Characterization of a New Epitope of IRBP That Induces Moderate to Severe Uveoretinitis in Mice With H-2b Haplotype

Abstract

Purpose: Experimental autoimmune uveitis (EAU) induced in mice using the retinal antigen interphotoreceptor retinoid binding protein (IRBP) is an animal model for posterior uveitis in humans. However, EAU induced by native IRBP protein or its widely used epitope amino acid residues 1 to 20 of human IRBP (hIRBP1-20) is inconsistent, often showing low scores and incidence. We found an urgent need to identify a better pathogenic epitope for the C57BL/6 strain.

Methods: Mice were immunized with uveitogenic peptides or with native bovine IRBP. Clinical and histological disease and associated immunological responses were evaluated. Truncated and substituted peptides, as well as bioinformatic analyses, were used to identify critical major histocompatibility complex (MHC)/T cell receptor (TCR) contact residues and the minimal core epitope.

Results: The new uveitogenic epitope of IRBP, amino acid residues 651 to 670 of human IRBP (LAQGAYRTAVDLESLASQLT [hIRBP651-670]) is uveitogenic for mice of the H-2b haplotype and elicits EAU with a higher severity and incidence in C57BL/6 mice than the previously characterized hIRBP1-20 epitope. Using truncated and substituted peptides, as well as bioinformatic analysis, we identified the critical contact residues with MHC/TCR and defined the minimal core epitope. This made it possible to design MHC tetramers and use them to detect epitope-specific T cells in the uveitic eye and in lymphoid organs of hIRBP651-670-immunized mice.

Conclusions: Data suggest that hIRBP651-670 is an epitope naturally processed from a conserved region of native IRBP, potentially explaining its relatively high uveitogenicity. This epitope should be useful for basic and preclinical studies of uveitis in the C57BL/6 model and gives access to genetically engineered mice available on this background.

Figure 1

hIRBP651-670 induces more severe EAU than hIRBP1-20. C57BL/6J mice were immunized with peptide hIRBP1-20 or hIRBP651-670 (300 μg per mouse) and 0.5 μg PTX. Disease was scored by histopathological analysis of eyes collected 21 days after immunization. (A) Average (±SE) scores and incidence from five individual experiments are shown (P < 0.0001, two-way ANOVA). (B) Combined scores are shown from all five experiments shown in Figure 1A (P = 0.0031, Mann-Whitney U test). (C) Retinal pathology in C57BL/6J mice immunized with hIRBP1-20 or hIRBP651-670. (D) Average scores (±SE) of EAU in strains C57BL/6J, C57BL/10J, and 129S1/SvImj immunized with 200 μg hIRBP651-670 and 1 μg PTX in eyes collected on day 21. Data are from two independent experiments.

Figure 2

Kinetics and dose-response of EAU induced with hIRBP651-670. (A) Experimental autoimmune uveitis was induced in C57BL/6J mice by using 300 μg hIRBP651-670 (n = 28) or 300 μg hIRBP1-20 (n = 28) or 150 μg native bovine IRBP protein (n = 12) and 0.5 μg PTX IP. Shown are average clinical scores as evaluated on the indicated days. Incidence rate is given in Table 2. Disease scores of hIRBP651-670 and hIRBP1-20 are significantly different (P = 0.0012 between groups by two-way ANOVA). (B) Dose response of hIRBP651-670–induced EAU. C57BL/6J mice were immunized with specified doses of hIRBP651-670 plus 0.5 μg PTX. Shown is the severity of disease as average (±SE) EAU score by histological analysis on day 21. Data are combined from three repeat experiments. (C) Lack of synergy is shown by mixing both of the uveitogenic peptides. C57BL/6J mice were immunized with 300 μg each of the individual peptides or 150 μg hIRBP651-670 plus 150 μg hIRBP1-20 (total: 300 μg of both of the peptides) and 0.5 μg PTX. Shown are average (±SE) histopathology scores compiled from three repeated experiments on day 21 post immunization (n = 17 per group).

Figure 3

Disease-associated immune responses. (A) C57BL/6J mice were immunized with 300 μg of one of the peptides or 150 μg IRBP and 0.5 μg PTX. Lymphocytes from draining lymph nodes of six mice per group were pooled and cultured with graded doses of each of the peptides or IRBP protein. Data are average (±SE) stimulation indexes from three independent experiments. (B) Levels of proinflammatory cytokines in eye extracts of EAU mice on day 14 after immunization. (C) Clinical scores of EAU by fundus examination of mice (those used in [B]) on day 13 post immunization. (D) Proinflammatory cytokine production by draining lymph node cells collected on day 9 or day 14 post immunization in response to immunizing peptide (20 μg/mL) measured in the culture supernatants after 48 hours of cell culture. Data are average (±SE) titers of cytokines from two independent experiments.

Figure 4

Identification of the minimal core epitope of hIRBP651-670 (residues 654–664). (A) C57BL/6J mice were immunized with 300 μg truncated versions of peptide hIRBP651-670 (listed in Table 1) plus 0.5 μg PTX. Average EAU scores of both of the eyes on day 21 are shown. Minimal epitope required for the induction of uveitis was residues 654 through 664. (B) Critical residues within the minimal core epitope were identified by inducing EAU in C57BL/6J mice, using 300 μg peptide hIRBP654-664 with alanine substitutions at each position (Table 1) plus 0.5 μg PTX. Shown are average EAU scores from both eyes of individual mice on day 21 after immunization compiled from two independent experiments (n = 12).

Figure 5

hIRBP651-670–specific T-cell line induces EAU in naïve mice. (A) Average (±SE) EAU scores of mice infused with the indicated numbers of cells from an hIRBP651-670–specific T-cell line after eight rounds of stimulation (eyes were collected on day 14 after transfer). (B) Histopathology of the eyes of mice that received 10 × 10⁶ activated hIRBP651-670–specific T-cell line on day 14 after adoptive transfer. Note destruction of photoreceptor layer, infiltration of inflammatory cells into the vitreous cavity, hemorrhage into subretinal space, and choroiditis compared to the healthy retinas in naïve mice. (C) hIRBP651-670–specific T-cell line stained with the fluorescently tagged tetramer I-A^b-hIRBP654-664. Approximately 29% of the live (7AAD-negative) CD4⁺ TCRα/β cells in the culture were tetramer-positive.

Figure 6

Identification of the I-A^b-hIRBP654-664 tetramer-positive cells in uveitic eyes of mice immunized with peptide hIRBP651-670 or the whole IRBP protein is shown. (A) Representative plot of CD4 T-cell population in the eyes (top) and peripheral lymphoid organs (bottom) of naive or immunized mice (day 21 after immunization). (B) Total number of tetramer-positive cells within the eyes (left) and percentage of tetramer-positive cells within the CD4 T-cell population in the peripheral lymphoid organs (right) of naive and immunized mice on day 21. Tetramer-positive cells could be detected in both the peptide-immunized and the native IRBP protein-immunized mice, showing that this sequence includes naturally processed epitope from IRBP protein and that epitope-specific T cells infiltrate the eyes of EAU mice.