Cells were sorted directly into RNA lysis buffer (Zymo Research) in FACS tubes, spun down briefly following completion of collection, and frozen at 80C until further processing. lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease. Keywords:Germinal Center, Autoreactive B Cell, Systemic Lupus Erythematosus, Epitope Spreading, Autoantibody, Break-of-Tolerance == INTRODUCTION == Systemic lupus erythematosus (SLE) is characterized by the production of antibodies to nucleic acid antigens (Ags) (Rahman and Isenberg, 2008), with >75% of patients having serum autoantibodies to double-stranded DNA (compared to ~0.5% of healthy controls), which typically appear a few years before SLE is diagnosed (Arbuckle et al., 2003). SLE patients at or after disease onset drift in their serological reactivities towards a variety of nuclear, nucleolar, and protein-DNA complexes: a process known as epitope spreading. Although the mechanism is not well understood, epitope spreading is thought to be driven by chronic immune responses causing inclusion of new autoreactive B cell clones (Arbuckle et al., 2003;Cornaby et al., 2015;Vanderlugt and Miller, 2002). Affinity-matured antibodies (Abs) arise in germinal centers (GCs), wherein B cell clones cycle between division, somatic hypermutation (SHM), and selection based on Ag affinity (Victora et al., 2010). This process of random mutation can generate BCRs that recognize self-Ags (Mietzner et al., 2008;Tiller et al., 2007); however, it has long been thought that GCs are able to limit the affinity maturation of autoreactive Oxcarbazepine cells (Han et al., 1995;Pulendran et al., 1995;Shokat and Goodnow, 1995;Vinuesa et Rabbit Polyclonal to Akt1 (phospho-Thr450) al., 2009). In fact, display of self-Ag by follicular dendritic cells (FDCs) within GCs can delete autoreactive cells (Yau et al., 2013), and autoreactive B cells can mutate away from autoreactivity (Reed et al., 2016;Sabouri et al., 2014). Therefore, it has remained questionable whether autoreactive B cells in GCs follow the Oxcarbazepine same rules of Ag engagement with FDCs, clonal evolution, and affinity maturation as those in foreign-Ag-elicited GCs. Many autoimmune mouse models have spontaneous GC formation (Luzina et al., 2001), but as these models are based on genetically modified B or T cells or have uncharacterized, complex genetic backgrounds, they are poorly suited to studying natural autoreactive GC behavior. Here, we developed a chimera mouse model that has spontaneous autoreactive GCs composed of self-reactive Oxcarbazepine B cells from the wild-type (WT) repertoire in a genetically normal context. Surprisingly, we find that there is no limit on the evolution of autoreactive GCs that is, they generate B cells targeting other self-Ags once tolerance is broken. == RESULTS == == 564Igi Mice Display Spontaneous GCs, VDJ diversification, SHM, and CSR == The 564Igi mouse is a murine model of SLE on C57BL/6 (B6) background, generated by knock-in of the heavy (H) and kappa light (K) chain of an autoreactive B cell clone targeting ribonuclear complexes (Berland et al., 2006). In heterozygous 564Igi mice, which carry a single copy of the knock-in H and K chain, ~50% of circulating B cells express the knock-in BCR (identified by anti-idiotype [Id] Ab staining). The remaining half are Id negative (Id) due to receptor editing or allelic inclusion (Berland et al., 2006;Chatterjee et al., 2013;Das et al., 2017;Luning Prak et al., 2011). The mice have high titers of IgG against nuclear-associated Ags, but do not exhibit disease until late in life. Already at 6 weeks of age, heterozygous 564Igi mice (564het) harbored robust GC B cell populations in spleen and cutaneous lymph nodes (LN) (Figure 1A). Littermates that had the H chain knock-in but lacked the K chain (564het K) had baseline GC B cell frequencies comparable to B6 controls (Figure 1A). The presence of spontaneous GC B cells correlated with the presence of circulating Id+(knock-in BCR) cells (Figure 1BandFigure S1A, Spearman correlation, p=0.0002.