APE2 Promotes AID-Dependent Somatic Hypermutation in Primary B Cell Cultures That Is Suppressed by APE1.

Somatic hypermutation (SHM) is necessary for Ab diversification and involves error-prone DNA repair of activation-induced cytidine deaminase-induced lesions in germinal center (GC) B cells but can also cause genomic instability. GC B cells express low levels of the DNA repair protein apurinic/apyrimidinic (AP) endonuclease (APE)1 and high levels of its homolog APE2. Reduced SHM in APE2-deficient mice suggests that APE2 promotes SHM, but these GC B cells also exhibit reduced proliferation that could impact mutation frequency. In this study, we test the hypothesis that APE2 promotes and APE1 suppresses SHM. We show how APE1/APE2 expression changes in primary murine spleen B cells during activation, impacting both SHM and class-switch recombination (CSR). High levels of both APE1 and APE2 early after activation promote CSR. However, after 2 d, APE1 levels decrease steadily with each cell division, even with repeated stimulation, whereas APE2 levels increase with each stimulation. When GC-level APE1/APE2 expression was engineered by reducing APE1 genetically (apex1+/-) and overexpressing APE2, bona fide activation-induced cytidine deaminase-dependent VDJH4 intron SHM became detectable in primary B cell cultures. The C terminus of APE2 that interacts with proliferating cell nuclear Ag promotes SHM and CSR, although its ATR-Chk1-interacting Zf-GRF domain is not required. However, APE2 does not increase mutations unless APE1 is reduced. Although APE1 promotes CSR, it suppresses SHM, suggesting that downregulation of APE1 in the GC is required for SHM. Genome-wide expression data compare GC and cultured B cells and new models depict how APE1 and APE2 expression and protein interactions change during B cell activation and affect the balance between accurate and error-prone repair during CSR and SHM.

Multiple endocrine neoplasia type 1 deletion in pancreatic alpha-cells leads to development of insulinomas in mice.

The pancreatic alpha- and beta-cells are critical components in regulating blood glucose homeostasis via secretion of glucagon and insulin, respectively. Both cell types are typically localized in the islets of Langerhans. However, little is known about the roles of paracrine interactions that contribute to their physiological functions. The lack of suitable cell lines to study alpha- and beta-cells interactions have led us to develop an alpha-cell-specific Cre-expressing transgenic line utilizing a glucagon promoter sequence, the Glu-Cre transgenic mouse. Here, we demonstrate that the Glu-Cre could specifically and efficiently excise floxed target genes in adult islet alpha-cells. We further showed that deletion of the tumor suppressor gene, multiple endocrine neoplasia type 1 (Men1), in alpha-cells led to tumorigenesis. However, to our surprise, the lack of Men1 in alpha-cells did not result in glucagonomas but rather beta-cell insulinomas. Because deletion of the Men1 alleles was only present in alpha-cells, our data suggested that cross communication between alpha- and beta-cells contributes to tumorigenesis in the absence of Men1. Together, we believed that the new model systems described here will allow future studies to decipher cellular interactions between islet alpha- and beta-cells in a physiological context.

Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection.

Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS. Applying the method to mouse pancreatic islet-cell subsets, we detected both expected and unknown lineage-specific gene expression patterns. Some beta cells from pregnant animals were found to express Mafb, previously observed only in immature beta cells during embryonic development. The four 'housekeeping' genes tested were expressed in purified islet-cell subpopulations with a notable variability, dependent on both cell lineage and developmental stage. Application of qNPA to intracellularly stained, FACS-sorted cells should be broadly applicable to the analysis of gene expression in subpopulations of any heterogeneous tissue, including tumors.

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors.

BACKGROUND: Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. RESULTS: We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgf beta 1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. CONCLUSION: AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.

Deciphering von Hippel-Lindau (VHL/Vhl)-associated pancreatic manifestations by inactivating Vhl in specific pancreatic cell populations.

The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in alpha-cells or an insulin promoter in beta-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL) in terminally differentiated alpha-cells or beta-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model systems reported here will provide the basis for further functional and genetic studies to define molecular mechanisms involved in VHL-associated pancreatic diseases.

Blood glucose levels regulate pancreatic beta-cell proliferation during experimentally-induced and spontaneous autoimmune diabetes in mice.

BACKGROUND: Type 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic beta-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of beta-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ss-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation. METHODOLOGY AND PRINCIPAL FINDINGS: We adapted multi-parameter flow cytometry techniques (including DNA-content measurements and 5'-bromo-2'-deoxyuridine [BrdU] incorporation) to study pancreatic islet single cell suspensions. These studies demonstrate that beta-cell proliferation rapidly increases at diabetes onset, and that this proliferation is closely correlated with the diabetic animals' elevated blood glucose levels. For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the beta-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs. In contrast, other-than-ss endocrine islet cells did not exhibit the same glucose-dependent proliferative responses. Rather, disease-associated alterations of BrdU-incorporation rates of delta-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (alpha-cells). CONCLUSION: We conclude that murine beta-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic beta-cell autoimmunity. These data suggest that timely control of the destructive immune response after disease manifestation could allow spontaneous regeneration of sufficient beta-cell mass to restore normal glucose homeostasis.

Dynamic changes in pancreatic endocrine cell abundance, distribution, and function in antigen-induced and spontaneous autoimmune diabetes.

OBJECTIVE: Insulin deficiency in type 1 diabetes and in rodent autoimmune diabetes models is caused by beta-cell-specific killing by autoreactive T-cells. Less is known about beta-cell numbers and phenotype remaining at diabetes onset and the fate of other pancreatic endocrine cellular constituents. RESEARCH DESIGN AND METHODS: We applied multicolor flow cytometry, confocal microscopy, and immunohistochemistry, supported by quantitative RT-PCR, to simultaneously track pancreatic endocrine cell frequencies and phenotypes during a T-cell-mediated beta-cell-destructive process using two independent autoimmune diabetes models, an inducible autoantigen-specific model and the spontaneously diabetic NOD mouse. RESULTS: The proportion of pancreatic insulin-positive beta-cells to glucagon-positive alpha-cells was about 4:1 in nondiabetic mice. Islets isolated from newly diabetic mice exhibited the expected severe beta-cell depletion accompanied by phenotypic beta-cell changes (i.e., hypertrophy and degranulation), but they also revealed a substantial loss of alpha-cells, which was further confirmed by quantitative immunohistochemisty. While maintaining normal randomly timed serum glucagon levels, newly diabetic mice displayed an impaired glucagon secretory response to non-insulin-induced hypoglycemia. CONCLUSIONS: Systematically applying multicolor flow cytometry and immunohistochemistry to track declining beta-cell numbers in recently diabetic mice revealed an altered endocrine cell composition that is consistent with a prominent and unexpected islet alpha-cell loss. These alterations were observed in induced and spontaneous autoimmune diabetes models, became apparent at diabetes onset, and differed markedly within islets compared with sub-islet-sized endocrine cell clusters and among pancreatic lobes. We propose that these changes are adaptive in nature, possibly fueled by worsening glycemia and regenerative processes.

Immunomodulation of autoimmune diabetes by dendritic cells.

The biology and properties of dendritic cells (DCs) have been intensely studied in the research areas of infectious diseases, tumor immunology, and vaccine development. This unique subset of immune cells has recently also moved to the center of interest for basic and clinical research in autoimmunity, owing not only to the extraordinary importance of DCs in the initiation and sustenance of adaptive immune responses, but also to more recent discoveries about their profound ability to control and downregulate ongoing T-cell responses. We review current progress of using DCs in mice for induction and propagation of autoimmune T-cell responses and their therapeutic potential to dampen or even stop beta-cell-specific autoimmunity. Finally, we offer our perspective on how basic research progress in DC technology, mostly from mouse models, may translate into emerging diagnostic and therapeutic applications for human type 1 diabetes.

No evidence for mouse pancreatic beta-cell epithelial-mesenchymal transition in vitro.

We used cre/loxP-based genetic lineage tracing analysis to test a previously proposed hypothesis that in vitro cultured adult pancreatic beta-cells undergo epithelial-mesenchymal transition (EMT) to generate a highly proliferative, differentiation-competent population of mesenchymal islet "progenitor" cells. Our results in the mouse that are likely to be directly relevant to the human system show that adult mouse beta-cells do not undergo EMT in vitro and that the mesenchymal cells that arise in cultures of adult pancreas are not derived from beta-cells. We argue that these cells most likely originate from expansion of mesenchymal cells integral to the heterogeneous pancreatic islet preparations. As such, these mesenchymal "progenitors" might not represent the best possible source for generation of physiologically competent beta-cells for treatment of diabetes.

Cytotoxic T cell-mediated diabetes in RIP-CD80 transgenic mice: autoantigen peptide sensitivity and fine specificity.

Rodent immune-mediated diabetes model studies have advanced understanding of beta cell-specific T cell responses, and the testing of therapeutic approaches. We have used an inducible diabetes model based on rat insulin promotor (RIP)-driven expression of CD80 (B7-1) on pancreatic beta cells. Using these mice, we have established that immunizing with a single autoantigen can promote progressive islet inflammation and eventually T cell-mediated diabetes. We now describe a potent immunization protocol using peptide-pulsed mature dendritic cells (DCs) to examine peptide epitopes derived from endogenous (preproinsulin) and transgenically expressed beta cell antigens, namely lymphocytic choriomeningitis virus glycoprotein (LCMV-GP). LCMV-GP epitopes efficiently promote beta cell destruction, and the autoantigenic peptide concentration used to load the DCs correlates directly with diabetes onset. The system allowed us to assess cytotoxic T cell (CTL) fine specificity by immunizing with DCs presenting altered peptide ligands (APLs) of the dominant LCMV-GP epitope, gp33. Finally, using an adoptive transfer system, we tested alternative in vitro T cell activation conditions, including APLs and mitogens, for their impact on T cell effector function and diabetes onset. Our studies revealed a marked discrepancy between (inflammatory) effector functions and diabetes progression, thus emphasizing the importance of structural identity between sensitizing and target epitope and the context of initial T cell activation.

Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus.

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.

Beta cell-specific CD80 (B7-1) expression disrupts tissue protection from autoantigen-specific CTL-mediated diabetes.

T cell responses toward pancreatic beta cell autoantigens arise spontaneously or on immunization in many mouse strains, yet sustained islet infiltration and progressive diabetes rarely ensues. Most mouse diabetes models overcome the innocuous coexistence of anti-islet specific T cells and endogenous islets via incompletely understood mechanisms (e.g. the spontaneous disease onset of the non-obese diabetic mouse) or depend on overwhelming numbers of peripheral islet-specific T cells. We report that insulin promoter murine CD80 (RIP-CD80) transgenic mice are extraordinarily susceptible to autoantigen-induced diabetes, while spontaneous disease is rare. Autoimmunity to the pancreatic beta cell-expressed glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) was elicited by a single injection of syngeneic fibroblastoid cell lines (FCL) loaded with the immunodominant LCMV-GP peptide, gp33. While both RIP-GP(+)and RIP-CD80(+)GP(+)mice mounted moderate CD4-independent CTL responses, only CD80(+)GP(+)mice developed severe insulitis and diabetes due to islet-infiltration of activated, gp33-specific, CD8(+)T cells. Strikingly, DNA immunization using plasmids encoding LCMV-GP or murine preproinsulin also efficiently induced Ag-specific RIP-CD80-dependent diabetes. We conclude that aberrant CD80-expression in a peripheral tissue disrupts that tissue's natural resistance to CD8 T cell-mediated autoimmune destruction. This rodent model thus represents a novel approach to identify beta cell-derived autoantigenic determinants involved in the pathogenesis of autoimmune diabetes, and may also serve as a prototype approach to uncover relevant autoantigens leading to a variety of organ-specific autoimmune disorders.

Induction of autoimmune diabetes through insulin (but not GAD65) DNA vaccination in nonobese diabetic and in RIP-B7.1 mice.

Insulin has been used to modify T-cell autoimmunity in experimental models of type 1 diabetes. In a large clinical trial, the effect of insulin to prevent type 1 diabetes is currently investigated. We here show that insulin can adversely trigger autoimmune diabetes in two mouse models of type 1 diabetes, using intramuscular DNA vaccination for antigen administration. In female nonobese diabetic (NOD) mice, diabetes development was enhanced after preproinsulin (ppIns) DNA treatment, and natural diabetes resistance in male NOD mice was diminished by ppIns DNA vaccination. In contrast, GAD65 DNA conferred partial diabetes protection, and empty DNA plasmid was without effect. In RIP-B7.1 C57BL/6 mice (expressing the T-cell costimulatory molecule B7.1 in pancreatic beta-cells), autoimmune diabetes occurred in 70% of animals after ppIns vaccination, whereas diabetes did not develop spontaneously in RIP-B7.1 mice or after GAD65 or control DNA treatment. Diabetes was characterized by diffuse CD4(+)CD8(+) T-cell infiltration of pancreatic islets and severe insulin deficiency, and ppIns, proinsulin, and insulin DNA were equally effective for disease induction. Our work provides a new model of experimental autoimmune diabetes suitable to study mechanisms and outcomes of insulin-specific T-cell reactivity. In antigen-based prevention of type 1 diabetes, diabetes acceleration should be considered as a potential adverse result.