Autoreactive B lymphocytes that are not culled by central tolerance in the bone marrow frequently enter the peripheral repertoire in a state of functional impairment, termed anergy. These cells are recognized as a liability for autoimmunity, but their contribution to disease is not well-understood. Insulin-specific 125Tg B cells support T cell-mediated Type 1 diabetes (T1D) in nonobese diabetic (NOD) mice, despite being anergic to B cell mitogens and T cell dependent immunization. Using this model, the potential of anergic, autoreactive B cells to present antigen and activate T cells was investigated. The data show that: a) insulin is captured and rapidly internalized by 125Tg BCRs, b) these antigen-exposed B cells are competent to activate both experienced and naïve CD4+ T cells, c) anergic 125Tg B cells are more efficient than naïve B cells at activating T cells when antigen is limiting, and d) 125Tg B cells are competent to generate low-affinity insulin B chain epitopes necessary for activation of diabetogenic anti-insulin BDC12-4.1 T cells, indicating the pathological relevance of anergic B cells in T1D. Thus, phenotypically tolerant B cells that are retained in the repertoire may promote autoimmunity by driving activation and expansion of autoaggressive T cells via antigen-presentation.

Autoreactive B lymphocytes are essential for the development of T cell–mediated type 1 diabetes (T1D). Cytoplasmic Bruton’s tyrosine kinase (BTK) is a key component of B cell signaling, and its deletion in T1D-prone NOD mice significantly reduces diabetes. However, the role of BTK in the survival and function of autoreactive B cells is not clear. To evaluate the contributions of BTK, we used mice in which B cells express an anti-insulin BCR (125Tg) and promote T1D, despite being anergic. Crossing Btk deficiency onto 125Tg mice reveals that, in contrast to immature B cells, mature anti-insulin B cells are exquisitely dependent upon BTK, because their numbers are reduced by 95%. BTK kinase domain inhibition reproduces this effect in mature anti-insulin B cells, with less impact at transitional stages. The increased dependence of anti-insulin B cells on BTK became particularly evident in an Igκ locus site–directed model, in which 50% of B cells edit their BCRs to noninsulin specificities; Btk deficiency preferentially depletes insulin binders from the follicular and marginal zone B cell subsets. The persistent few Btk-deficient anti-insulin B cells remain competent to internalize Ag and invade pancreatic islets. As such, loss of BTK does not significantly reduce diabetes incidence in 125Tg/NOD mice as it does in NOD mice with a normal B cell repertoire. Thus, BTK targeting may not impair autoreactive anti-insulin B cell function, yet it may provide protection in an endogenous repertoire by decreasing the relative availability of mature autoreactive B cells.

Expansion of autoimmune-prone marginal zone (MZ) B cells has been implicated in type 1 diabetes (T1D). To test disease contributions of MZ B cells in NOD mice, Notch2 haploinsufficiency (Notch2+/−) was introduced, but failed to eliminate the MZ, as it does in C57BL/6 mice. Notch2+/−/NOD have MZ B cell numbers similar to WT C57BL/6, yet still develop diabetes. To test whether BCR-signaling supports Notch2+/−/NOD MZ B cells, Bruton's tyrosine kinase (Btk)-deficiency was introduced. Surprisingly, MZ B cells failed to develop in Btk-deficient Notch2+/−/NOD mice. Expression of Notch2 and its transcriptional target, Hes5, were increased in NOD MZ B cells compared with C57BL/6 MZ B cells. Btk-deficiency reduced Notch2+/− signaling exclusively in NOD B cells, suggesting that BCR-signaling enhances Notch2 signaling in this autoimmune model. The role of BCR-signaling was further investigated using an anti-insulin transgenic BCR (125Tg). Anti-insulin B cells in 125Tg/Notch2+/−/NOD mice populate an enlarged MZ, suggesting that low level BCR signaling overcomes reliance on Notch2. Tracking clonotypes of anti-insulin B cells in H chain only VH125Tg/NOD mice showed that BTK-dependent selection into the MZ depends on strength of antigenic binding, while Notch2-mediated selection does not. Importantly, anti-insulin B cell numbers were reduced by Btk-deficiency, but not Notch2-haploinsufficiency. These studies show that: 1) Notch2-haploinsufficiency limits NOD MZ B cell expansion without preventing T1D, 2) BTK supports the Notch2 pathway in NOD MZ B cells, and 3) autoreactive NOD B cell survival relies on BTK more than Notch2, regardless of MZ location, which may have important implications for disease-intervention strategies.

Type 1 diabetes (T1D) results from adaptive autoimmune destruction of insulin-producing beta cells in pancreatic islets. Macrophages and dendritic cells (DCs) have also been implicated in the disease process, although their roles are less well-studied than those of T cells. S100A4 is a small calcium-activated molecule that contributes to cancer metastases and fibrosis, and has recently been linked to autoimmunity. We have discovered S100A4 among inflammatory infiltrates in pancreatic islets in a T1D model. Cells expressing S100A4 bear surface markers associated with antigen-presentation, such as MHCII and CD86. These cells include CD11c+ DCs, CD11b+ macrophages, and CD11b+/CD11c+ myeloid populations. Neither plasmacytoid DCs (PDCA1+/Ly6C+/B220+) nor lymphoid DCs (CD8+) express S100A4. S100A4-deficiency was engineered by GFP-targeting and introgressed onto the nonobese diabetic (NOD) mouse model of T1D for >10 generations. S100A4-/-/NOD mice are protected from the development of T1D, with a concomitant reduction in insulitis. However, GFP-expression in S100A4-deficient cells indicates that these cell populations remain in the inflamed islets. When compared to S100A4-sufficient counterparts, S100A4-deficient bone marrow-derived DCs have decreased ability to upregulate CD86, or to present antigen and activate T cells in vitro. Thus, S100A4 may contribute to the development of T1D by supporting myeloid cell antigen-presentation to autoreactive T cells.