Endogenous retrovirus Gag antigen and its gene variants are unique autoantigens expressed in the pancreatic islets of non-obese diabetic mice.

Endogenous retrovirus (ERV) are remnants of ancient retroviruses that have been incorporated into the genome and evidence suggests that they may play a role in the etiology of T1D. We previously identified a murine leukemia retrovirus-like ERV whose Env and Gag antigens are involved in autoimmune responses in non-obese diabetic (NOD) mice. In this study, we show that the Gag antigen is present in the islet stromal cells. Although Gag gene transcripts were present, Gag protein was not detected in diabetes-resistant mice. Cloning and sequencing analysis of individual Gag genes revealed that NOD islets express Gag gene variants with complete open-reading frames (ORFs), in contrast to the diabetes-resistant mice, whose islet Gag gene transcripts are mostly non-ORFs. Importantly, the ORFs obtained from the NOD islets are extremely heterogenous, coding for various mutants that are absence in the genome. We further show that Gag antigens are stimulatory for autoreactive T cells and identified one islet-expressing Gag variant that contains an altered peptide ligand capable of inducing IFN-gamma release by the T cells. The data highlight a unique retrovirus-like factor in the islets of the NOD mouse strain, which may participate in key events triggering autoimmunity and T1D.

Autoimmune Responses to Exosomes and Candidate Antigens Contribute to Type 1 Diabetes in Non-Obese Diabetic Mice.

PURPOSE OF REVIEW: The initial autoimmune trigger of type 1 diabetes (T1D) remains unclear. In non-obese diabetic (NOD) mice, islet inflammation starts early in life, suggesting the presence of an endogenous trigger for the spontaneous autoimmune response in this T1D mouse model. In this review, we argue that abnormal release of exosomes might be the trigger of the early inflammatory and autoimmune responses in the islets. RECENT FINDINGS: Exosomes are nano-sized membrane complexes that are secreted by cells following fusion of late endosomes and/or multivesicular bodies with the plasma membrane. They are known extracellular messengers, communicating among neighboring cells via transporting large molecules from parent cells to recipient cells. Recent evidence demonstrates that these extracellular vesicles can modulate immune responses. It has been shown that insulinoma and islet mesenchymal stem cell-released exosomes are potent immune stimuli that can induce autoreactive B and T cells. Searching for candidate antigens in the exosomes identified endogenous retrovirus (ERV) Env and Gag antigens, which are homologous to an endogenous murine leukemia retrovirus. Autoantibodies and autoreactive T cells spontaneously developed in NOD mice can react to these retroviral antigens. More importantly, expression of the retroviral antigens in the islet mesenchymal stem cells is associated with disease susceptibility, and the expression is restricted to T1D-susceptible but not resistant mouse strains. Exosomes are novel autoimmune targets, carrying autoantigens that can stimulate innate and adaptive immune responses. An abnormal or excess release of exosomes, particularly those ones containing endogenous retroviral antigens might be responsible for triggering tissue-specific inflammatory and autoimmune responses.

Type 1 diabetes pathogenesis is modulated by spontaneous autoimmune responses to endogenous retrovirus antigens in NOD mice.

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non-obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well-characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus-like particles produced by co-expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes-resistant mice developed anti-Env autoantibodies that increase in titer as disease progresses. A lentiviral-based RNA interference knockdown of Gag revealed that Gag contributes to the MV-induced T-cell response, whose diabetogenic function can be demonstrated via cell-transfer into immune-deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet-derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes-resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti-retroviral responses to trigger autoimmunity.

Exosomes released by islet-derived mesenchymal stem cells trigger autoimmune responses in NOD mice.

Exosomes (EXOs) are secreted, nano-sized membrane vesicles that contain potent immunostimulatory materials. We have recently demonstrated that insulinoma-released EXOs can stimulate the autoimmune responses in nonobese diabetic (NOD) mice, a spontaneous disease model for type 1 diabetes. To investigate whether primary islet cells can produce EXOs, we isolated cells from the islet of Langerhans of NOD mice and cultured them in vitro. Interestingly, cultured islets release fibroblast-like, fast-replicating cells that express mesenchymal stem cell (MSC) markers, including CD105 and stem-cell antigen-1. These islet MSC-like cells release highly immunostimulatory EXOs that could activate autoreactive B and T cells endogenously primed in NOD mice. Serum EXO levels and EXO-induced interferon-γ production were positively correlated with disease progression at the early prediabetic stage. Consistent with these observations, immunohistological analysis of pancreata showed that CD105(+) cells are restricted to the peri-islet area in normal islets but penetrate into the ß-cell area as lymphocyte infiltration occurs. Immunization with EXOs promoted expansion of transferred diabetogenic T cells and accelerated the effector T cell-mediated destruction of islets. Thus, EXOs could be the autoantigen carrier with potent adjuvant activities and may function as the autoimmune trigger in NOD mice.

Insulinoma-released exosomes activate autoreactive marginal zone-like B cells that expand endogenously in prediabetic NOD mice.

Exosomes (EXOs) are nano-sized secreted microvesicles that can function as potent endogenous carriers of adjuvant and antigens. To examine a possible role in autoimmunity for EXOs, we studied EXO-induced immune responses in nonobese diabetic (NOD) mice, an autoimmune-prone strain with tissue-specific targeting at insulin-secreting beta cells. EXOs released by insulinoma cells can activate various antigen-presenting cells to secrete several proinflammatory cytokines and chemokines. A subset of B cells responded to EXO stimulation in culture by proliferation, and expressed surface markers representing marginal zone B cells, which was independent of T helper cells. Importantly, splenic B cells from prediabetic NOD mice, but not diabetic-resistant mice, exhibited increased reactivity to EXOs, which was correlated with a high level of serum EXOs. We found that MyD88-mediated innate TLR signals were essential for the B-cell response; transgenic B cells expressing surface immunoglobulin specific for insulin reacted to EXO stimulation, and addition of a calcineurin inhibitor FK506 abrogated the EXO-induced B-cell response, suggesting that both innate and antigen-specific signals may be involved. Thus, EXOs may contribute to the development of autoimmunity and type 1 diabetes in NOD mice, partially via activating autoreactive marginal zone-like B cells.