Inhibitory peptide analogs derived from a major uveitogenic epitope protect from antiretinal autoimmunity by inducing type 2 and regulatory T cells.

We identified inhibitory peptide analogs (IPAs), capable of immunomodulating experimental autoimmune uveitis (EAU), induced in B10.RIII mice by immunization with the retinal antigen interphotoreceptor-binding protein in CFA. Alanine-substituted peptides of the major pathogenic epitope, residues 161-180, were synthesized. They were tested for immunogenicity, cross-reactivity with the native 161-180 epitope, pathogenicity, and ability to prevent EAU when given in IFA before EAU challenge with native murine (m)161-180. Two peptides, 169A and 171A, were unable to elicit disease but cross-reacted with m161-180 by lymphocyte proliferation. Mice pretreated with either of the substituted peptides failed to develop EAU after challenge with the native epitope, m161-180, and had reduced cellular responses by lymphocyte proliferation and by delayed hypersensitivity. Their cytokine response profile to m161-180 showed reduced antigen-specific IFN-gamma and IL-17, whereas IL-4, IL-5, IL-10, and IL-13 from IPA-protected mice were increased, and serum antibody titers to m161-180 revealed reduced IgG2a and elevated IgG1 isotypes, suggesting a Th2 shift in the response. Protection was transferable with lymphoid cells from protected donors to naïve recipients, who were subsequently immunized for EAU. Thus, IPA pretreatment prevents induction of EAU by skewing the response to a subsequent uveitogenic challenge with the native peptide to a nonpathogenic phenotype, as well as by eliciting transferable regulatory cells.

An immunologically privileged retinal antigen elicits tolerance: major role for central selection mechanisms.

Immunologically privileged retinal antigens can serve as targets of experimental autoimmune uveitis (EAU), a model for human uveitis. The tolerance status of susceptible strains, whose target antigen is not expressed in the thymus at detectable levels, is unclear. Here, we address this issue directly by analyzing the consequences of genetic deficiency versus sufficiency of a uveitogenic retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). IRBP-knockout (KO) and wild-type (WT) mice on a highly EAU-susceptible background were challenged with IRBP. The KO mice had greatly elevated responses to IRBP, an altered recognition of IRBP epitopes, and their primed T cells induced exacerbated disease in WT recipients. Ultrasensitive immunohistochemical staining visualized sparse IRBP-positive cells, undetectable by conventional assays, in thymi of WT (but not of KO) mice. IRBP message was PCR amplified from these cells after microdissection. Thymus transplantation between KO and WT hosts demonstrated that this level of expression is functionally relevant and sets the threshold of immune (and autoimmune) reactivity. Namely, KO recipients of WT thymi generated reduced IRBP-specific responses, and WT recipients of KO thymi developed enhanced responses and a highly exacerbated disease. Repertoire culling and thymus-dependent CD25+ T cells were implicated in this effect. Thus, uveitis-susceptible individuals display a detectable and functionally significant tolerance to their target antigen, in which central mechanisms play a prominent role.

Interphotoreceptor retinoid-binding protein (IRBP)-deficient C57BL/6 mice have enhanced immunological and immunopathogenic responses to IRBP and an altered recognition of IRBP epitopes.

Experimental autoimmune uveitis (EAU) and pinealitis (EAP) can be induced in susceptible mice by immunization with immunologically privileged retinal antigens. In the present study, we analyzed the immunologic and immunopathologic responses of mice deficient in the retinal autoantigen interphotoreceptor retinoid-binding protein (IRBP). The consequences of IRBP deficiency on the T-cell repertoire were also investigated. IRBP+/+, IRBP+/- and IRBP-/- mice on the C57BL/6 background were immunized with IRBP or with a pathogenic epitope, IRBP(1-20) peptide in adjuvant, and were evaluated for disease severity and immunological responses. C57BL/6 IRBP-/- mice were completely resistant to EAU and EAP, and had enhanced immunological responses to IRBP and to its pathogenic peptide 1-20, as compared to their IRBP+/+ counterparts. IRBP-/- mice exhibited an altered IRBP epitope recognition. T cell epitope mapping revealed a response to IRBP peptide 271-290 in IRBP-/- mice, that was absent in the wild type. Primed T cells of IRBP-/- mice transferred an exacerbated form of EAU to nai;ve wild type recipients. A gene-dose effect was evident in that C57BL/6 IRBP+/- mice, exhibited intermediate immunological responses and lower disease scores compared to wild type. We conclude that expression of IRBP in target tissues is a necessary prerequisite for disease induction, excluding other retinoid-binding or vision-related proteins as surrogate targets. Furthermore, endogenous expression of IRBP is directly responsible for lowering the threshold of susceptibility to uveitic disease.

A humanized model of experimental autoimmune uveitis in HLA class II transgenic mice.

Experimental autoimmune uveitis (EAU) is a disease of the neural retina induced by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein (IRBP) and arrestin (retinal soluble antigen, S-Ag). EAU serves as a model for human autoimmune uveitic diseases associated with major histocompatibility complex (HLA) genes, in which patients exhibit immunological responses to retinal antigens. Here we report the development of a humanized EAU model in HLA transgenic (TG) mice. HLA-DR3, -DR4, -DQ6, and -DQ8 TG mice were susceptible to IRBP-induced EAU. Importantly, HLA-DR3 TG mice developed severe EAU with S-Ag, to which wild-type mice are highly resistant. Lymphocyte proliferation was blocked by anti-HLA antibodies, confirming that antigen is functionally presented by the human MHC molecules. Disease could be transferred by immune cells with a Th1-like cytokine profile. Antigen-specific T cell repertoire, as manifested by responses to overlapping peptides derived from S-Ag or IRBP, differed from that of wild-type mice. Interestingly, DR3 TG mice, but not wild-type mice, recognized an immunodominant S-Ag epitope between residues 291 and 310 that overlaps with a region of S-Ag recognized by uveitis patients. Thus, EAU in HLA TG mice offers a new model of uveitis that should represent human disease more faithfully than currently existing models.