SUMMARY On the lupus-prone MRL-lpr/lpr (MRL-lpr) background AM14 rheumatoid factor (RF) B cells are activated, differentiate into plasmablasts, and undergo somatic hypermutation outside of follicles. Using multiple strategies to impair T cells, we found that such AM14 B cell activation did not require T cells, but could be modulated by them. In vitro, the signaling adaptor MyD88 is required for IgG anti-chromatin to stimulate AM14 B cell proliferation when T cells are absent. However the roles of Toll-like receptors (TLRs) in AM14 B cell activation in vivo have not been investigated. We found that activation, expansion and differentiation of AM14 B cells depended on MyD88; however, mice lacking either TLR7 or TLR9 displayed partial defects, indicating complex roles for these receptors. T-independent activation of certain autoreactive B cells, which instead can gain stimuli via endogenous TLR ligands, may be the initial step in the generation of canonical autoantibodies.

A dominant type of spontaneous autoreactive B cell activation in murine lupus is the extrafollicular generation of plasmablasts. The factors governing such activation have been difficult to identify due to the stochastic onset and chronic nature of the response. Thus, the ability to induce a similar autoreactive B cell response with a known autoantigen in vivo would be a powerful tool in deciphering how autoimmune responses are initiated. We report here the establishment and characterization of a system to initiate autoreactive extrafollicular B cell responses, using IgG anti-chromatin antibodies, that closely mirrors the spontaneous response. We demonstrate that exogenously administered anti-chromatin antibody, presumably by forming immune complexes with released nuclear material, drives activation of rheumatoid factor B cells in AM14 Tg mice. Anti-chromatin elicits autoreactive B cell activation and development into antibody-forming cells at the T zone/red pulp border. Plasmablast generation occurs equally in BALB/c, MRL/+ and MRL/lpr mice, indicating that an autoimmuneprone genetic background is not required for the induced response. Importantly, infused IgG anti-chromatin induces somatic hypermutation in the absence of a GC response, thus proving the extrafollicular somatic hypermutation pathway. This system provides a window on the initiation of an autoantibody response and reveals authentic initiators of it.See accompanying commentary: http://dx

Several reports have emerged demonstrating that many insect species have unanticipated “trained” immune responses, where immunization with a pathogen has a significant and lasting protective effect. This is particularly interesting considering that many of the well-characterized vertebrate adaptive cell subsets and humoral tools associated with immunological memory are absent in invertebrates. Here we report our investigations of immunity in Periplaneta americana, the cockroach, and the use of imaging flow cytometry to investigate cellular changes in response to pathogen exposure. Staining for cellular features such as nuclear and cell membrane morphology, lysosomes, and other subcellular structures were used to categorize cells and monitor their changes over the course of an immune response. Stains are also used to reveal proliferation of certain cell subsets. As cockroaches are worldwide pests in both homes and hospitals, how they manage infection will be of broad interest. Additionally, elucidating rudimentary systems of adaptive immunity in invertebrates may facilitate our development of therapeutics for immunocompromised vertebrates whose well-understood mechanisms of defense are dysfunctional.

Memory B cells could be responsible for chronic autoimmunity, as suggested by results in SLE patients and SLE-prone mice: The frequency of AM14 RF transgenic B cells fluctuates in the peripheral blood of aged MRL/lpr mice. Activation of RF B cells in autoimmune MRL/lpr mice results in an extrafollicular response that undergoes somatic mutation. Lineage trees of RF clones suggest mutation upon secondary antigen exposure. We recently found that this response can be recapitulated in AM14 Tg mice that were exposed to IgG2a anti-chromatin Abs. The mechanism controlling extrafollicular activation and whether this response leads to a form of memory is unclear. When AM14 heavy chain knock-in B cells are transferred into non-transgenic recipients, followed by exposure to IgG2a anti-chromatin, an extrafollicular response that includes class switch ensues. We are tracking the development and function of these cells over time to determine if autoreactive resting memory B cells form, or alternatively, whether the maintenance of autoreactive B cell clones requires continuous autoantigen exposure and activation. Supported by 1F31AI071694-01 (RAS) and AI36529 (MJS)