Sequence 168 to 177 of interphotoreceptor retinoid-binding protein (IRBP) is an antigenic epitope for autoreactive CD8 T cells in the B10RIII mouse

Abstract

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.

Fig. 1. Detection of CD8⁺ IRBP161-180-specific T cells using CFSE staining

(A): Nylon wool-enriched T cells, prepared from IRBP161-180-immunized B10RIII mice on day 13 p.i., were stained with CFSE, then stimulated in vitro with or without IRBP161-180 for 2 days, and the activated T cell blasts separated on a Ficoll gradient and re-cultured in IL-2-containing medium. For FACS analysis, the T cells were stained separately with PE-labeled antibodies against mouse CD8. The days indicated include 2 in culture with antigen and the remainder in IL-2-containing medium. (B): Antigen-dependent proliferative response to IRBP161-180. Nylon wool-enriched T cells from IRBP161-180-immunized B10RIII mice were cultured at 37° C for 48 h in 96-well microtiter plates with syngeneic APC with graded doses of IRBP161-180, and [³H] thymidine incorporation during the last 8 h assessed. The proliferative response is expressed as the mean cpm ± SD for triplicate wells.

Fig. 2. Response of in vivo IRBP161-180-primed T cells to truncated IRBP peptides

Purified CD4⁺ and CD8⁺ T cells were prepared from the spleens of immunized B10RIII mice at day 13 p.i. using StemSep columns (see Materials and Methods) and were subjected to FACS analysis using FITC-labeled antibodies against mouse CD4 or CD8 as described previously (Shao et al., 2005a). Unfractionated splenic T cells were also prepared by passage through a nylon wool column. The proliferative response (upper panel) and IFN-γ production (lower panel) of purified CD4⁺ and CD8⁺ IRBP-specific T cells or unfractionated T cells from IRBP161-180-immunized mice to a panel of truncated IRBP peptides were tested. The results of the proliferation assay are shown are the mean cpm and are representative of those for 5 separate experiments, each involving pooled T cells from 8–10 IRBP161-180-immunized B10RIII mice; the SD was always less than 15%. IFN-γ was evaluated by ELISA and the results are representative of those for 3 assays.

Fig. 3. Binding of complexes containing H-2D^r and various IRBP161-180-derived peptides to IRBP161-180-specific T cells

IRBP161-180-specific T cells, prepared from immunized B6 mice at day 13 p.i., were stimulated in vitro with 20 μg/ml of IRBP161-180 and APCs, then T cell blasts were separated by Ficoll gradient centrifugation and cultured in IL-2 containing medium for a week. The cells were then incubated with complexes containing H-2D^r and the indicated IRBP-derived peptide or no peptide (control), then were incubated with PE-labeled anti-human IgG1 antibody (detecting bound H-2D^r fusion protein, see Methods) (y axis) and FITC-labeled anti-mouse CD8 antibody (x axis).

Fig. 4. Response of CD8⁺ IRBP161-180-specific T cells to H-2D^r/IRBP-derived peptide complexes in vitro

The proliferative response of 4 × 10⁵ in vivo primed CD8⁺ IRBP161-180-specific T cells to IRBP-derived peptide-complexed or non-complexed recombinant H-2D^r molecules (10 μg/ml) was tested in the absence or presence of B7 molecules in the thymidine incorporation assay. The results shown are representative of those for 4 separate tests.

Fig. 5. Uveitogenic activity of IRBP168-177-activated IRBP161-180-specific T cells

Nylon wool-enriched splenic T cells, prepared from IRBP161-180-immunized B10RIII mice at day 13 p.i., were subjected to in vitro stimulation with 20 μg/ml of IRBP161-180 or IRBP168-177 and APCs (the control group was exposed to APC alone in the absence of peptide), then 5 ×10⁶ T cell blasts, separated by Ficoll gradient centrifugation, were transferred to each groups (n=4) of recipient mouse. The clinical score was monitored by fundoscopy and the eyes of the recipient mice subjected to pathological examination 15 days later. Eye histology showed that IRBP168-177-stimulated T cells induced modest, but significant, EAU upon adoptive transfer to naïve B10RIII mice.