A shared epitope of the interphotoreceptor retinoid-binding protein recognized by the CD4+ and CD8+ autoreactive T cells

Abstract

We previously demonstrated that cultures of rat uveitogenic T cells rapidly become dominated by CD4+ cells, but activation of CD8+ autoreactive T cells also occurred during the in vitro culture of in vivo-primed T cells. In the present study, we show that the commonly used uveitogenic peptide, interphotoreceptor retinoid-binding protein (IRBP) 1-20, generated both CD4+ and CD8+ autoreactive T cells in the C57BL/6 (B6) mouse and that this 20-mer contains at least two distinct antigenic epitopes. To determine whether the CD8 response was Ag-specific and whether CD4+ and CD8+ IRBP1-20-specific T cells recognize distinct antigenic epitopes, we prepared highly purified CD4+ and CD8+ T cells from IRBP1-20-primed mice and tested their proliferative response to a large panel of truncated peptides derived from IRBP1-20. The results showed that both CD4+ and CD8+ T cells recognized the same spectrum of peptides. In addition, peptides P10-18 were found to bind effectively to CD8+ IRBP1-20-specific T cells when complexed with recombinant H-2K(b) and also stimulate the proliferation and cytokine production of CD4+ IRBP1-20-specific T cells. Our results document for the first time that CD8+ and CD4+ autoreactive T cells display characteristic epitope recognition and they both recognize the same core epitope.

FIGURE 1

Detection of CD8⁺ IRBP1–20-specific T cells using CFSE staining. A, Nylon wool-enriched T cells, prepared from IRBP1–20-immunized B6 mice 13 days p.i., were stained with CFSE, then stimulated with IRBP1–20 and APCs, and the activated T cell blasts separated on a Ficoll gradient and recultured in IL-2-containing medium for 2–3 days. For FACS analysis, the T cells were stained with PE-labeled Abs against mouse CD4 or CD8. B, Nylon wool-enriched T cells, prepared from IRBP1–20-immunized B6 mice 13 days p.i., were stimulated with IRBP1–20 and APCs. The activated T cell blasts were separated on a Ficoll gradient, recultured in IL-2-containing medium for 2–3 days, and dually stained with PE-anti-mouse TCR (H57) and FITC-anti-mouse CD8. C, Specificity of the T cell proliferative response to IRBP1–20. Nylon wool-enriched T cells from IRBP1–20-immunized B6 mice were cultured at 37°C for 48 h in 96-well microtiter plates with syngeneic APC with or without IRBP1–20, and [³H]thymidine incorporation during the last 8 h was assessed. The proliferative response is expressed as the mean cpm ± SD for triplicate wells.

FIGURE 2

Response of in vivo IRBP1–20-primed T cells to truncated IRBP peptides. A, Purity of the CD4 and CD8 T cell preparations. CD4⁺ and CD8⁺ T cells were prepared from the spleens of immunized B6 mice 13 days p.i. using StemSep columns (see Materials and Methods) and subjected to FACS analysis using FITC-labeled Abs against mouse CD4 or CD8. Controls were stained with FITC conjugate only. Nonfractionated splenic T cells enriched by passage through a nylon wool column are shown in the bottom panel. The histogram shows fluorescence on an arbitrary log scale on the x-axis. B, The proliferative response of purified CD4⁺ and CD8⁺ IRBP-specific T cells from IRBP1–20-immunized mice to a panel of truncated IRBP peptides was tested. The results shown are the mean cpm values and are representative of those for five separate experiments, each involving pooled T cells from 8 to 10 IRBP1– 20-immunized B6 mice; the SD was always <15%.

FIGURE 3

Residues that are crucial for the activation of IRBP1–20-primed T cells in vitro. The proliferative response of purified CD4⁺ and CD8⁺ IRBP-specific T cells from IRBP1–20-immunized mice to the indicated IRBP peptides was tested. The results shown are the mean cpm values and are representative of those for three separate experiments, each involving pooled T cells from six to eight IRBP1–20-immunized B6 mice; the SD was always <15%.

FIGURE 4

The response of CD8⁺ IRBP1–20-specific T cells to P10–18 is H-2D^b/K^b restricted. In thymidine incorporation assays, the proliferative response of 4 × 10⁵ in vivo-primed CD8⁺ IRBP-specific T cells to a suboptimal dose (5 μg/ml) of P10–18 was tested in the absence or presence of Abs against H-2D^b, K^b, or both (final concentration 10 μg/ml). The results shown are representative of those for four separate tests.

FIGURE 5

Binding to complexes containing H-2K^b and various IRBP-derived peptides to IRBP1–20-specific T cells. IRBP1–20-specific T cells, prepared from immunized B6 mice 13 days p.i., were stimulated in vitro with 20 μg/ml IRBP1–20 and APCs, then the 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-2K^b and IRBP- peptides (see Materials and Methods) (y-axis) and FITC-labeled anti-mouse CD8 (x-axis).

FIGURE 6

Uveitogenic activity of P10–18- and P3– 13-activated IRBP1–20-specific T cells. Nylon wool-enriched splenic T cells, prepared from IRBP1–20-immunized B6 mice 13 days p.i., were subjected to in vitro stimulation with 20 μg/ml IRBP1–20, P10–18, or P3–13 and APCs, then 5 × 10⁶ T cell blasts, separated by Ficoll gradient centrifugation, were transferred to each recipient mouse. The clinical score was then monitored by fundoscopy, and the eyes of the recipient mice were subjected to pathological examination 15 days later. A, Mean clinical score of uveitis after adoptive transfer (n = 5). B, Eye histology shows that P10–18-and P3–13-stimulated IRBP-specific T cells are potently pathogenic.