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Ocular immune privilege preserves the visual axis by preventing the induction of sight-threatening nonspecific inflammation. Although privilege is essential for maintaining visual integrity, intraocular tumors exploit the privileged environment and grow progressively within the anterior chamber of the eye. Recently, a large number of laboratories have constructed genetically engineered tumor cell vaccines that express high levels of costimulatory signals. These vaccines are designed to bypass the normal pathways of T-cell activation and directly activate CD8+ tumor-specific T cells. In the following series of experiments, we determined whether a tumor cell vaccine that uses costimulatory signals (CD80 and IL-12) is capable of eliminating tumors within the immune-privileged anterior chamber. As expected, vaccine-immunized mice rejected subcutaneous flank tumors (a non-privileged site). However, the vaccine failed to protect mice from even a small number of tumor cells transplanted into the immune-privileged anterior chamber. Surprisingly, immunized mice that were simultaneously challenged with subcutaneous and anterior chamber tumors were unable to eliminate tumors at either site. The failure of systemic protective immunity coincided with the loss of tumor-specific delayed hypersensitivity and cytotoxic T cells. We conclude that tumor cell vaccines that induce complete protection against tumors in non-immune-privileged sites fail to protect against the same tumor within an ocular immune-privileged site. Moreover, a tumor that escapes elimination within the eye can terminate systemic protective immunity that is induced by the tumor cell vaccine.

Unlabelled: Corneal graft rejection can be prevented by local macrophage depletion, via subconjunctival injections with clodronate liposomes. To unravel the underlying immunological mechanism responsible for prolonged graft survival in this circumstance, the effect of this regimen on induction of donor-specific delayed type hypersensitivity (DTH) and anterior chamber associated immune deviation (ACAID) was determined. The study showed that although subconjunctival clodronate liposome-treatment failed to alter systemically induced DTH and ACAID, both types of immune response were absent in clodronate liposome-treated rats after corneal transplantation. Thus, elimination of macrophages from the corneal transplant site renders corneal allografts immunologically "invisible" to the recipient., Purpose: To determine whether local macrophage depletion, via administration of clodronate liposomes, alters delayed type hypersensitivity (DTH) responses and induction of anterior chamber associated immune deviation (ACAID) after corneal allotransplantation., Methods: Clodronate liposome-treated and untreated rats received orthotopic corneal allografts and were tested for DTH responses and induction of ACAID towards donor antigens. Also in subconjunctivally treated and untreated rats, DTH responses were measured after subcutaneous immunization or induction of ACAID with allogeneic spleen cells., Results: Subconjunctival injected clodronate liposomes prevented grafted rats from developing donor-specific DTH as well as ACAID. By contrast, subconjunctivally injected clodronate liposomes had no effect on donor-specific DTH responses after systemic immunization or on the induction of ACAID with allogeneic cells., Conclusions: Depletion of macrophages at the time of corneal allografting seems to render the grafts immunologically invisible to the recipients. This could explain why these grafts survive "indefinitely" without any other form of therapy.

Purpose: To examine the effect of donor-specific anterior chamber-associated immune deviation (ACAID) induction on the survival of orthotopic corneal allografts in neovascularized graft beds., Methods: To induce donor-specific ACAID in recipients, peritoneal exudate cells (PEC) from C57BL/6 mice were incubated overnight with transforming growth factor (TGF)-beta. Cultured PEC were injected intravenously (i.v.) into BALB/c mice, and, 1 week later, these animals received orthotopic corneal allografts from C57BL/6 donors into neovascularized graft beds. Control mice received i.v. injection of syngeneic (BALB/c) PEC, cultured overnight with TGF-beta, and then received orthotopic corneal allografts from C57BL/6 donors., Results: All corneal allografts (15 out of 15) were rejected within 2 weeks after grafting in the neovascularized graft beds of control animals. However, only 6 out of 16 (37.5%) of corneal allografts were rejected in recipients in which donor-specific ACAID had been induced by injection of allogeneic PEC cultured with TGF-beta., Conclusion: Previous studies revealed that rejection of orthotopic corneal allografts in neovascularized graft beds in mice correlated with acquisition of donor-specific delayed hypersensitivity (DH). The results of this study suggest that induction of donor-specific ACAID, which selectively impairs DH responses to donor antigens, effectively prolongs corneal allograft survival in "high-risk" eyes.

The present study examined the role of tumor infiltrating lymphocytes (TIL) in the rejection of intraocular tumors from SV40 transgenic mice. Tumor cells from an intraocular tumor arising in an SV40 transgenic FVB/N mouse were transplanted into the eyes of syngeneic FVB/N mice and the TIL isolated. TIL were assessed for direct cytolytic activity in vitro. TIL were also transferred passively to immunosuppressed FVB/N mice to determine if they could mediate intraocular tumor rejection. The role of CD4+ and CD8+ T cells in intraocular tumor rejection was evaluated by depleting the respective cell populations in FVB/N hosts prior to intraocular tumor challenge. The results showed that intraocular tumors undergoing rejection in immunocompetent syngeneic hosts became infiltrated with T cells, with the CD8+ subset predominating at the time of rejection. By contrast, athymic nude mice did not reject the intraocular tumors nor did the tumors become infiltrated with TIL. TIL displayed direct, tumor-specific cytolytic activity immediately after isolation from the tumor-containing eyes. FVB/N hosts depleted of CD4+ T cells were unable to reject their intraocular tumors. In vivo depletion of CD8+ T cells delayed, but did not prevent tumor rejection. Adoptively transferred TIL mediated swift rejection of intraocular tumors in immunoincompetent recipients. Recipients of TIL, but not recipients of normal spleen cells, acquired significant tumor-specific CTL activity that was demonstrable in vitro. The results strongly suggest, but do not prove, that TIL mediate rejection of intraocular tumors from transgenic mice by direct cytolysis. Although CD4+ T cells are necessary for tumor rejection and are capable of direct cytolysis, the predominant effector cells are CD8+ CTL.

Achieving the long-term goal of successful engraftment of retinal tissues into eyes blinded by endogenous retinal failure will depend, at least in part, on controlling the immune response to antigens expressed on retinal grafts. Since histoincompatible tissues will have to be used for such transplants, methods to control immune rejection directed at transplantation alloantigens must be devised. In addition, it has recently been observed in mice that developing neural retinal tissues express retina-specific antigens that have the potential to contribute to graft failure. To examine the potential risk of this contribution, syngeneic neonatal neural retinas were implanted into the anterior chamber, vitreous cavity, and subconjunctival space of adult BALB/c mice. During two weeks post-implantation, the fate of these grafts was observed clinically and histologically, and the retina-specific immune responses of the recipients were evaluated. Whereas grafts placed intraocularly thrived, carried out their inherent developmental potential, and were healthy and intact at the end of the observation interval. Grafts placed extraocularly expressed their differentiation program poorly. These grafts underwent rapid decline and attrition, although the process was not accompanied by significant inflammation. Recipients of subconjunctival, but not AC or VC, implants developed retina-specific delayed hypersensitivity. Alternatively, mice bearing AC and VC implants of neonatal neural retina developed retina-specific anterior chamber associated immune deviation. It is concluded that retinal autoantigens are expressed on developing neural retinal tissues and these antigens are highly immunogenic when retinal grafts are placed at conventional body sites. When similar grafts are placed in immune privileged compartments of the normal eye, they enjoy significant survival. Circumstantial evidence indicates that intraocular retinal grafts are protected and maintained in part by the emergence of systemic retinal auto-antigen-specific immune suppression.

Experimental Autoimmune Uveitis (EAU) was induced by immunization with bovine interphotoreceptor retinoid binding protein (IRBP) in B10.A mice. The experiments were performed to evaluate whether Anterior Chamber Associated Immune Deviation (ACAID) can be induced by IRBP when injected intracamerally. The results indicate that anterior chamber (AC) injection of IRBP impaired the development of IRBP-specific delayed hypersensitivity and prevented the expression of EAU following immunization with IRBP-CFA. Adoptive transfer of spleen cells obtained from mice that received IRBP into AC suppressed EAU, whether administered prior to or after the uveitogenic regimen. Most important, IRBP-specific suppressor cells from AC-IRBP treated mice when injected into IRBP-EAU mice suppressed and eliminated already established intraocular inflammation. IRBP-specific, ACAID-inducing suppressor T cells act on the efferent limb of the immune response, and represent ideal modalities for treating already established EAU.

The normal aqueous humor contains a variety of soluble immunosuppressive factors, including transforming growth factor-beta, alpha-melanocyte stimulating hormone, and vasoactive intestinal peptide. These factors are largely the secretory products of parenchymal cells of the iris and ciliary body. TGF beta has recently been shown to alter the functional capacity of intraocular antigen presenting cells, such that they are capable of inducing Anterior Chamber Associated Immune Deviation (ACAID). This deviant systemic immune response is characterized by an impaired capacity to mount an effective cell-mediated immune attack directed at antigens that are placed in, or arise within, the eye. A second property of immunosuppressive factors in aqueous humor is to suppress directly the expression of delayed hypersensitivity in the anterior chamber. In fact, even when the intraocular microenvironment is disturbed by local instillation of gamma-interferon, making it possible for limited expression of cell-mediated immunity in the eye, the microenvironment of the anterior chamber remains profoundly immunosuppressive. In this latter instance, prostaglandins replace TGF beta as the major molecular mediators of suppression in the aqueous humor. In the aggregate, factors present in the normal (or perturbed) intraocular microenvironment have the capacity to modify both the afferent and efferent limbs of the systemic immune response, and this accounts for the longstanding observation that the anterior chamber is an immunologically privileged site. Since evidence suggests that the eye can mobilize more than one molecular mechanism in its effort to limit the sight-destroying potential of immunogenic inflammation, we believe that elucidation of intraocular cytokines and factors that create and maintain an immunosuppressive microenvironment will contribute to a better understanding of the pathogenesis of the acute and chronic uveitides, especially those of autoimmune and infectious etiology.

Immunologic privilege of the anterior chamber has been associated with the capacity to induce a unique form of deviant systemic immunity after anterior chamber (AC) immunization. However, the capacity of privilege to suppress expression of immunity in the AC has not been examined. We studied the ability of the AC to sustain immunogenic inflammation after direct antigen challenge (delayed hypersensitivity-DH) in C57BL/6 mice primed to M tuberculosis (MT) antigens. Compared to subcutaneous and subconjunctival sites where primed mice demonstrated vigorous and significant DH, the anterior chambers of these mice failed to develop signs of inflammation unless toxic doses of antigen were injected. In an attempt to promote intraocular DH, the AC's of MT-primed mice were pre-treated with subinflammatory intracameral injections of IFN-gamma, a cytokine that antagonizes TGF-beta, recruits antigen presenting cells (APC) from the blood and activates resident APC precursors. It was observed that AC injection of IFN-gamma, followed 3 days later by AC challenge with 200 ng of MT, resulted in severe intraocular inflammation only in primed (but not naive) mice. We conclude that the normal mouse AC resists DH unless its immunosuppressive microenvironment is abolished, as in these experiments by IFN-gamma. We propose that impaired expression of cell-mediated immunity is an important component of immune privilege of the AC.

After uniocular anterior chamber inoculation of the KOS strain of HSV-1 in BALB/c mice, acute retinal necrosis develops in the uninoculated eye. These mice exhibit suppression of virus-specific delayed hypersensitivity which is the hallmark of anterior chamber associated immune deviation (ACAID). In contrast, inoculation of C57BL/6 mice with KOS induces vigorous virus-specific DTH, and there is no retinal necrosis in the uninoculated eye. We performed experiments using RH116, a mutant of KOS and SC16, a neurovirulent strain of HSV-1, in BALB/c and C57BL/6 mice to examine whether virus or host factors determine the outcome of anterior chamber inoculation of HSV-1 (ACAID or DTH, retinal necrosis or retinal preservation). We found that high titers of virus (approximately 10(5)) within the first 24 hours in the inoculated eye were linked to the induction of ACAID and that the induction of ACAID following anterior chamber inoculation of HSV-1 is neither an exclusive feature of a strain of virus nor is the ability to develop ACAID a distinguishing characteristic of an individual strain of mice. Taken together, our findings suggest that the interplay of both virus and host factors results ultimately in acute retinal necrosis following anterior chamber inoculation of HSV-1 in the mouse.

Following uniocular anterior chamber injection of HSV-1 (KOS) in BALB/c and A/J mice, it is paradoxical that (a) acute retinal necrosis (ARN) develops only in the uninjected eyes, and (b) ARN occurs only in mice that are immunocompetent, event though these mice invariably display anterior chamber associated immune deviation (ACAID), wherein delayed hypersensitivity to HSV antigens is grossly impaired. Previous studies have revealed that ARN develops only if the titer of infectious virus in the contralateral eye exceeds 4 Log10 PFU, indicating that virus infection is essential to ARN. However, HSV-1 titers in contralateral eyes of similarly infected athymic mice also exceed 4 Log10 PFU, even though these mice never develop ARN - implying that virus alone is insufficient to cause retinal cell destruction. In an effort to define the pathogenic immune component of ARN, we have evaluated in vitro viral antigen-specific T cell responses in mice with ARN and ACAID. We found that T cells, harvested from draining ipsilateral cervical lymph nodes and contrateral eye proliferate in response to viral antigens, express IL-2 receptors, and include HSV-1-specific precursor cytotoxic T cells (pTc), but not direct cytotoxic T cells (Tc). Since the time of appearance of HSV-specific pTc in the contralateral eye coincides with entry of infectious virus into that eye, and since their mutual appearance heralds the onset of retinal necrosis, we conclude that destruction of the retina is initiated by virus-specific cytotoxic T cells that lyse HSV-infected retinal cells.

The eye enjoys a privileged immunological status, in contrast to most sites in the body. Historically, it was thought that the eye, due to its lack of lymphatics, was not subject to the same immune surveillance as other tissues. In opposition to this, recent study has revealed that presenting foreign material via the eye produces marked effects on systemic immunity, leading to a state of tolerance when the same foreign antigen is presented systemically. The immunology of the eye is now known to consist of a myriad of local and systemic effects.

A secondary immune response in the rabbit eye can be provoked by intravenous injection of the antigen, previously injected intravitreally. The inflammation process consists of iris hyperemia, pericorneal redness and cellular and proteinous exudates in the anterior chamber and vitreous. The influence of vitrectomy combined with lensectomy or preceded by extracapsular lens extraction on the character of the secondary immune response has been studied in the rabbit eye. Both eyes of adult rabbits were injected intravitreally with human serum albumin. Five to ten weeks later, when the primary inflammation process had vanished, the vitreous and the lens were removed from the right eye in one group of animals. In a second group of rabbits, an extracapsular lens extraction was performed upon both eyes, later followed by vitrectomy of the right eye. Four weeks later all animals received an intravenous booster injection with human serum albumin. The eyes which still contained the vitreous developed a secondary inflammation consisting predominantly of cells and fibrin clots in the anterior chamber however without flare. On the contrary, the eyes which underwent vitrectomy developed a flare in the anterior chamber with only a few cells detectable. Analysis of cells and protein in the anterior chamber and histological evaluation supported the clinically observed differences between both eyes. These results suggest that the persistence of the vitreous is associated with the reactivation of a secondary immune response. Vitrectomy combined with lensectomy or preceded by extracapsular lens extraction leads to increased passive transfer of proteinous material through the vessels.

The immune response to antigens within the anterior chamber is deviant (anterior chamber associated immune deviation - ACAID) in that delayed hypersensitivity is deficient, whereas other immune effector modalities are intact. Experimental evidence indicates that the eye itself is critical to the induction of ACAID. We have examined the antigen processing and presenting potential of cells within the anterior segment of the eye, and have analyzed the potential immunoregulatory properties of these cells, their secretory products, and the aqueous humor itself. Evidence indicates that bone marrow-derived cells within the stroma of the iris and ciliary body inhibit antigen-driven T lymphocyte activation, although they themselves lack the capacity to present antigens to T lymphocytes. The mechanism is in part through secretion of immunosuppressive cytokines. Since aqueous humor contains similar cytokines, it is inferred that these molecules are constitutively secreted. We have determined that a major inhibitory molecule within normal aqueous humor is transforming growth factor-beta (TGFB), which inhibits antigen processing and presentation, and suppresses both T lymphocyte activation and certain aspects of non-specific inflammation. These effects also turn out to be properties of normal aqueous humor. These findings support the hypothesis that local features of the eye modify intraocular antigens such that an ACAID-inducing signal is produced. Experimental evidence suggests that these same properties may play a major role in suppressing efferent immune responses in the eye.

Herpes simplex virus Type 1 (HSV-1) inoculated intracamerally (IC) into the anterior chamber of BALB/c eyes induces anterior chamber associated immune deviation (ACAID) in which T cell-mediated delayed type hypersensitivity (DTH) responses to HSV-1 are impaired while B cell-mediated anti-HSV neutralizing antibody responses are intact or enhanced.

To test the hypothesis that ACAID induction is instrumental in producing the distinctive pattern of retinal pathology that follows anterior chamber inoculation of HSV-1 in BALB/c mice, panels of mice received uniocular anterior chamber, uniocular intravitreal, and bilateral anterior chamber inoculations of HSV-1. It was found that contralateral retinitis developed after the first two routes, and ACAID was induced by all three. Enucleation of eyes inoculated with HSV-1 before 3 days post-inoculation (but not thereafter) prevented both ACAID and contralateral retinitis. Intracameral inoculations of HSV-2 induced vigorous delayed hypersensitivity and failed to incite contralateral retinitis. It is concluded that ACAID induction plays a crucial role in the pathogenesis of contralateral retinitis following anterior chamber inoculation of HSV-1.

The injection of a variety of antigens, including transplantation antigens, haptens, and viral glycoproteins, into the anterior chamber of eyes of mice produces a characteristic spectrum of immune responses in which suppressed cell mediated immunity dominates. This phenomenon is called Anterior Chamber Associated Immune Deviation (ACAID). Having recently demonstrated that soluble antigens also can induce ACAID in mice, we examined the possibility that an important soluble ocular antigen, retinal S antigen (S Ag) may be capable of inducing ACAID in rats. We have found that injection of S Ag with adjuvant into the anterior chamber of eyes of adult Lewis rats evokes neither a humoral nor a cell mediated immune response that can be detected. However, animals that received S Ag intracamerally in this manner did respond to a subsequent immunogenic dose of S Ag in CFA injected into the hind foot pads, by producing serum anti S Ag antibodies. Importantly, these rats failed to display antigen-specific delayed hypersensitivity. Moreover, lymphoid cells from anterior chamber pre-treated animals, when adoptively transferred, inhibited the development of S Ag-specific delayed hypersensitivity in naive recipients indicating that an active suppression mechanism had been generated. Thus, a soluble antigen injected intracamerally in adult Lewis rats can generate ACAID. The possibility that ACAID may be relevant to the development of S Ag induced uveitis is discussed.

We investigated the role of various immune components in the pathogenesis of immune-mediated experimental herpetic uveitis. Inbred III/J strain of rabbits were sensitized with an intravitreal injection of 10(3) PFU of type 1 herpes simplex virus (HSV), and sensitized cervical lymph node (LN) cells were obtained on postinfection day 12. Intravitreal injection to the normal III/J rabbit eye of HSV antigen with either sensitized LN cells or anti-HSV serum failed to induce uveitis, whereas intravitreal injection of HSV-antigen with both sensitized LN cells and anti-HSV serum produced severe uveitis within six hours. The combination of sensitized LN cells, HSV-antigen and normal rabbit serum, or that of normal LN cells, HSV antigen and anti-HSV serum, did not induce uveitis. Further studies using B lymphocyte and T lymphocyte fractions from sensitized LN showed that only the combination of sensitized T lymphocytes, HSV antigen and anti-HSV serum regularly produced uveitis following intravitreal injection. These results indicate that the interaction of HSV antigen, sensitized T lymphocytes and anti-HSV antibody may play a role in the pathogenesis of immune-mediated herpetic uveitis.

In order to determine whether antiviral immunity is pathogenic in mouse eyes, HSV-1 was injected into the anterior chamber of one eye of adult athymic BALB/c mice. The eyes of these T cell deficient mice were examined clinically and histopathologically for ocular disease. The anterior segment of injected eyes developed progressive inflammatory reactions that eventually destroyed the ciliary body and then progressed to the posterior compartment where partial necrosis occurred, but only in the inner layers of the retina. A milder form of the same process developed between 7 and 10 days in the contralateral eye. Uninoculated eyes displayed little evidence of choroiditis, hemorrhage, massive necrosis, or disintegration of the architecture of the retina. Since these are features that are found in contralateral retinas of euthymic BALB/c mice infected in one eye via the anterior chamber route, it is concluded that acute retinitis found in contralateral eyes of immunocompetent mice has an immunopathogenic basis. However since euthymic mice develop anterior chamber associated immune deviation (ACAID) (and therefore do not display virus-specific delayed hypersensitivity), the identity of the relevant immune effector remains unknown. Based on these observations and our previous ocular findings following intracameral inoculation of HSV-2, we suggest that in susceptible mice, herpes simplex viruses can induce several pathogenetically distinct forms of retinitis, some of which are mediated by virus-specific immune effector cells.

Inoculation of herpes simplex virus Type 1 into the anterior chamber of BALB/c mice induces a characteristic ocular infection involving primarily the anterior segment of virus-injected eyes and the retina of contralateral uninjected eyes. In the BALB/c strain, which is susceptible to herpetic retinitis, anti-HSV delayed hypersensitivity responses are actively suppressed, although anti-HSV antibody responses remain intact. We previously demonstrated that C57B1/6 mice, as well as (BALB/c X C57B1/6) F1 are highly resistant to HSV-1 retinitis. Studies have now been performed to determine the relationship between systemic immune responses to HSV and the resistance of C57B1/6 and the (BALB/c X C57B1/6) F1 to develop retinitis after inoculation of HSV into one anterior chamber. Despite the resistance to herpetic retinitis by B6 and F1 mice, both strains developed a pattern of systemic immune responses to HSV similar to those observed in the susceptible BALB/c mice. Therefore, resistance to retinal infection with HSV-1 does not appear to correlate with the pattern of systemic cell-mediated or humoral immune responses to intracameral HSV-1.