Induction Of Anterior Chamber-associated Immune Deviation Research Articles

Pigment Dispersion Contributes to Ocular Immune Privilege in a DBA/2J Mouse Model of Pigmentary Glaucoma.

To investigate the effects of anterior chamber pigment dispersion on ocular immune privilege and the possible mechanisms involved in a DBA/2J mouse model of pigmentary glaucoma. DBA/2J mice were utilized as a pigment dispersion model, and age-matched C57BL/6J mice were used as the control group in this study. Proteins in the aqueous humor (AH) and serum were quantified using the bicinchoninic acid assay. Immune cells in the AH were detected using hematoxylin and eosin staining and immunocytochemistry. The expression of TGF-β2 in the AH and cytokine levels (IL-10, IFN-γ) in serum were measured using ELISA. Anterior chamber-associated immune deviation (ACAID) was induced in DBA/2J mice by injecting antigens into the anterior chamber. Delayed-type hypersensitivity (DTH) assays were used to assess the induction of ACAID. In DBA/2J mice, before and after pigment dispersion, following anterior chamber injection of pigment particles, and after ACAID modeling, the expression of regulatory T cells (Tregs) was detected using flow cytometry. Compared to C57BL/6J mice, the protein concentration, immune cell count, and TGF-β2 levels in the AH were elevated in DBA/2J mice. Protein concentration and IL-10 levels in serum were increased, while IFN-γ levels were decreased in DBA/2J. Additionally, the expression of Treg cells in the spleen of DBA/2J mice was significantly increased after pigment dispersion and anterior chamber injection of pigment particles. At 3 and 6 months, DTH responses in DBA/2J mice were not inhibited, thus preventing ACAID induction. However, the opposite was observed at 9 months in DBA/2J mice. Furthermore, the ACAID group exhibited an augmented expression of Treg cells. Dispersion of pigment particles in the anterior chamber of the eye enhances the state of ocular immune privilege by influencing the immunosuppressive microenvironment and inducing more Treg cells to reestablish ACAID.

Anterior chamber associated immune deviation used as a neuroprotective strategy in rats with spinal cord injury.

The inflammatory response is probably one of the main destructive events occurring after spinal cord injury (SCI). Its progression depends mostly on the autoimmune response developed against neural constituents. Therefore, modulation or inhibition of this self-reactive reaction could help to reduce tissue destruction. Anterior chamber associated immune deviation (ACAID) is a phenomenon that induces immune-tolerance to antigens injected into the eye´s anterior chamber, provoking the reduction of such immune response. In the light of this notion, induction of ACAID to neural constituents could be used as a potential prophylactic therapy to promote neuroprotection. In order to evaluate this approach, three experiments were performed. In the first one, the capability to induce ACAID of the spinal cord extract (SCE) and the myelin basic protein (MBP) was evaluated. Using the delayed type hypersensibility assay (DTH) we demonstrated that both, SCE and MBP were capable of inducing ACAID. In the second experiment we evaluated the effect of SCE-induced ACAID on neurological and morphological recovery after SCI. In the results, there was a significant improvement of motor recovery, nociceptive hypersensitivity and motoneuron survival in rats with SCE-induced ACAID. Moreover, ACAID also up-regulated the expression of genes encoding for anti-inflammatory cytokines and FoxP3 but down-regulated those for pro-inflamatory cytokines. Finally, in the third experiment, the effect of a more simple and practical strategy was evaluated: MBP-induced ACAID, we also found significant neurological and morphological outcomes. In the present study we demonstrate that the induction of ACAID against neural antigens in rats, promotes neuroprotection after SCI.

Tolerogenic dendritic cells suppress murine corneal allograft rejection by modulating CD28/CTLA‐4 expression on regulatory T cells

Anterior chamber-associated immune deviation (ACAID) is initiated when dendritic cells (DCs) capture intraocular antigen and induce regulatory T cells (Tregs) in the spleen. We investigated whether DCs could be used as an immunotherapy to prevent murine corneal allograft rejection by inducing Tregs. Bone marrow-derived DCs (BMDCs) were differentiated with transforming growth factor-β2 (TGF-β2) in the presence of donor-derived allopeptide in vitro (TGFβ2-DCs), and adoptively transferred to BALB/c mice. Three days later a corneal allograft transplant was carried out. Graft rejection, as well as the number and the phenotype of splenic Tregs, were determined after transplant. CD86, CD80, CD40 were present, and MHC class II expression were significantly reduced on TGFβ2-DCs compared to BMDCs not exposed to TGF-β2. TGFβ2-DCs increased the number of splenic Tregs (CD4(+)CD25(+)) in recipient mice prior to transplant by modulating CD28/CTLA-4 expression. These induced Tregs suppressed proliferation of CD4(+)CD25(-) T cells ex vivo. TGFβ2-DCs delayed corneal allograft rejection and TGFβ2-DC recipient mice had significantly more splenic Tregs expressing Foxp3 and CTLA-4, but fewer CD28+ Tregs. Expression of GITR, CD69, and CD45RB were similar in TGFβ2-DC and control mice. Therefore, the phenotype of TGFβ2-DCs suggests they are tolerogenic DCs (tolDCs). In vivo, these cells increased the number and function of Tregs by modulating CD28/CTLA-4 expression. The suppressor Tregs induced by TGFβ2-DCs may be involved in the induction of ACAID, which helps to suppress corneal allograft rejection. Our results provide proof of principle for the use of tolDCs as immunotherapy during transplant.

Effect of CD4(+)CD25(+) regulatory T cells in the development of anterior chamber-associated immune deviation.

To investigate whether CD4(+)CD25(+) regulatory T (Treg) cells play a role in the development of anterior chamber-associated immune deviation (ACAID). The dynamic changes in the frequency of CD4(+)CD25(+) T cells, CD4(+)CD25(+) FoxP3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells from spleens of mice with ACAID were analyzed by flow cytometry. Foxp3 mRNA expression in purified CD4(+)CD25(+) T cells was analyzed using real-time PCR. The suppressive effect of purified CD4(+)CD25(+) T cells on the proliferation of CD4(+)CD25(-) T cells was evaluated by [(3)H] thymidine incorporation. A blocking experiment was performed to further address the role of CD4(+)CD25(+) T cells in ACAID. The expression of IL-10 in purified CD4(+)CD25(+) T cells was evaluated by ELISA. Increased frequencies of CD4(+)CD25(+) T cells, CD4(+)CD25(+) FoxP3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells were observed in ACAID. The CD4(+)CD25(+) T cells from mice with ACAID showed enhanced suppressive effect on the proliferation of CD4(+)CD25(-) T cells. Treatment of BALB/c mice with anti-CD25 antibody after injection of OVA into the anterior chamber significantly inhibited the induction of ACAID. Furthermore, purified CD4(+)CD25(+) T cells from ACAID mice secreted IL-10. Our results demonstrate that Treg cells are induced in the mice undergoing ACAID. These Treg cells may play a role in the development of ACAID.

Role of NKT cells in anterior chamber-associated immune deviation

Cells in the eye have a limited capacity for regeneration and, as such, immune-mediated inflammation can lead to blindness. The eye is designed to quench immune-mediated inflammation – a condition known as immune privilege. An important component of immune privilege is the dynamic immunoregulatory process termed anterior chamber-associated immune deviation (ACAID), which is initiated when antigens enter the eye. ACAID suppresses the initiation of antigen-specific inflammation in the eye and the effector stages of immune reactions. Four organ systems are crucial for the induction of ACAID: the eye, thymus, spleen and sympathetic nervous system. Multiple cell populations contribute to ACAID, with natural killer T cells playing a crucial role in the thymic and splenic phases of ACAID. Interactions between natural killer T cells and multiple cell populations in the spleen culminate in the tight regulation of immune-mediated inflammation in the eye and the preservation of vision.

Anterior Chamber-Associated Immune Deviation (ACAID): An Acute Response to Ocular Insult Protects from Future Immune-Mediated Damage?

The "immune privilege" that inhibits immune defense mechanisms that could lead to damage to sensitive ocular tissue is based on the expression of immunosuppressive factors on ocular tissue and in ocular fluids. In addition to this environmental protection, the injection of antigen into the anterior chamber or infection in the anterior chamber induces a systemic suppression of potentially damaging cell-mediated and humoral responses to the antigen. Here we discuss evidence that suggests that Anterior Chamber-Associated Immune Deviation (ACAID)(a) is initiated by an ocular response to moderate inflammation that leads to a systemic immunoregulatory response. Injection into the anterior chamber induces a rise in TNF-α and MCP-1 in aqueous humor and an infiltration of circulating F4/80(+) monocytes that home to the iris. The induction of ACAID is dependent on this infiltration of circulating monocytes that eventually emigrate to the thymus and spleen where they induce regulatory T cells that inhibit the inductive or effector phases of a cell-mediated immune response. ACAID therefore protects the eye from the collateral damage of an immune response to infection by suppressing a future potentially damaging response to infection.

Eye‐derived systemic immune regulation : ACAID

AbstractA form of systemic tolerance is created when antigenic material is placed in the anterior chamber of the eye, an immune privileged site. Termed anterior chamber associated immune deviation (ACAID), this form of tolerance insures that the systemic immune response to eye‐derived antigens is devoid of T cells that mediate delayed hypersensitivity and antibodies that fix complement. ACAID arises when antigen is captured by intraocular antigen presenting cells, then carried to the spleen where a microenvironment is created that activates antigen‐specific T cells to differentiate into regulatory cells that interfere with the induction of delayed hypersensitivity as well as its expression. Several cytokines and neuropeptides, constitutively present in the aqueous humor of the eye, imposes distinctive properties on antigen presenting cells, which play primary roles to the induction of ACAID. In this SIS, I would like to provide up‐to‐date information of what kinds of diseases ACAID can be effective for and whether human monocytes can acquire the specific functions as ACAID‐inducing antigen presenting cells, in addition to molecules, cells, and concepts newly recognized as contributing to tolerance induction induced in ACAID. Evidence is given to support the idea that application of such information may lead to potential for therapeutic applications of ACAID mechanisms in prevention of progression of immune‐inflammatory diseases in humans.

Expression of programmed death 1 and its ligands in iris-ciliary body from mice with anterior chamber-associated immune deviation

To study the expression and possible implication of programmed death 1 (PD-1) and its ligands in the iris-ciliary body from mice with anterior chamber-associated immune deviation (ACAID). This was an experimental study. Twenty-four BALB/c mice were divided into ACAID group, negative controls, positive controls and phosphate-buffer saline controls. ACAID was evaluated by delayed-type hypersensitivity response. Expressions of PD-1 and its ligands (PD-L1, PD-L2) in the iris-ciliary body from ACAID mice were determined by real-time polymerase chain reaction and immunohistochemical studies. Delayed-type hypersensitivity was not detected in ACAID group, indicating that ACAID was induced successfully. The expression of PD-1 mRNA in ACAID group was higher than that in all other groups (F = 248. 109, P < 0.05). The positive controls showed a high expression of PD-L1 mRNA (F = 179. 033, P < 0.05). PD-1 and PD-L1 positive cells with round or oval shapes were found in whole iris-ciliary body, especially in the iris base and pupil margin. Expression of PD-1 and PD-L1 proteins were similar to their expression at mRNA levels. Neither mRNA nor protein of PD-L2 was detected in the iris-ciliary body in all groups. An increased expression of PD-1 in the iris-ciliary body of ACAID mice indicates that this is involved in the induction of ACAID.

The effects of glaucoma filtering surgery on anterior chamber-associated immune deviation and contribution of lymphatic drainage in rats

To investigate whether glaucoma filtering surgery (GFS) in rats would impair the eye's capacity to induce anterior chamber-associated immune deviation (ACAID) and assess the possible mechanism involved. Rats subjected to GFS were injected with bovine serum antigen (BSA) into the anterior chamber to induce ACAID. Animals that had their cervical lymph nodes (CLNs) excised before filtering surgery and those that had sham filtering surgery served as control comparison groups. Antigen-specific delayed-type hypersensitivity (DTH) was used to identify the induction of ACAID. Antigen level in the CLNs was indicated by the percentage of FITC-positive cells in CLNs after FITC dextran was injected into the anterior chamber. Statistical analyses were performed using the student's t-test for comparison of data between control and experimental groups. A P<0.05 was required for results to be considered statistically significant. Rats undergoing GFS demonstrated antigen-specific DTH, while those in the sham filtering surgery or CLNs excised groups failed to acquire an antigen-specific DTH response. The percentage of FITC-positive cells in CLNs was significantly increased (P=0.001) in GFS (mean+/-SD: 2.96+/-0.67%) vs controls (1.57+/-0.48%) at 1 day, but not at 3, 5, 7, or 12 days post-antigen injection. GFS prevents the induction of ACAID in rats, and the antigen drainage to CLNs plays a critical role in this process. The results suggest that the ocular immune status might be altered by GFS.

Latanoprost does not affect immune privilege of corneal allografts

The present study determined whether topical latanoprost, a prostaglandin (PG) F2α analog, influences the induction of anterior chamber-associated immune deviation (ACAID), corneal neovascularization (NV) or survival of corneal allografts in mice. BALB/c mice received topical latanoprost or PGE2 once or multiple times daily starting 4weeks prior to or the day of anterior chamber injection of C57BL/6 splenocytes. Induction of allo-specific ACAID was assessed by ear challenge with C57BL/6 splenocytes 1week after subcutaneous immunization. In a separate experiment, orthotopic corneal transplantation was performed using C57BL/6 mice as donors and BALB/c mice as recipients. Recipients were randomized in a masked fashion to receive topical latanoprost or PGE2. Graft fate was assessed clinically under surgical microscopy. Presence of MHC class II+ CD11c+ or CD11b+ cells in normal BALB/c mouse eyes following latanoprost or PGE2 administration was assessed immunohistochemically. Control mice received topical 20% dimethyl sulfoxide or no treatment. Allo-specific ACAID was induced after 2 or 6weeks of once daily treatment with latanoprost, and was induced even after 6weeks of multiple treatments with latanoprost. Conversely, mice receiving PGE2 failed to develop ACAID. Opacity and corneal NV scores for allografts treated with latanoprost were statistically indistinguishable from those for control allografts (p>0.05), whereas all allografts treated with PGE2 were rejected. Opacity and NV scores were significantly higher in these allografts than in controls (p<0.05). A number of MHC class II+ CD11c+ cells were present in the central cornea after PGE2 treatment. Topical application of latanoprost does not influence induction of ACAID or graft outcomes including opacity and NV, whereas PGE2 does. Immune privilege of corneal allograft is maintained after topical latanoprost application in mice.

CD4+PD-1+T Cells Acting as Regulatory Cells during the Induction of Anterior Chamber-Associated Immune Deviation

To study the expression and functional characteristics of programmed death-1 (PD-1) and its ligands in the spleens of mice undergoing anterior chamber-associated immune deviation (ACAID). ACAID was induced in BALB/c mice by intracameral injection of ovalbumin (OVA). The expression of PD-1 and its ligands in the spleens of ACAID mice was determined by quantitative real-time PCR, Western blotting, and flow cytometry. In vitro proliferation assays, enzyme-linked immunosorbent assays, and adoptive transfer assays were used to investigate the functional characteristics of splenic CD4+PD-1+ T cells of ACAID mice. Both mRNA and protein of PD-1, PD-L1, and PD-L2 were markedly upregulated in the spleens of ACAID mice compared with controls. CD4+PD-1+ T cells from ACAID mice produced large amounts of IL-10 and exhibited in vitro antigen-specific suppressive activity. CD4+PD-1+ T cells from ACAID mice were able to significantly inhibit the antigen-specific, delayed-type hypersensitivity response when adoptively transferred to naive mice. CD4+PD-1+ T cells from ACAID mice, as regulatory cells, are involved in the induction of antigen-specific suppression in association with enhanced expression of IL-10. CD4+PD-1+ T cells in the murine spleen may represent a substantial population of regulatory T cells possibly responsible for the induction of ACAID after intracameral injection of antigen.

Induction of Eye-Derived Tolerance Does Not Depend on Naturally Occurring CD4+CD25+T Regulatory Cells

Regulatory CD4+ T cells (T regs) arise in the spleens of mice with anterior chamber-associated immune deviation (ACAID), an eye-derived tolerance evoked by injection of antigen into the ocular anterior chamber (AC). The current study was conducted to investigate the possibility that these T regs express CD25 and are derived from natural CD4+CD25+ T cells. Naïve T cells from DO11.10 mice were activated in vitro by ovalbumin (OVA)-pulsed, TGFbeta-treated antigen-presenting cells (APCs), and the expression of CD25 assayed by flow cytometry. OVA-specific ACAID T regs were obtained from the spleens of DO11.10 mice with ACAID to OVA. Immunomagnetic enrichment was used to sort out CD4+CD25+, and CD4+CD25- ACAID T cells before they were injected into OVA-immunized mice or examined for mRNA expression of the regulatory T-cell transcription factor Foxp3. In addition, before AC injection of OVA, systemic depletion of CD25+ T cells was performed with injections of anti-IL-2 receptor antibody into the mice. OVA-specific T cells from DO11.10 mice expressed CD25 when exposed to OVA-pulsed, TGFbeta-treated APCs, even when the DO11.10 T cells were depleted of CD25+ cells before their in vitro stimulation. In addition, DH was suppressed in naïve mice that were injected with CD4+CD25+ or CD4+CD25- ACAID T cells. The CD4+CD25+, but not the CD4+CD25-, ACAID T regs expressed Foxp3. Finally, OVA induced ACAID in mice depleted of CD25+ cells. Some of the CD4+ T regs of ACAID arise from CD25- precursors, and the induction of ACAID is not dependent on the presence of natural CD4+CD25+ T regs.

Critical contribution of CD80 and CD86 to induction of anterior chamber-associated immune deviation

Intraocular inoculation of antigens induces anterior chamber-associated immune deviation (ACAID), which is mediated by development of regulatory T cells in response to antigen-presenting cells (APC) pre-conditioned by intraocular transforming growth factor-beta (TGF-beta). In this study, we examined the involvement of T-cell co-stimulatory molecules in this process. To mimic the intraocular APC, thioglycollate-elicited peritoneal exudate cells (PEC) were pre-treated with TGF-beta in vitro. Expression of CD80, CD86, OX40 ligand (OX40L) and CD70 was analyzed by flow cytometry. Contribution of these molecules to co-stimulatory activity of TGF-beta-treated PEC on antigen-stimulated T-cell proliferation and cytokine production was determined by inhibition with blocking antibodies in vitro. Contribution of CD80 and CD86 to induction of ACAID was determined by the administration of blocking antibodies at intraocular antigen inoculation in vivo. TGF-beta-treated PEC expressed CD80 and CD86 but not OX40L or CD70. Antigen-stimulated T cells proliferated and produced IL-10, but not IFN-gamma, in response to co-stimulation by TGF-beta-treated PEC, which was abrogated by blocking antibodies against CD80 and CD86. Induction of regulatory cells mediating ACAID was abolished by in vivo blockade of CD80 and CD86. The present results indicated that CD80 and CD86 play a critical role in induction of ACAID, possibly by co-stimulating expansion and IL-10 production of regulatory T cells in response to TGF-beta-conditioned APC.

Analysis of Immune Privilege in Eyes with Mycobacteria tuberculosa Adjuvant-Induced Uveitis

Purpose: To examine the effects of intravitreal Mycobacteria tuberculosa adjuvant (MTA) on ocular immune privilege. Methods: MTA was injected into the vitreous cavity of BALB/c mouse eyes to induce anterior uveitis. The inflamed eyes were then examined for their capacity to afford immune privilege to injected allogeneic tumor cells and to promote anterior chamber-associated immune deviation (ACAID). Aqueous humor (AqH) was tested for IL-12 content and for its ability to inhibit T-cell activation. Results: AqH removed from MTA-inflamed eyes at 6 and 12 h contained high levels of IL-12, which then fell almost to baseline at 24 h. This is relevant to the finding that the inflamed eye failed to support ACAID induction at an early time period and then regained the ACAID-induction capability at a later time. Nonetheless, AqH removed from MTA-inflamed eyes retained its capacity to suppress T-cell activation, and MTA-inflamed eyes afforded extended survival to alloantigenic tumor cells implanted into the anterior chamber. Conclusion: Intraocular inflammation evoked by MTA causes the local accumulation of IL-12 and simultaneously robs the eye of its capacity to promote systemic immune tolerance to eye-derived antigens. However, MTA-inflamed eyes retain immune privilege, as indicated by their support of the progressive growth of allogeneic tumor cells.

Role of tumor necrosis factor receptor expression in anterior chamber-associated immune deviation (ACAID) and corneal allograft survival.

To determine the role of tumor necrosis factor receptors (TNFRs) in corneal allograft rejection. Corneal epithelial and endothelial cells were examined by flow cytometry for the expression of TNFRI and TNFRII and their susceptibility to TNF-alpha-induced apoptosis. Corneal allografts from normal and TNFRI and TNFRII knockout (KO) C57BL/6 mice were transplanted to BALB/c hosts, and the fate of the allografts was monitored. C57BL/6 spleen cells were injected into the anterior chamber (AC) of BALB/c mice to induce anterior chamber-associated immune deviation (ACAID) and promote corneal allograft survival. The presence of ACAID suppressor cells in corneal allograft recipients was tested using a local adoptive transfer (LAT) assay. Murine corneal epithelial and endothelial cells expressed TNFRI and TNFRII and were susceptible to TNF-alpha-induced apoptosis, yet corneal allografts from either TNFRI or TNFRII donors did not enjoy a lower incidence of rejection or a prolongation in survival time compared to corneal allografts from normal C57BL/6 donors. Moreover, all 31 of the TNFRII KO corneal grafts were rejected by naïve BALB/c hosts. Rejection of TNFRII KO corneal grafts occurred even though suppressor cells developed in the hosts and inhibited the expression of delayed-type hypersensitivity to donor alloantigens. Expression of TNFRII on corneal cells conveys a degree of protection against immune rejection of corneal allografts by a mechanism that is independent of ACAID. Moreover, induction of ACAID before the application of TNFRII KO corneal allografts fails to improve survival and does not replace the TNFRII-dependent protective mechanism.

Splenic B cells act as antigen presenting cells for the induction of anterior chamber-associated immune deviation.

To characterize the role of B cells in the induction of anterior chamber-associated immune deviation (ACAID). An in vitro model of the ACAID spleen was used to recapitulate the events that occur when antigen is introduced into the anterior chamber of the eye and culminates in the appearance of antigen-specific, CD8(+) suppressor cells. In vitro-generated suppressor cells mimicked those produced by anterior chamber injection of antigen, as shown by their antigen specificity, surface expression of CD8, and capacity to suppress DTH, which is mediated by previously immunized T cells. B cells were found to be necessary for suppressor cell development. The B cell receptor (BCR) was necessary for the induction of ACAID and conveyed antigen specificity to the suppressor T cells. Lysosomal acidification of internalized antigen was necessary for B cells to induce ACAID; however, transporter of antigen processing (TAP) was not required for the generation of ACAID. The results suggest that B cells use the BCR to capture and internalize antigen from ACAID-inducing macrophages. Lysosomal acidification of the captured antigen is essential for the processing of the ACAID antigen before TAP-independent presentation to suppressor cells.

Effect of local macrophage depletion on cellular immunity and tolerance evoked by corneal allografts

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.

The eye’s view of antigen presentation

A form of systemic tolerance is created when antigenic material is placed in the anterior chamber of the eye, an immune privileged site. Termed anterior chamber associated immune deviation (ACAID), this form of tolerance insures that the systemic immune response to eye-derived antigens is devoid of T cells that mediate delayed hypersensitivity and antibodies that fix complement. As a consequence, the eye is spared the disruptive blinding influence of immunogenic inflammation. ACAID arises when antigen is captured by intraocular antigen presenting cells, then carried to the marginal zone of the spleen where, in the presence of natural killer T cells and marginal zone B cells, a microenvironment is created that activates antigen-specific T cells to differentiate into regulatory cells that interfere with the induction of delayed hypersensitivity as well as its expression. Transforming growth factor beta-2 (TGFβ-2), constitutively present in the aqueous humor of the eye, imposes distinctive properties on antigen presenting cells. Thrombospondin is an early activated gene following TGFβ-2 treatment and as a consequence the eye-derived antigen presenting cells fail to secrete IL-12 or express CD40. The lack of these potent stimulators of Th1 cell differentiation is central to the induction of ACAID.

Th1/Th2-balance in anterior chamber-associated immune deviation by alloantigen.

Immune deviation induced by an injection of antigens (Ags) in the anterior chamber of the eye has been termed anterior chamber-associated immune deviation (ACAID). Inoculated Ag induces the generation of T cells that down-regulate delayed-type hypersensitivity (DTH). The induction mechanism may well involve various cytokines. BALB/c mice were inoculated with C3H/He splenocytes. After a week, subcutaneous immunization was performed in mice with (ACAID group) or without (positive control group) intracameral inoculation of splenocytes. DTH responses were determined by ear-swelling assay a week after subcutaneous immunization. To ascertain which cytokines suppress or promote ACAID induction, the gene transcription levels of various cytokines were evaluated by ribonuclease protection assay in alloantigen-pulsed splenocytes. Enzyme-linked immunosorbent assay was performed to quantitate cytokine production in the culture supernatants. Alloantigen-specific DTH was suppressed in the ACAID group. Interleukin (IL)-2 and interferon-gamma (IFN-gamma) gene transcription levels in the ACAID-induced group were significantly suppressed, but IL-4, IL-5, IL-6, IL-9, and IL-10 gene transcription levels were not different from those in the positive control group. IFN-gamma and IL-2 production in the ACAID group was significantly suppressed compared with that in the positive control group. Increased expression of IL-4 and IL-10 was not detected in the ACAID group compared with the positive control group. The results suggest that Th1 suppression of cytokine secretion in the splenic phase plays a role in ACAID induction and Th2-secreting cytokines do not particularly affect ACAID induction by alloantigen.

Requirement for splenic CD4+ T cells in the immune privilege of the anterior chamber of the eye.

Injection of antigen into the anterior chamber of the eye induces suppression of antigen-specific DTH, called anterior chamber-associated immune deviation (ACAID). It has been shown that the spleen is required for the induction of ACAID and detecting the ACAID-inducing signal from the eye. To examine the in vivo role of spleen cells, fractions of spleen cells were adoptively transferred into splenectomized mice. The present study showed that DTH was not suppressed in splenectomized mice, but was inhibited in splenectomized mice transferred with a primed CD4+ T cell-containing fraction of spleen cells. This indicates that the splenic CD4+ T cells comprise the regulatory T cells for the DTH response. When we examined the cytokine profile of the infiltrating T cells in the eye of primed mice by reverse transcriptase-polymerase chain reaction (RT-PCR), we found that they expressed IL-4, IL-10 mRNA (Th2 type), but not IL-2 and interferon-gamma (IFN-gamma) mRNA (Th1 type). By contrast, T cells which can elicit normal DTH response expressed IL-2 and IFN-gamma mRNA. These results suggest that splenic CD4+ T cells comprising the regulatory phenotype are required for the induction of ACAID, and that a DTH response to the antigen may be prevented by Th2-dominant CD4+ T cells.