Reversing Tolerance in Isotype Switch–Competent Anti-Insulin B Lymphocytes

Abstract

Autoreactive B lymphocytes that escape central tolerance and mature in the periphery are a liability for developing autoimmunity. IgG insulin autoantibodies that predict type 1 diabetes and complicate insulin therapies indicate that mechanisms for tolerance to insulin are flawed. To examine peripheral tolerance in anti-insulin B cells, we generated C57BL/6 mice that harbor anti-insulin VDJH-125 site directed to the native IgH locus (VH125SD). Class switch–competent anti-insulin B cells fail to produce IgG Abs following T cell–dependent immunization of VH125SD mice with heterologous insulin, and they exhibit markedly impaired proliferation to anti-CD40 plus insulin in vitro. In contrast, costimulation with LPS plus insulin drives robust anti-insulin B cell proliferation. Furthermore, VH125SD mice produce both IgM and IgG2a anti-insulin Abs following immunization with insulin conjugated to type 1 T cell–independent Brucella abortus ring test Ag (BRT). Anti-insulin B cells undergo clonal expansion in vivo and emerge as IgM+ and IgM− GL7+Fas+ germinal center (GC) B cells following immunization with insulin-BRT, but not BRT alone. Analysis of Igκ genes in VH125SD mice immunized with insulin-BRT reveals that anti-insulin Vκ from the preimmune repertoire is selected into GCs. These data demonstrate that class switch–competent anti-insulin B cells remain functionally silent in T cell–dependent immune responses, yet these B cells are vulnerable to reversal of anergy following combined BCR/TLR engagement that promotes Ag-specific GC responses and Ab production. Environmental factors that lead to infection and inflammation could play a critical yet underappreciated role in driving loss of tolerance and promoting autoimmune disease.