Injury of Müller cells increases the incidence of experimental autoimmune uveoretinitis

Müller cells have been shown to have a dual effect in vitro on autoimmune T helper lymphocytes. In a coculture system, Müller cells have a primary inhibitory effect on the proliferation of T lymphocytes. In conditions where their inhibitory action is suppressed, Müller cells can, however, stimulate T cells. In the present study we evaluated the in vivo effect of Müller cells on actively induced experimental autoimmune uveoretinitis (EAU). Ten millimoles of L-alpha-aminoadipic acid (L-AAA), a specific gliotoxic agent, was injected into the vitreous of one eye of Wistar-Furth (WF) rats (a low EAU responder) on the day of immunization. Control rats were injected similarly with phosphate-buffered saline alone. The rats were immunized with S-antigen in CFA or in CFA alone. The results demonstrate that the incidence of EAU increases twofold in the eyes receiving an intravitreal injection of L-AAA in comparison to the contralateral eyes not receiving an injection. No such difference in EAU incidence was observed in control animals. Some rats that had been immunized with CFA alone after an intravitreal injection of L-AAA demonstrated a small amount of retinal perivascular inflammatory cell infiltrate but did not develop typical EAU lesions. The retinal vasculature was normal on examination by fluorescein angiography after injection of L-AAA. These data suggest that Müller cells can influence the course of uveoretinitis through their interaction with T cells.     

Major histocompatibility complex-controlled protective influences on experimental autoimmune encephalomyelitis are peptide specific

The myelin basic protein (MBP) peptide 63–88-induced experimental autoimmune encephalomyelitis (EAE) and its associated T cell cytokine profile are influenced by the rat major histocompatibility complex (MHC). There is an allele-specific protective influence of the MHC class I region, whereas the MHC class II region display either disease-protective or -promoting effects. To investigate if the MHC-associated protection is dependent on certain combinations of MBP peptide and MHC molecules, we have now used another peptide (MBP 89–101). A broader and different set of rat MHC alleles were associated with EAE induced with MBP 89–101 as compared to MBP 63–88. All EAE-susceptible strains mounted peptide-specific strong T helper (Th) 1-like immune responses in vitro. Immunization of rats with an extended peptide (MBP 87–110) induced EAE associated with the same MHC haplotypes as the 89–101 peptide, except in LEW.1N (RT1ⁿ) rats which were relatively resistant. Only this strain responded with additional Th2-like and transforming growth factor-β responses to the peptide in vitro. In vivo depletion of CD8⁺ cells aggravated the disease in this strain. We conclude that both MHC-controlled promoting and protective influences on EAE are dependent on certain MHC/MBP peptide combinations, and that the 87–110 region of MBP contains a major MHC-associated encephalitogenic epitope in the rat.     

Leflunomide: Inhibition of S-antigen induced autoimmune uveitis in Lewis rats

Leflunomide (LEF) is a novel immunomodulator which has been reported to be efficacious in experimental models of systemic autoimmune diseases and in treating rheumatoid arthritis in man. Leflunomide's ability to ameliorate ocular disease processes was investigated in a model of autoimmune eye disease, experimental autoimmune uveitis (EAU). EAU was induced by the injection of retinal S-antigen (S–Ag) into the foot-pad of Lewis rats. Leflunomide, or the reference compound cyclosporin A (CSA), was administered orally or topically (to one eye) each day beginning on the day of S–Ag injection. Drug efficacy was measured by the suppression in clinical signs of ocular inflammation and confirmed by histology. Both oral and topical ocular treatment with LEF suppressed the ocular disease signs and symptoms and retinal necrosis and reduced the S–Ag antibody levels associated with EAU in a dose-dependent manner. Both LEF and CSA were able to inhibit totally the disease manifestations of EAU; however, a comparison of the IC₅₀ and IC₉₀ values indicate that LEF is more potent than CSA in inhibiting EAU. These results suggest that leflunomide may be useful for treating autoimmune diseases of the eye.     

The major histocompatibility complex influences myelin basic protein 63-88-induced T cell cytokine profile and experimental autoimmune encephalomyelitis

Polymorphism of the major histocompatibility complex (MHC) influences susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP) in rats. Current concepts relate such influences to the capacity of class II molecules to present relevant peptides to autoreactive T cells. We have here analyzed the MHC influence on the immune response and the development of EAE after immunization with the immunodominant peptide MBP-63–88. Analysis of MHC-congenic LEWIS strains showed that RT1^a, RT1^c and RT1^l haplotypes are permissive for disease induction, whereas RT1^d and RT1^u are resistant. All EAE responding strains showed peptide-specific proliferation and interferon (IFN)-γ secretion, but no early significant tendency to express interleukin (IL-4) or transforming growth factor (TGF)-β mRNA in lymphocytes in response to the MBP 63–88, 7 days post immunization (p.i.). Later, 14 days p.i., peptide-specific induction of IL-4 and TGF-β occurred in RT1^l rats. Among the EAE non-responders strains, only the RT1^u rats showed an immune response to MBP 63-88. This response, however, was qualitatively different from the immune response in the EAE-susceptible strains. Thus, there was no proliferation and only moderate IFN-γ production in response to peptide, but in contrast, a significant and early peptide-induced IL-4 and TGF-β response was observed. The data suggest that the MHC-associated susceptibility to EAE is partly related to the ability to mount a TH1-like immune response while the MHC-associated EAE resistance may either be related to MBP peptide non-responsiveness or to peptide recognition and induction of a qualitatively different and disease down-regulatory immune response.     

Staphylococcal enterotoxin B and tumor-necrosis factor-α-induced relapses of experimental allergic encephalomyelitis: Protection by transforming growth factor-β and interleukin-10

A study was made of the ability of the superantigen staphylococcal enterotoxin B (SEB) to induce relapses of experimental allergic encephalomyelitis (EAE) in SJL mice that had partially or completely recovered from acute EAE. We find that a single injection of 0.05 mg SEB i.v. induces mild relapses in 50% of such mice. In addition, tumor necrosis factor (TNF)-α (0.2 μg, i.p.) also induces EAE relapses in 43% of SJL mice when injected 1–2 months after recovery. SEB does not induce a second relapse if reinjected when Vβ17a⁺ T cells are still partially deleted. In these mice, however, TNF-α is equally effective in inducing relapses as in mice that did not receive SEB previously. We showed earlier that transforming growth factor (TGF)-β and TNF-α have antagonistic effects on experimental autoimmune diseases; e.g., in spontaneously relapsing EAE, TGF-β and anti-TNF were protective, while anti-TGF-β caused disease exacerbation. Interleukin (IL)-10 is also known to counteract certain TNF effects. We now find that both human IL-10 and TGF-β2 lower the incidence of EAE relapses when given simultaneously with SEB or TNF-α. The protective effect of TGF-β is significant only against relapses induced by SEB (reduced to 9%), and that of IL-10 only against relapses induced by TNF (reduced to 0%) with the treatment regimens employed. Neutralizing anti-TGF-β does not increase the incidence of SEB-induced EAE relapses. In contrast, anti-IL-10 increases both the incidence and the severity of such relapses. We conclude that TNF production is probably important in causing EAE relapses, but that other aspects of the SEB-induced reactivation of myelin-specific T cells also contribute. Furthermore, endogenous IL-10 rather than TGF-β production appears to limit the susceptibility to induction of EAE relapses in this model.     

Immunostimulatory and immunomodulatory peptides derived from the alpha1 domain of HLA-B27 in experimental autoimmune diseases in Lewis rats

Peptides derived from amino acid sequence 60-80 of HLA-B27 (B27PA, aa 60-72 and B2702PA, aa 60-80) mimic cytokeratin and are able to induce in vitro proliferation of human peripheral blood lymphocytes as well as arthritis in Lewis rats. Here we show that the pathogenic epitope recognized by autoaggressive rat T cells is located at the N-terminus of the sequence, between aa 60 and 72. A C-terminally elongated 25mer peptide (B2702.60-84) showed increased pathogenicity, indicating either a second arthritogenic epitope or an immunomodulatory region within this peptide. B2702.60-84 has been described to inhibit murine and human CD8 + cytotoxic T cells (CTL) and was even successfully used for the treatment of allograft rejection. In addition to pathogenicity we have investigated the immunomodulatory effect of peptide B2702.60-84 in our rat model of experimental autoimmune uveitis (EAU), induced with retinal S-Antigen peptide PDSAg. We found that disease exacerbated following coimmunization of PDSAg with B2702.60-84. In vitro, the B27-peptide enhanced the proliferation of CD4+ T cell lines specific for retinal autoantigen peptides during coincubation of B2702.60-84 with the respective antigen. Oral tolerance induction, an effective mechanism to prevent uveitis in Lewis rats, is abrogated by cofeeding peptide B2702.60-84 with the tolerogen PDSAg. In rat EAU, naturally occurring regulatory T cells and orally induced gamma deltaTCR+ suppressor cells are CD8+ which might be impeded by peptide B2702.60-84. As a consequence of their abrogated suppressive capacity disease was exacerbated. We propose a similar role of HLA-B27 in man: disturbing the mechanisms down-regulating self-responses might lead to autoimmune diseases. This could explain the high association of HLA-B27 with a variety of autoimmune diseases targeting different organs or tissues.     

Ex‐vivo tolerogenic F4/80+ antigen‐presenting cells (APC) induce efferent CD8+ regulatory T cell‐dependent suppression of experimental autoimmune uveitis

It is known that inoculation of antigen into the anterior chamber (a.c.) of a mouse eye induces a.c.‐associated immune deviation (ACAID), which is mediated in part by antigen‐specific local and peripheral tolerance to the inciting antigen. ACAID can also be induced in vivo by intravenous (i.v.) inoculation of ex‐vivo‐generated tolerogenic antigen‐presenting cells (TolAPC). The purpose of this study was to test if in‐vitro‐generated retinal antigen‐pulsed TolAPC suppressed established experimental autoimmune uveitis (EAU). Retinal antigen‐pulsed TolAPC were injected i.v. into mice 7 days post‐induction of EAU. We observed that retinal antigen‐pulsed TolAPC suppressed the incidence and severity of the clinical expression of EAU and reduced the expression of associated inflammatory cytokines. Moreover, extract of whole retina efficiently replaced interphotoreceptor retinoid‐binding protein (IRBP) in the preparation of TolAPC used to induce tolerance in EAU mice. Finally, the suppression of EAU could be transferred to a new set of EAU mice with CD8⁺ but not with CD4⁺regulatory T cells (T_reg). Retinal antigen‐pulsed TolAPC suppressed ongoing EAU by inducing CD8⁺ T_reg cells that, in turn, suppressed the effector activity of the IRBP‐specific T cells and altered the clinical symptoms of autoimmune inflammation in the eye. The ability to use retinal extract for the antigen raises the possibility that retinal extract could be used to produce autologous TolAPC and then used as therapy in human uveitis.     

Treg and T-effector cells in autoimmune CNS inflammation: a delicate balance, easily disturbed

EAE is the primary pre‐clinical disease for modelling the autoimmune/inflammatory component of multiple sclerosis. In fact, EAE is the primordial CD4⁺ T‐cell‐driven autoimmune disease model. It is striking (although perhaps unsurprising) that more than 10 000 publications over seven decades have provided a confusing, rather than a satisfying, picture of etiopathology. In the current issue of the European Journal of Immunology, an analysis of mice lacking LFA‐1 is reported. Given the role of this integrin in T‐cell activation and effector cell migration, one might predict resistance of LFA‐1^?/? mice to EAE induction. Instead, this study unexpectedly reports that EAE was exacerbated in the absence of LFA‐1, and that this correlated with a decrease in the steady‐state numbers of Foxp3⁺ Treg in the LFA‐1^?/? mice. Previous studies on the role of LFA‐1 in EAE have been reviewed recently. This Commentary focuses on our current understanding of Treg function in the development and resolution of EAE and discusses how the absence of LFA‐1 might unhinge these, possibly by altering the generation of Treg in the thymus, their expansion in response to autoantigen immunization, or their infiltration of the CNS.     

Combined anti-interleukin-2 receptor and low-dose cyclosporine therapy in experimental autoimmune uveoretinitis.

The effect of combination treatment with anti-interleukin-2 (IL-2)-receptor monoclonal antibody (ART18) and cyclosporine A (CsA) on the effector stage of experimental autoimmune uveoretinitis (EAU) was examined. Efferent-stage EAU was induced in Lewis rats by adoptive transfer of a T-helper cell line specific to retinal soluble antigen (SAg). Rats were treated with ART18 (0.5 mg/kg/day), low dose CsA (1.5 mg/kg/day), or a combination of both. The results were compared to groups treated with high dose CsA (10 mg/kg/day) and to sham-treated animals, with respect to clinical and histological EAU, lymphocyte proliferative responses to SAg, and the ability to transfer EAU to secondary recipients. Ten-day combination therapy with ART18 and low-dose CsA was more effective than high-dose CsA and almost completely suppressed EAU development. ART18 as sole therapy was partially effective, and was better than low dose CsA as sole therapy. Splenocytes of protected animals did not transfer EAU to secondary recipients, while splenocytes of sham-treated controls did, suggesting that the number of uveitogenic lymphocytes in the treated host was reduced by the therapy. In contrast, this therapy was completely ineffective against EAU induced by active immunization. The possible reasons for this discrepancy between the two respective models of EAU are discussed.     

Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis

We have shown previously that complement factor H (CFH) and complement factor B (CFB) are constitutively expressed by retinal pigment epithelial cells and their production is regulated by inflammatory cytokines, suggesting that the alternative pathway (AP) of complement activation might play a role in retinal inflammation. In this study, we further investigated the role of the AP in retinal inflammation using experimental autoimmune uveoretinitis (EAU) as a model. Mice with EAU show increased levels of C3d deposition and CFB expression in the retina. Retinal inflammation was suppressed clinically and histologically by blocking AP‐mediated complement activation with a complement receptor of the Ig superfamily fusion protein (CRIg‐Fc). In line with reduced inflammation, C3d deposition and CFB expression were markedly decreased by CRIg‐Fc treatment. Treatment with CRIg‐Fc also led to reduced T‐cell proliferation and IFN‐γ, TNF‐α, IL‐17, and IL‐6 cytokine production by T cells, and reduced nitric oxide production in BM‐derived macrophages. Our results suggest that AP‐mediated complement activation contributes significantly to retinal inflammation in EAU. CRIg‐Fc suppressed retinal inflammation in EAU by blocking AP‐mediated complement activation with probable direct effects on C3/C5 activation of macrophages, thus leading to reduced nitric oxide production by infiltrating CRIg^? macrophages.     

Experimental autoimmune uveoretinitis (EAU) induced by retinal interphotoreceptor retinoid-binding protein (IRBP): differences between EAU induced by IRBP and by S-antigen

Rats immunized with the retinal interphotoreceptor retinoid-binding protein (IRBP) develop an inflammatory eye disease, "experimental autoimmune uveoretinitis" (EAU). The ocular changes which characterize the EAU induced by IRBP resemble those seen in rats which develop EAU by immunization with another retinal protein, S-antigen (S-Ag). Yet, the two antigens do not cross-react antigenically and the two diseases differ by several features: At low doses (less than or equal to 4 micrograms/rat), IRBP was more uveitogenic in Lewis rats than was S-Ag, inducing disease more reproducibly and with earlier onset time. On the other hand, at higher doses (greater than or equal to 20 micrograms/rat) the disease induced by S-Ag was more severe than that induced by the same doses of IRBP. Rats of various inbred strains differed in their susceptibility to EAU induced by these two antigens. In particular, BN rats were more susceptible to IRBP-induced EAU than to the S-Ag-induced disease, while WF and RCS-rdy+ rats developed severe EAU when immunized with S-Ag but showed minimal or no ocular change when immunized with IRBP.     

A novel pathogenic RBP‐3 peptide reveals epitope spreading in persistent experimental autoimmune uveoretinitis

Experimental autoimmune uveoretinitis (EAU) in the C57BL/6J mouse is a model of non‐infectious posterior segment intraocular inflammation that parallels clinical features of the human disease. The purpose of this study was to analyse the immune response to the four murine subunits of retinol binding protein‐3 (RBP‐3) to identify pathogenic epitopes to investigate the presence of intramolecular epitope spreading during the persistent inflammation phase observed in this model of EAU. Recombinant murine subunits of the RBP‐3 protein were purified and used to immunize C57BL/6J mice to induce EAU. An overlapping peptide library was used to screen RBP‐3 subunit 3 for immunogenicity and pathogenicity. Disease phenotype and characterization of pathogenic subunits and peptides was undertaken by topical endoscopic fundal imaging, immunohistochemistry, proliferation assays and flow cytometry. RBP‐3 subunits 1, 2 and 3 induced EAU in the C57BL/6J mice, with subunit 3 eliciting the most destructive clinical disease. Within subunit 3 we identified a novel uveitogenic epitope, 629–643. The disease induced by this peptide was comparable to that produced by the uveitogenic 1–20 peptide. Following immunization, peptide‐specific responses by CD4⁺ and CD8⁺ T‐cell subsets were detected, and cells from both populations were present in the retinal inflammatory infiltrate. Intramolecular epitope spreading between 629–643 and 1–20 was detected in mice with clinical signs of disease. The 629–643 RBP‐3 peptide is a major uveitogenic peptide for the induction of EAU in C57BL/6J mice and the persistent clinical disease induced with one peptide leads to epitope spreading.     

Cytokines and apoptotic molecules in experimental melanin-protein induced uveitis (EMIU) and experimental autoimmune uveoretinitis (EAU).

The cytokine profile and occurrence of apoptosis during experimental melanin-protein induced uveitis (EMIU) were investigated and compared with that of experimental autoimmune uveoretinitis (EAU). EMIU or EAU was induced in Lewis rats. Eyes were collected at different time points after immunization. Cytokine mRNA expression was identified in the inflammatory cells in the uvea of EMIU rats; IL-2, IFN-gamma and IL-12 increased at the peak of the inflammation, and then tapered off as inflammation subsided. IL-4 and IL-10 increased at the peak of ocular inflammation, and persisted with inflammation resolved. Fas and FasL were expressed consistently in ocular resident cells of EMIU, but were elevated in EAU. In EAU, Bcl-2 expression showed a sharp peak in inflammatory cells but not in the resident cells. In EMIU, high levels of Bcl-2 were present and persisted in both ocular resident and inflammatory cells. Expression of Bax was relatively stable in both EAU and EMIU. Cellular DNA fragmentation was detected in the retinal glial cells of EAU and some inflammatory cells of EMIU. In EMIU, the dynamics of Th1 cytokines were consistent with the ocular inflammation, whereas persistent expression of Th2 cytokines was consistent with their known regulatory role. The continuous high expression of Bcl-2 and the high ratio of Bcl-2 to Bax in the eyes of EMIU may possibly contribute to prevention of ocular tissue damage, and of inflammatory cells from undergoing apoptosis, thus resulting in chronic recurrent inflammation.     

TH2 activated cells prevent experimental autoimmune uveoretinitis,a TH1-dependent autoimmune disease

Mercuric chloride (HgCl₂) injections protect (Lewis x Brown-Norway) F1 (F1) rats against experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal S antigen (S-Ag); in contrast HgCl₂-injected F1 rats develop EAU following transfer of lymph node (LN) cells from rats immunized with S-Ag alone. In the present study we demonstrate that the ability of LN cells from rats protected against EAU to transfer the disease into naive F1 rats was considerably reduced. These LN cells neither produced interleukin (IL)-2 nor (interferon (IFN)-γ but exhibited mRNA for IL-4. In contrast, LN cells from diseased rats easily transferred EAU into naive F1 rats, produced significant IL-2 and IFN-γ levels but barely exhibited mRNA for IL-4. Furthermore protected rats predominantly produced IgG2b anti-S-Ag antibodies, while diseased rats produced IgG2b anti-S-Ag antibodies and the increase in expression of MHC class II molecules on B cells was higher in protected rats than in diseased rats. These data suggest that (1) to exert a protective effect, HgCl₂ must act at an early stage of differentiation of precursors of S-Ag specific T cells, and (2) this effect is related to the preferential activation of TH2 cells to the detriment of uveitogenic TH1 cells. Finally, these results indicate that activation of TH2 cells protect from a TH1-dependent autoimmune disease.     

Induction of experimental autoimmune uveoretinitis in Lewis rats with purified recombinant human retinal S-antigen fusion protein.

Full-length human retinal cDNA for S antigen (S-ag) and for the alpha subunit of transducin (alpha-Td) were subcloned into a bacterial expression plasmid vector to generate recombinant fusion proteins with glutathione-S-transferase (GST). The recombinant GST-S-ag and rGST-alpha-Td fusion proteins were purified from bacterial extracts by continuous flow preparative gel electrophoresis under denaturing conditions, and were assessed for their ability to induce experimental autoimmune uveoretinitis (EAU). Immunization of Lewis rats with single doses of 10 micrograms-100 micrograms rGST-S-ag in Freund's complete adjuvant supplemented with Bordetella pertussis readily induced clinical signs of EAU. Immunization with GST alone did not induce EAU indicating that disease activity was ascribable to the S-ag residues in the fusion protein. Although the alpha-Td shares limited sequence homology with S-ag, the rGST-alpha-Td fusion protein was also not uveitogenic in Lewis rats. The clinical severity of EAU in Lewis rats sensitized with rGST-S-ag was found to be milder than that induced with native S-ag preparations purified from human retina. However, humoral antibody responses to sensitization with the recombinant S-ag fusion protein were of a higher magnitude than with native S-ag. The availability of recombinant preparations of human S-ag protein will be of value in studying its processing and presentation to T cells derived from patients with autoimmune retinal vasculitis.     

Transgenic expression of an immunologically privileged retinal antigen extraocularly enhances self tolerance and abrogates susceptibility to autoimmune uveitis

Interphotoreceptor retinoid-binding protein (IRBP) is an immunologically privileged retinal antigen that can elicit experimental autoimmune uveitis (EAU). The nature and extent of tolerance to immunologically privileged self antigens is poorly understood. To investigate whether transgenic expression of IRBP extraocularly enhances tolerance and protects from EAU we prepared mice that express half of the mouse IRBP gene, containing a potent uveitogenic epitope (residues 161 - 180), under control of MHC class II promoter. Transgene mRNA was detectable in many tissues. Transgenic protein was undetectable by conventional assays, but was detected in thymic tissue by lymphocyte proliferation assay after induction of the promoter. Transgenic mice challenged with p161 - 180 did not develop EAU and had reduced immunological responses, but remained susceptible to EAU induced by whole IRBP, that contains additional uveitogenic epitopes. Disease was also induced by wild type T cells specific to p161 - 180. Thus, extraocular expression of a privileged retinal antigen enhances self tolerance, supporting the notion that sequestration contributes to immune privilege. Exceedingly low levels of transgene expression result in tolerance that is both profound and epitope specific, implying anergy or deletion of the endogenous uveitogenic repertoire. The same level of expression is, however, insufficient to tolerize wild-type effector T cells in the periphery.     

IL‐33 attenuates the development of experimental autoimmune uveitis

Interleukin‐33 (IL‐33) is associated with several important immune‐mediated disorders. However, its role in uveitis, an important eye inflammatory disease, is unknown. Here, we investigated the function of IL‐33 in the development of experimental autoimmune uveitis (EAU). IL‐33 and IL‐33 receptor (ST2) were expressed in murine retinal pigment epithelial (RPE) cells in culture, and IL‐33 increased the expression of Il33 and Mcp1 mRNA in RPE cells. In situ, IL‐33 was highly expressed in the inner nuclear cells of the retina of naïve mice, and its expression was elevated in EAU mice. ST2‐deficient mice developed exacerbated EAU compared with WT mice, and administration of IL‐33 to WT mice significantly reduced EAU severity. The attenuated EAU in IL‐33‐treated mice was accompanied by decreased frequency of IFN‐γ⁺ and IL‐17⁺ CD4⁺ T cells and reduced IFN‐γ and IL‐17 production but with increased frequency of IL‐5⁺ and IL‐4⁺ CD4 T cells and IL‐5 production in the draining lymph node and spleen. Macrophages from the IL‐33‐treated mice show a significantly higher polarization toward an alternatively activated macrophage phenotype. Our results therefore demonstrate that the endogenous IL‐33/ST2 pathway plays an important role in EAU, and suggest that IL‐33 represents a potential option for treatment of uveitis.     

Induction of oral tolerance to S-antigen induced experimental autoimmune uveitis by a uveitogenic 20mer peptide

Experimental autoimmune uveitis (EAU) in Lewis rats is a T-cell dependent disease, which is induced by immunization with retinal S-Antigen (S-Ag) or its unveitogenic peptides. We have recently reported that the oral administration of S-Ag prior to the uveitogenic challenge results in suppression of the disease and of the cellular responses. We examined the effect of oral administration of a recently described uveitogenic peptide (Peptide 35) on the development of EAU induced with the whole protein. The severity of inflammation and retinal destruction, as well as the cellular proliferative responses, were suppressed compared to controls fed with Keyhole Limpet Hemocyanin (KLH). In addition, the levels of antigen-specific antibody isotypes in the serum of gavaged rats were determined. Although (IgA) levels were reduced in rats gavaged with S-Ag or the uveitogenic peptide, IgM and IgG levels were unaltered. Thus, the oral administration of Peptide 35 approaches the state of unresponsiveness seen in S-Ag feeding. In addition, the unresponsiveness can be demonstrated on both the cellular and humoral level.