Silencing of high-affinity insulin-reactive B lymphocytes by anergy and impact of the NOD genetic background in mice

Abstract

Aims/hypothesis Previous studies have demonstrated that high-affinity insulin-binding B cells (IBCs) silenced by anergy in healthy humans lose their anergy in islet autoantibody-positive individuals with recent-onset type 1 diabetes, and in autoantibody-negative first-degree relatives carrying certain risk alleles. Here we explore that hypothesis that IBCs are found in the immune periphery of disease-resistant C57BL/6-H2g7 mice, where, as in healthy humans, they are anergic, but that in disease-prone genetic backgrounds (NOD) become activated and migrate to the pancreas and pancreatic lymph nodes, where they participate in the development of type 1 diabetes. Methods We compared the status of high-affinity IBCs in disease-resistant VH125.C57BL/6-H2g7 and disease-prone VH125.NOD mice. Results Consistent with findings in healthy humans, high-affinity IBCs reach the periphery in disease-resistant mice and are anergic, as indicated by a reduced expression of membrane IgM, unresponsiveness to antigen and failure to become activated or accumulate in the pancreatic lymph nodes or pancreas. In NOD mice, high-affinity IBCs reach the periphery early in life and increase in number prior to the onset of hyperglycaemia. These cells are not anergic; they become activated, produce autoantibodies and accumulate in the pancreas and pancreatic lymph nodes prior to disease development. Conclusions/interpretation These findings are consistent with genetic determination of the escape of high-affinity IBCs from anergy and their early contribution to the development of type 1 diabetes.

A Precision B Cell–Targeted Therapeutic Approach to Autoimmunity Caused by Phosphatidylinositol 3-Kinase Pathway Dysregulation

“…NOD mice have increased susceptibility to additional autoimmune diseases, such as rheumatoid arthritis, SLE, and the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis (60). Reduced B cell expression of PTEN has been reported in lupus patients (22), and we have observed reduced PTEN levels in the B cells of both T1D and AITD patients (29,61). Thus, loss of B cell tolerance in both human and mouse may be driven in part by PI3K pathway dysregulation.…”

“…We, and others, have shown that acute deletion of SHIP-1 or PTEN and expression of a constitutively active catalytic subunit of PI3K in anergic B cells leads to immediate loss of anergy, followed by cell proliferation, differentiation, and production of autoantibodies, thus demonstrating the importance of these proteins and their regulation of the PI3K pathway in maintaining B cell anergy (19,27,28). Importantly, B cells from SLE, type 1 diabetes (T1D) and autoimmune thyroiditis (AITD) patients express reduced levels of PTEN, consistent with a possible role in autoimmunity (22,29). The apparent inability to regulate the PI3K pathway in these patients suggests that inhibition of PI3K could, by compensating for reduced inositol lipid phosphatase activity, be an affective therapeutic.…”

Defining B cell tolerance checkpoints and their modulation by immunotherapy

“…Autoreactive B cells which developed in these mice had a range of affinities for self-antigen and developed at much lower frequencies than the conventional BCR transgenic systems [27]. These studies revealed a deletional landscape that appears to preserve low-affinity autoreactive BCRs while deleting high affinity ones [29,31,170,185,590]. In the present study, a contribution from central deletion could not be detected.…”

“…For example, crossing actin.mOVA mice to mice with genetic lesions in TLR7, as used in BCR transgenic models [125,588], may be an effective model to study SLE given the role of TLR7 in loss of tolerance [232]. Similar studies on insulin-specific diabetogenic B cells are currently being carried out on a NOD background [590] and humans with autoimmune diabetes [165,166].…”

Techniques to Study Antigen-Specific B Cell Responses

“…Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61). The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79,81,82). Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity.…”