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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.

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic β-cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire-expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the β-cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID-TEC mice for insulin-deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. β-cell-specific autoimmune destruction was observed, as well as islet-specific T cell infiltration. The presence of insulin-specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti-insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes-resistant HLA class II alleles. ID-TEC mice could serve as a new model for studying this pathology.

Chronic pancreatitis is an inflammatory disease of the pancreas that causes permanent changes in the function and structure of the pancreas. It is most commonly a complication of cystic fibrosis or due to a genetic predisposition. Chronic pancreatitis generally presents symptomatically as recurrent abdominal pain, which becomes persistent over time. The pain eventually becomes disabling. Once specific medical treatments and endoscopic interventions are no longer efficacious, total pancreatectomy is the alternative of choice for helping the patient achieve pain control. While daily administrations of digestive enzymes cannot be avoided, insulin-dependent diabetes can be prevented by transplanting the isolated pancreatic islets back to the patient. The greater the number of islets infused, the greater the chance to prevent or at least control the effects of surgical diabetes. We present here a technical approach for the isolation and preservation of the islets proven to be efficient to obtain high numbers of islets, favoring the successful treatment of young patients.

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 therapy of type 1 diabetes is an open challenging problem. The restoration of normoglycemia and insulin independence in immunosuppressed type 1 diabetic recipients of islet allotransplantation has shown the potential of a cell-based diabetes therapy. Even if successful, this approach poses a problem of scarce tissue supply. Xenotransplantation can be the answer to this limited donor availability and, among possible candidate tissues for xenotransplantation, porcine islets are the closest to a future clinical application. Xenotransplantation, with pigs as donors, offers the possibility of using healthy, living, and genetically modified islets from pathogen-free animals available in unlimited number of islets. Several studies in the pig-to-nonhuman primate model demonstrated the feasibility of successful preclinical islet xenotransplantation and have provided insights into the critical events and possible mechanisms of immune recognition and rejection of xenogeneic islet grafts. Particularly promising results in the achievement of prolonged insulin independence were obtained with newly developed, genetically modified pigs islets able to produce immunoregulatory products, using different implantation sites, and new immunotherapeutic strategies. Nonetheless, further efforts are needed to generate additional safety and efficacy data in nonhuman primate models to safely translate these findings into the clinic.

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.

One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids effectively promoted homing of lymphocyte progenitors and supported thymopoiesis. Nude mice transplanted with thymus organoids promptly rejected skin allografts and were able to mount antigen-specific humoral responses against ovalbumin on immunization. Notably, tolerance to skin allografts was achieved by transplanting thymus organoids constructed with either thymic epithelial cells coexpressing both syngeneic and allogenic major histocompatibility complexes, or mixtures of donor and recipient thymic epithelial cells. Our results demonstrate the technical feasibility of restoring thymic function with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in organ transplantation and regenerative medicine.