Human immune system development and rejection of human islet allografts in spontaneously diabetic NOD-Rag1null IL2rgammanull Ins2Akita mice

Abstract

Objective: To create an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell-derived beta-cells in the absence or presence of a functional human immune system.

Research design and methods: We backcrossed the Ins2(Akita) mutation onto the NOD-Rag1(null) IL2rgamma(null) strain and determined 1) the spontaneous development of hyperglycemia, 2) the ability of human islets, mouse islets, and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts.

Results: We confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2rgamma(null), Rag1(null), and Ins2(Akita) genes in NOD-Rag1(null) IL2rgamma(null) Ins2(Akita) (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets, required to restore euglycemia in chemically diabetic NOD-scid IL2rgamma(null) and spontaneously diabetic NRG-Akita mice, were quantified following transplantation via the intrapancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with >50% of engrafted NRG-Akita mice capable of rejecting human islet allografts.

Conclusions: NRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell-derived beta-cells in the absence or presence of an alloreactive human immune system.

FIG. 1.

Onset of hyperglycemia and islet morphology in male and female NRG-Akita mice. Male (A) and female (C) NRG-Akita and NOD-Rag1^null IL2r γ^null littermate control mice were followed for >200 days with blood glucose monitored at the days of age indicated as described in research design and methods. Data points shown are of individual animals. Pancreata from male (B) and female (D) NRG-Akita and NOD-Rag1^null IL2rγ^null littermate control mice at the indicated ages were stained with H-E and immunohistochemically for insulin and glucagon. Young and older control NRG (+/+) mice and young NRG-Akita (+/Akita) mice displayed normal architecture and histochemical structure with insulin-positive cells throughout the islets and a peripheral rim of glucagon-positive cells. Older NRG-Akita mice displayed collapsed islet structure with fewer insulin-positive β-cells and numerous glucagon positive cells scattered throughout the islets. Blood glucose levels of the mice at the time of recovery of the pancreas are shown at the bottom of each panel. Magnification ×400. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Transplantation of mouse and human islets into diabetic NRG-Akita mice. Diabetic NRG-Akita mice were transplanted in the renal subcapsular space with 4,000 IEQ human islets or with 20 islets/g body wt mouse islets as described in research design and methods. Blood glucose levels were determined, and the kidney bearing the islet transplant and the host pancreas were recovered at the end of the experiment for histological and immunohistological analyses. A: H-E, insulin, and glucagon staining of transplanted mouse (left panel) and human (right panel) islets and host pancreas of the NRG-Akita transplant recipient at the times indicated after islet transplantation. In the renal subcapsular space, there was robust engraftment of mouse and human islets. Magnification ×200. B: Frequency of diabetes in mouse or human islet recipients. No significant differences were observed between recipients of mouse or human islets. Small vertical bars indicate censored data, i.e., mice that were found dead or were removed from the study for other analyses. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Transplantation of human islet allografts into diabetic NRG-Akita mice engrafted with human HSC. Diabetic NRG-Akita and HSC-engrafted NRG-Akita mice were transplanted subrenally with 4,000 human IEQ as described in research design and methods. A: Frequency of diabetes in islet allograft recipients. NRG-Akita vs. HSC-engrafted NRG-Akita, P = 0.03. B: Representative histology and immunochemical staining patterns are shown. Note the abundance of insulin-positive cells and the absence of human CD45-positive cells in non–HSC engrafted NRG-Akita mice (left panel). Note the presence of fewer insulin-positive cells and islet graft infiltration by human CD45⁺ cells in HSC-engrafted NRG-Akita mice that were normoglycemic at the end of the experiment (middle panel). Note the scarcity of insulin-positive cells and moderate numbers of human CD45⁺ cells in HSC-engrafted NRG Akita mice that were hyperglycemic at the end of the experiment and had rejected their human islet allografts (right panel). Magnification ×200. huCD45, human CD45 staining; Insulin, insulin staining. (A high-quality digital representation of this figure is available in the online issue.)