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For reasons not fully understood, patients with an organ-specific autoimmune disease have increased risks of developing autoimmune responses against other organs/tissues. We identified ICA69, a known β-cell autoantigen in Type 1 diabetes, as a potential common target in multi-organ autoimmunity. NOD mice immunized with ICA69 polypeptides exhibited exacerbated inflammation not only in the islets, but also in the salivary glands. To further investigate ICA69 autoimmunity, two genetically modified mouse lines were generated to modulate thymic ICA69 expression: the heterozygous ICA69(del/wt) line and the thymic medullary epithelial cell-specific deletion Aire-ΔICA69 line. Suboptimal central negative selection of ICA69-reactive T-cells was observed in both lines. Aire-ΔICA69 mice spontaneously developed coincident autoimmune responses to the pancreas, the salivary glands, the thyroid, and the stomach. Our findings establish a direct link between compromised thymic ICA69 expression and autoimmunity against multiple ICA69-expressing organs, and identify a potential novel mechanism for the development of multi-organ autoimmune diseases.

Anti-insulin autoimmunity is one of the primary forces in initiating and progressing β-cell destruction in type 1 diabetes. While insulin expression in thymic medullary epithelial cells has been shown to be essential for establishing β-cell central tolerance, the function of insulin expression in antigen-presenting cells (APCs) of hematopoietic lineage remains elusive. With a Cre-lox reporter approach, we labeled Aire-expressing cells with enhanced yellow fluorescent proteins, and found that insulin expression in the spleen was restricted predominantly to a population of Aire(+)CD11c(int)B220(+) dendritic cells (DCs). Targeted insulin deletion in APCs failed to induce anti-islet autoimmunity in B6 mice. In contrast, elevated levels of T cell infiltration into islets were observed in B6(g7) congenic mice when insulin was specifically deleted in their CD11c-expressing DCs (B6(g7)·CD11c-ΔIns mice). Thus, insulin expression in BM-derived, Aire(+) tolerogenic DCs may play an essential role to prevent the activation and expansion of insulin-reactive T cells in the periphery.

The essential role of tissue specific antigen (TSA) expression in medullary thymic epithelial cells (mTECs) in mediating central tolerance has been well-established. We have previously shown that mice with mTEC-specific insulin deletion develop autoimmune diabetes within 3 weeks postnatal. To further understand the mechanisms of mTEC-mediated self-tolerance, we generated the Aire-YFP mice by crossing R26R-YFP reporters with Aire-Cre transgenic mice. Three subsets of mTECs were isolated from these mice: the YFPLOWCD80LOWClass IILOW pre-Aire cells; the YFPHICD80HIClass IIHI Aire+ cells; and the YFPHICD80INTClass IIINT post-Aire cells. Diverse TSA transcripts were detected in Aire+ mTECs, but not, or more restricted in pre- and post-Aire subsets, respectively. To investigate the temporal differentiation of mTECs, we generated the Aire-Cre:R26R-DTR (Aire-DTR) mice, which marked both Aire+ and post-Aire mTECs with diphtheria toxin (DT) receptors. Consecutive administration of DT resulted in ablation of both subsets, whereas pre-Aire mTECs remained intact. Upon DT withdrawal, transcripts of Aire and TSAs remained absent at day 3, but became detectable at day 5. Interestingly, no autoimmunity was observed in Aire-DTR mice treated weekly with a single dose of DT, suggesting that the newly generated Aire+ mTECs could reestablish self-tolerance in a timely manner. Our data provide new insights into the temporal regeneration of mTECs and its importance in maintaining central tolerance.