Effective central tolerance is required to control the large extent of autoreactivity normally present in the developing B cell repertoire. Insulin-reactive B cells are required for T1D in the NOD mouse, as engineered mice lacking this population are protected from disease. The VH125Tg model is used to define this population, which is found with increased frequency in the periphery of NOD vs. non-autoimmune C57BL/6 VH125Tg mice, but the ontogeny of this disparity is unknown. To better understand the origins of these pernicious B cells, anti-insulin B cells were tracked during development in the polyclonal repertoire of VH125Tg mice. An increased proportion of insulin-binding B cells is apparent in NOD mice at the earliest point of antigen commitment in the bone marrow. Two predominant light chains were identified in B cells that bind heterologous insulin. Interestingly, Vκ4-57-1 polymorphisms that confer a CDR3 Pro-Pro motif enhance self-reactivity in VH125Tg/NOD mice. Despite binding circulating autoantigen in vivo, anti-insulin B cells transition from the parenchyma to the sinusoids in the bone marrow of NOD mice and enter the periphery unimpeded. Anti-insulin B cells expand at the site of autoimmune attack in the pancreas and correlate with increased numbers of IFN-γ producing cells in the repertoire. These data identify failure to cull autoreactive B cells in the bone marrow as the primary source of anti-insulin B cells in NOD mice, and suggest that dysregulation of central tolerance permits escape into the periphery to promote disease.

BackgroundPressure overload and prolonged angiotensin II (Ang II) infusion elicit cardiac hypertrophy in Ang II receptor 1 (AT1) null mouse, whereas Ang II receptor 2 (AT2) gene deletion abolishes the hypertrophic response. The roles and signals of the cardiac AT2 receptor still remain unsettled. Promyelocytic leukemia zinc finger protein (PLZF) was shown to bind to the AT2 receptor and transmit the hypertrophic signal. Using PLZF knockout mice we directed our studies on the function of PLZF concerning the cardiac specific transcription factor GATA4, and GATA4 targets.Methodology and Principal FindingsPLZF knockout and age-matched wild-type (WT) mice were treated with Ang II, infused at a rate of 4.2 ng·kg−1·min−1 for 3 weeks. Ang II elevated systolic blood pressure to comparable levels in PLZF knockout and WT mice (140 mmHg). WT mice developed prominent cardiac hypertrophy and fibrosis after Ang II infusion. In contrast, there was no obvious cardiac hypertrophy or fibrosis in PLZF knockout mice. An AT2 receptor blocker given to Ang II-infused wild type mice prevented hypertrophy, verifying the role of AT2 receptor for cardiac hypertrophy. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that PLZF bound to the GATA4 gene regulatory region. A Luciferase assay verified that PLZF up-regulated GATA4 gene expression and the absence of PLZF expression in vivo produced a corresponding repression of GATA4 protein.ConclusionsPLZF is an important AT2 receptor binding protein in mediating Ang II induced cardiac hypertrophy through an AT2 receptor-dependent signal pathway. The angiotensin II-AT2-PLZF-GATA4 signal may further augment Ang II induced pathological effects on cardiomyocytes.

To clarify the relationship between Angiotensin II AT 1 and AT 2 receptors, we studied AT 1 receptor mRNA and binding expression in tissues from AT 2 receptor gene-disrupted (AT 2 −/−) female mice, where AT 2 receptors are not expressed in vivo, using in situ hybridization and quantitative autoradiography. Wild type mice expressed AT 1A receptor mRNA and AT 1 receptor binding in lung parenchyma, the spleen, predominantly in the red pulp, and in liver parenchyma. In wild type mice, lung AT 2 receptors were expressed in lung bronchial epithelium and smooth muscle, and were not present in the lung parenchyma, the spleen or the liver. This indicates that AT 1 and AT 2 receptors were not expressed in the same cells. In AT 2 −/− mice, we found higher AT 1A receptor mRNA and AT 1 receptor binding in lung parenchyma and in the red pulp of the spleen, but not in the liver, when compared to littermate wild-type controls. Our results suggest that impaired AT 2 receptor function upregulates AT 1 receptor transcription and expression in a tissue-specific manner and in cells not expressing AT 2 receptors. AT 1 upregulation explains the increased sensitivity to Angiotensin II characteristic of the AT 2 −/− phenotype, consistent with enhanced AT 1 receptor activation in a number of tissues.

Immune tolerance breakdown precedes autoimmune disease. Receptor editing is an immune tolerance mechanism that alters BCR specificity to combat frequent autoreactivity in the developing B cell repertoire. Much of what is known about receptor editing derives from multivalent antigens, or soluble antigens which interact with the BCR with supra-physiologic affinity (1010 M-1). A system was therefore developed in which the BCR interacts with insulin autoantigen with a more physiologic affinity (107 M-1) to better reflect B cells that arise in the developing bone marrow repertoire. Surprisingly, circulating insulin was found to elicit receptor editing in non-autoimmune mice. This process occurred less efficiently in the type 1 diabetes-prone NOD strain, consistent with enhanced anti-insulin B cell escape into the periphery from the bone marrow observed in this strain. Receptor editing efficiency was enhanced by treatment with an insulin-targeting mAb that selectively eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. A F(ab’)2 of this antibody also depleted developing anti-insulin B lymphocytes, confirming receptor editing as a central tolerance mechanism targeted by this approach. These studies show that a small protein hormone of modest affinity elicits receptor editing, but with less efficiency in the context of autoimmunity. This defect can be overcome with autoantigen-targeted therapy, which offers promise for the treatment of autoimmune disease.