Figure 2. Presence of anti-IRBP antibodies and IRBP-reactive T cells in Aire^GW/+ Lyn^–/– mice with uveitis.

(A) The sera of 2- to 6-month-old Aire^GW/+ Lyn^–/– mice with (n = 14) and without (n = 9) uveitis, or of WT (n = 8), Aire^GW/+ (n = 36), or Lyn^–/– (n = 31) mice, were analyzed for the presence of anti-IRBP antibodies with a radioligand binding assay and normalized to a commercially available anti-IRBP antibody to determine autoantibody index (AI). Each dot represents an individual mouse, and the horizontal lines show mean ± SD. The dashed line represents the limit of detection, calculated as an average of AI values for all WT samples plus 3 SDs. (B) Representative flow cytometric analysis of IRBP P2 tetramer–binding CD4 T cells in pooled spleen and cervical lymph nodes (top) and funduscopy (bottom) from 2- to 5-month-old Aire^GW/+ Lyn^–/– mice with and without uveitis. Plots were pregated for CD4⁺ T cells as described in Methods. The calculated total number of tetramer-positive cells in the whole sample is shown on the plot. (C) Numbers of P2-specific CD3⁺ CD4 T cells gated as in B from individual Aire^GW/+ Lyn^–/– mice with and without uveitis, and from WT and single-mutant control mice. Each dot represents an individual mouse, and the horizontal lines show mean ± SD. The dashed line represents the limit of detection, calculated as average number of P2-binding CD3⁺CD8⁺ T cells plus 3 SDs. Data are pooled from 3 to 5 independent experiments. ***P < 0.001, 1-way ANOVA.