(A) Schematic of the first 8 exons of targeted G228W allele. The asterisk marks the site of the missense mutation. Arrowheads flanking the neo cassette represent loxP sites. The targeting construct is indicated by the dotted line, and the 5′ and 3′ ends are marked with vertical dotted lines. A single loxP remained after Cre-mediated neo excision in ES cells (bottom). (B) Southern blot of electroporated ES cell clones using a probe external to the construct (shown in A) to ascertain homologous recombination. Banding pattern of genomic DNA cut with KpnI from WT (left) and a targeted ES cell clone (right) are shown. Lanes were run on the same gel but were noncontiguous. (C) DNA (first line) and amino acid (second line) sequences around G228W point mutation. The mutated codon is outlined with a rectangle. (D) Immunohistochemical staining of thymi for cytokeratin 5 (red) and Aire (green). Scale bar: 100 μm. (E) Top: Quantitation of number of Aire-positive cells per area of thymic medulla in thymic sections. Bottom: Quantitation of the thymic medullary areas per section of Aire^+/+ (n = 17) and Aire^GW/+ (n = 13) thymi. Sections were randomly selected from at least 3 different mice per genotype. Averages ± SD are shown. (F) Histogram of Aire expression by flow cytometry in mTEChi (red) cells, cortical thymic epithelial cells (cTECs) (green) cells, and isotype control (blue). Numbers represent average ± SD within gated region of mTEChi cells; MFI is presented as mean ± SD (n = 3 for each genotype).

(A) Representative H&E-stained sections of lacrimal (top row) and salivary glands (bottom row) in Aire^+/+ (left column) and Aire^GW/+ (right column) mice at 20 weeks of age. Arrows indicate areas of lymphocytic infiltration seen in Aire^GW/+ mice. Images were taken at ×20 magnification. (B) Infiltration scores for lacrimal gland in bone marrow chimeras aged 10 weeks after bone marrow transplantation. The genotypes of the bone marrow recipients (either Aire^+/+ or Aire^GW/+) and donors (either Aire^+/+ or Aire^GW/+) are shown for each cohort. Bars represent average infiltrate score for each group. *P < 0.003 between Aire^+/+ recipients and Aire^GW/+ recipients. Each circle represents an individual mouse. (C) Infiltration scores for salivary gland of thymic transplants into nude C57BL/6 mice aged 12 weeks after transplantation. The genotypes of thymic donors (either Aire^+/+ or Aire^GW/+) are shown. Bars represent average infiltrate score for each group. *P = 0.016 between Aire^+/+ and Aire^GW/+ thymic donors. Each circle represents an individual mouse.

(A) Infiltration scores of NOD.+/+, NOD.+/o, NOD.GW/+, NOD.GW/o, and NOD.o/o cohorts at 10 (top) and 25–30 weeks (bottom) for organs indicated. Each circle represents an individual mouse. Bars indicate average infiltrate score for each cohort. **P ≤ 0.001 compared with both Aire^+/+ and Aire^+/o. NA, not available due to death. (B) Top: Thyroid infiltrate scores of 15- to 25-week-old mice in the NOD background of given genotype. Each circle represents an individual mouse. Bottom: H&E-stained sections (original magnification, ×5) of thyroid gland from 20-week-old NOD.+/+ (left) and NOD.GW/+ (right) mice. (C) Top: H&E-stained sections (original magnification, ×20) of sciatic nerve from 25-week-old NOD.+/+ (left) and NOD.GW/+ (right) littermates. Bottom: H&E-stained pancreas sections (original magnification, ×5) from NOD.o/o (left), NOD.GW/+ (middle), and NOD.GW/o (right). Arrows point to islets; arrowhead points to intra-islet infiltration. (D) Percentage of activated CD4⁺ splenocytes (CD62L^lo, CD44^hi) in 6- to 10-week-old mice of given genotype in the NOD background by flow cytometry. Averages ± SD of 5 independent experiments are shown. **P ≤ 0.03 compared with both NOD.+/+ and NOD.+/o. (E) Weight curves of NOD.+/+ (n = 10), NOD.GW/+ (n = 11), and NOD.GW/o (n = 4) cohorts, showing average weight ± SD. (F) Neuropathy (red squares) and diabetes (blue circles) incidence curves of NOD.+/+ (n = 10, open symbols) and NOD.GW/+ (n = 11, filled symbols) littermates. *P = 0.0035, difference in neuropathy between NOD.+/+ and NOD.GW/+ mice.

(A) Representative flow cytometric plots of thymocytes. CD4 versus CD8 plots (top row) and Vα2 versus Vβ5 plots (bottom row) are shown. Numbers indicate percentage of lymphocytes in each gate. DP, double positive. Plots of OTII single-Tg mice are shown in the left column. Plots of OTII × Rip-mOVA double-Tg mice are shown in the second column. Plots of OTII × RIP-mOVA double-Tg mice in the Aireo/o setting are shown in the third column. Plots of OTII × RIP-mOVA double-Tg mice in the G228W heterozygous (Aire^GW/+) setting are shown in the right column. (B) Average ± SD thymocyte numbers of CD4⁺CD8⁺ clonotype-positive for each genotype. n = 7 for OTII alone; n = 9 for Aire^+/+ OTII × RIP-mOVA; Aire^+/o OTII × RIP-mOVA; n = 6 for Aire^o/o OTII × RIP-mOVA; n = 9 for Aire^GW/+ OTII × RIP-mOVA. (C) Relative expression of thymic mOVA as determined by real-time RT-PCR on whole thymic stromal preparations of WT (Aire^+/+) and Aire^GW/+ mice carrying the RIP-mOVA transgene. Data shown are representative of 2 experiments.

(A) Heat map of genes previously shown to be downregulated in Aire^o/o mTECs (26), comparing 3 replicates of Aire^+/+ versus Aire^GW/+ mTECs. Scale bar: log₂ ratio of a given sample relative to the average abundance across all 6 samples. (B) Twenty-one most downregulated genes in Aire^GW/+ compared with Aire^+/+ as determined by fold change (FC, defined as expression in Aire^GW/+ divided by that in WT). All genes shown were differentially expressed between Aire^GW/+ and Aire^+/+ mTECs (P ≤ 0.01). Genes are highlighted in yellow if they were expressed in only 1 tissue; in orange if expressed in several tissues; and in blue if ubiquitously expressed. (C and D) Relative expression levels by real-time RT-PCR of genes known to be downregulated in Aire^o/o mice (C) or newly discovered to be downregulated in Aire^GW/+ by microarray analysis (D). Representative results for insulin 2 (Ins 2), spermine binding protein (Sbp), IRBP, hemoglobin Y (Hbby), salivary protein 2 (Spt2), and Spt1 are shown in C. Representative results for prostaglandin D2 synthase (Ptdgs), similar to common salivary protein 1 (Dcpp), G protein–coupled receptor 50 (Gpr50), and cellular retinoic acid binding protein I (Crabp1) are shown in D. Relative expression levels in Aire^+/o, Aire^o/o, and Aire^GW/+ compared with Aire^+/+ thymi are shown for each gene. A representative experiment is shown, with each measurement done in quadruplicate. At least 2 independent experiments were performed.

(A and B) Eye infiltration scores for Aire^+/+, Aire^GW/+, and Aire^o/o mice in the C57BL/6 (A) and NOD (B) backgrounds. Mice were aged to 15–25 weeks, except for NOD.o/o mice (rightmost cohort in B), which were sacrificed when requiring euthanasia (≤10 weeks). *P ≤ 0.01 compared with Aire^+/+ littermate controls. (C) Immunoblot of whole eye extract using sera from Aire^o/o in the C57BL/6 background (B6.o/o); WT mice in the C57BL/6 background (B6.+/+); G228W heterozygous mice in the C57BL/6 background (B6.GW/+); Aire^o/o in the NOD background (NOD.o/o); WT mice in the NOD background (NOD.+/+), and G228W heterozygous mice in the NOD background (NOD.GW/+). NOD.o/o mice were 6–12 weeks of age; all others were 15–25 weeks of age. The arrow indicates band of the expected size for IRBP. (D) Results of real-time RT-PCR for IRBP in Aire^GW/+ compared with Aire^+/+ mice are shown. Mice from the C57BL/6 background (left) and the NOD background (right) were tested.

(A and B) Immunohistochemistry of Aire^+/+ (A) and Aire^GW/+ (B) thymi stained with the indicated nuclear marker (green; left columns), anti-Aire antibody (red; middle columns) and DAPI (blue). Arrows indicate nuclear inclusion bodies. Scale bar: 3.17 μm. (C) Coimmunoprecipitation of G228W and WT AIRE. myc-tagged WT AIRE (m.AIRE WT), G228W AIRE (m.AIRE GW), and/or FLAG-tagged G228W AIRE (f.AIRE GW) were transfected into 1C6 cells, immunoprecipitated with anti-FLAG antibody, and subjected to Western blotting with either anti-myc (top row) or FLAG antibody (second row). To ensure protein expression, 10% of lysates were blotted with anti-myc (third row) or anti-FLAG (fourth row) antibody. (D) Insulin promoter activation by G228W and WT AIRE. m.AIRE WT, f.AIRE GW, or both (GW/WT of 1:5, 1:2, and 1:1 in columns 3–5, respectively) were transfected with the HIP339Luc insulin promoter–luciferase reporter into 1C6 cells. Protein expression using anti-FLAG (top row), anti-myc (middle row), or anti-GAPDH (bottom row) is shown by Western blot. (E) Chromatin immunoprecipitation of 1C6 cells transfected with empty vector (vector), WT AIRE (WT), G228W AIRE (GW), or the latter 2 in combination (WT+GW) with anti-AIRE antibody (black bars) or isotype control (white bars). Average amount (±SD) of mouse insulin 2 promoter (normalized as percentage of input) in the immunoprecipitates is shown. Relative amounts of AIRE input and GAPDH (loading control) are shown by Western blot (bottom panel). Measurements were done in triplicate in 2 independent experiments, with results of a representative experiment shown.