Aire-deficient mice have a limited autoreactive repertoire and predominantly recognize IRBP. (A) Immunoblotting of whole-eye extracts with aire-deficient (individual animals listed by number) and strain- and age-matched wild types for BALB/c, C57BL/6, and NOD mice. (B) C57BL/6 and NOD aire-deficient animals were bled by tail vein at various time points, and sera were immunoblotted against whole-eye extracts to determine kinetic changes in autoantibody reactivity. Arrows in A and B indicate the IRBP band. (C) Purified, full-length bovine IRBP was immunoblotted with sera derived from aire-deficient animals. (D) Sera from individual animals were preincubated with bovine IRBP or S-Ag before use as primaries in an immunoblot against whole-eye extract. S-Ag, another photoreceptor-specific antigen, was included as a control. (E) Sera preincubated with bovine IRBP or S-Ag were also used for immunohistochemistry and visualized with DAB chromogen.

Uveitis in aire-deficient animals is T cell dependent, and increased frequencies of IRBP-specific T cells arise in aire-deficient animals. Immunostaining of frozen eye sections from aire-deficient mice with cell surface markers identifies CD4⁺ T cells as the predominant cells within the mononuclear infiltrate. (A) CD8⁺ T cells and IgD⁺ B cells are also present in the retina. (B) No infiltrate was observed in the eyes of age-matched, aire-sufficient animals. (C) Eye disease can be transferred with a pooled population of splenocytes and cervical lymph node cells from aire-deficient animals. Cells devoid of (E) CD8⁺ T cells but not (D) CD4⁺ T cells are capable of transferring disease into immunodeficient hosts. T cells were purified from cervical lymph nodes from unimmunized aire-deficient and age- and sex-matched aire wild-type controls in the C57BL/6 background (18–20 wk old). (F) T cells were assayed in an ELISPOT assay for IFN-γ production in the presence of 10 μg/ml IRBP, media alone, or 10 μg/ml OVA in the presence of APCs (ConA was also tested as a positive control; not depicted). Open circles represent individual aire-deficient animals; the solid line indicates the mean value. Closed circles represent individual aire-sufficient animals; the dashed line indicates the mean value. For IRBP and media alone, n = 7 animals per group; the number of spots for IRBP was statistically significant (P = 0.0023) for aire-deficient compared with aire-sufficient animals. For the OVA control, n = 3 animals per group.

IRBP is an aire-regulated antigen expressed in the thymus. (A) cDNA derived from thymic stroma of aire-deficient and aire- sufficient animals was prepared. Quantitative real-time PCR was used to determine the expression of known TSAs (insulin and Gad67) and IRBP. Values are normalized to cyclophilin and are relative to expression in the WT TEC stroma. Data represent the mean of three independent experiments. A representative amplification plot of (B) cyclophilin, (C) IRBP, and (D) insulin are shown for wild-type and aire-deficient thymic stroma. (E) Summaries of genes shown in two publicly available datasets, GSE85 (reference 3) and GSE2585 (reference 13), to be aire-regulated in the thymus were each queried for retinal-expressed transcripts. The top nine transcripts fitting this profile are shown (all statistically significant; P < 0.05). (F) Quantitative real-time PCR was used to confirm the expression of the microarray-identified genes. Values are normalized to cyclophilin and are relative to expression in the WT TEC stroma. Error bars represent mean ± SD.

Uveitis in aire-deficient animals is IRBP dependent. The aire mutation was bred with IRBP-deficient animals (both backcrossed >10 generations back to C57BL/6) to produce animals devoid of aire and IRBP expression. 18–20-wk-old DKOs and aire single knockouts were analyzed for disease. (A) Aire single knockouts, but not DKOs, displayed a characteristic mononuclear cell infiltrate (indicated with an arrow) in the retina, as shown in representative histology from the indicated groups. However, both sets of animals showed infiltrates in numerous other organs, as described for aire-deficient mice. (B) Blue-shaded sections of the pie graphs indicate the presence of mononuclear infiltrates in the designated organ, and each circle and corresponding number represent an individual mouse for that group. (C) Similarly, aire single knockouts, but not DKOs, displayed an autoantibody reactivity to the photoreceptor cell layer, as shown in representative staining. Both sets of animals had autoantibodies present against other organs, summarized in (D), where circles/numbers represent individual mice and red-shaded wedges represent positive staining for a particular autoantibody. (E) Immunoblotting of whole-eye extracts prepared from immunodeficient scid animals with sera from 18–20-wk-old aire-deficient, IRBP-deficient, or aire- and IRBP-deficient animals in the C57BL/6 background. (F) Flow cytometry was used to assess the presence or absence of CD4⁺ T cells within the retina. Ocular cells from aire-deficient, IRBP-sufficient (thin black line), aire-deficient/IRBP-deficient (DKO; thick black line), or C57BL/6 wild-type (red line) mice were gated on lymphocytes and stained with CD4.

The absence of IRBP within the thymic compartment is, by itself, sufficient for autoimmunity. Thymi from IRBP-deficient or wild-type control mice in the C57BL/6 background were isolated and cultured for 8 d in 2-deoxyguanosine to deplete hematopoietic cells. Thymic stroma was then transferred to individual C57BL/6 nude congenic recipients (n = 5 for recipients in each group) under the kidney capsule. (A) Reconstituted mice were aged 12 wk after transfer and were analyzed for the presence of eye-specific autoantibodies by indirect immunofluorescence. (B) Hematoxylin and eosin–stained sections from recipients of IRBP-sufficient or IRBP-deficient thymic stroma. (C) Flow cytometry was used to assess the presence or absence of CD4⁺ T cells within the retina of nude recipients. Ocular cells from IRBP-deficient thymic stroma recipients (thin black line), IRBP-sufficient thymic stroma recipients (DKO; thick black line), or C57BL/6 wild-type (red line) mice were gated on lymphocytes and stained with CD4.