum-Hudson JA: An immunogenetic analysis of resistance to herpes simplex virus in inbred strains of mice. Invest Ophthalmol Vis Sci 28:1549, 1987.

228 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1991 Vol. 32 4. Foster CS, Monroe JG, Campbell R, Kalpaxis J, Wetzig R, and Greene MI: Ocular immune responses. II.

5 228 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1991 Vol Foster CS, Monroe JG, Campbell R, Kalpaxis J, Wetzig R, and Greene MI: Ocular immune responses. II. Priming of A/J mice in the vitreous induces either enhancement of or suppression of subsequent hapten-specific DTH responses. J Immunol 136:2787, Streilein JW and Niederkorn JY: Characterization of the suppressor cell(s) responsible for Anterior Chamber Associated Immune Deviation (ACAID) induced in BALB/c mice by P815 cells. J Immunol 134: 1381, Francois J, Victoria-Troncoso V, and Maudgal PC: Immunology of the vitreous body. Modern Problem Ophthalmology 16:196, Brinkman CJJ, Winkens JH, and Broekhuyse RM: Immune response evoked by antigen injection in the rabbit vitreous combined with immunopotentiation. Graefes Arch Clin Exp Ophthalmol 217:213, Cousins SW, and Streilein JW: Aqueous humor inhibits alloantigen-driven lymphocyte proliferation in vivo. Invest Ophthalmol Vis Sci (Suppl) 30:440, Mizuno K, Clark AF, and Streilein JW: Induction of anterior chamber associated immune deviation in rats receiving intracameral injections of retinal S antigen. Curr Eye Res 7:627, Lutty GA, Thompson DC, Gallup JY, Mello RJ, Patz A, and Fenselau A: Vitreous: an inhibitor of retinal extract-induced neovascularization. Invest Ophthalmol Vis Sci 24:52, 1983.

T Cells in the Uninjected Eye after Anterior Chamber Inoculation of Herpes Simplex Virus Type 1

T Cells in the Uninjected Eye after Anterior Chamber Inoculation of Herpes Simplex Virus Type 1 Atsushi Azumi and Sally S. Atherton PURPOSE. TO investigate T cell infiltration in the posterior segment