Interphotoreceptor Retinoid Binding Protein Fragment (IRBP)

We previously demonstrated that a significant proportion of interphotoreceptor retinoid-binding protein (IRBP)-specific uveitogenic T cells in the C57BL/6 mouse and Lewis rat express CD8. The aims of this study were to determine whether some of the IRBP-specific T cells in the B10RIII mouse also express CD8 and whether CD8 and CD4 IRBP-specific T cells in the B10RIII mouse recognize a different or the same antigenic epitope. Our results show that autoreactive CD8 T cells were abundant in B10RIII mice immunized with the uveitogenic peptide IRBP161-180. Using multimers of recombinant H-2D(r) molecules, we also showed that the binding of the H-2D(r) fusion protein to IRBP161-180-expanded CD8 T cells was dependent on the peptide complexed with the recombinant molecules. The use of a panel of truncated peptides showed that the truncated 10-mer peptide, IRBP168-177, retained the ability to bind to, and stimulate, IRBP161-180-specific CD8 T cells after complexing with a dimeric MHC class I (H-2D(r)) molecule. Finally, adoptive transfer of IRBP161-180-specific T cells stimulated with IRBP168-177 consistently induced mild, but significant, EAU in naïve B10RIII mice.

The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene.

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.