Signaling through the B cell receptor (BCR) can drive B cell activation and contribute to B cell differentiation into antibody-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. Here, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is down regulated in B cells by BCR or TLR signaling through a pathway dependent on PI3 kinase, Btk, IKK2 and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase SHP1 or membrane receptors CD22 and/or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings indicate that downregulation of Ets1 occurs in response to B cell activation via either BCR or TLR signaling thereby allowing B cell differentiation and that the maintenance of Ets1 expression is an important function of the inhibitory Lyn → CD22/SiglecG → SHP1 pathway in B cells.

Accumulation of plasma cells and autoantibodies against nuclear antigens characterize both human and murine lupus. Understanding how these processes are controlled may reveal novel therapeutic targets for this disease. Mice deficient in Lyn, a negative regulator of B and myeloid cell activity, develop lupus-like autoimmune disease. Here, we show that lyn À/À mice exhibit increased splenic plasmablasts and plasma cells and produce IgM against a wide range of self-antigens. Both events require Btk, a target of Lyn-dependent inhibitory pathways. A Btk-dependent increase in the expression of the plasma cell survival factor IL-6 by lyn À/À splenic myeloid cells was also observed. Surprisingly, IL-6 was not required for plasma cell accumulation or polyclonal IgM autoreactivity in lyn À/À mice. IL-6 was, however, necessary for the production of IgG autoantibodies, which we show are focused towards a limited set of nucleic acid-containing and glomerular autoantigens in lyn À/À mice. A similar uncoupling of plasma cell accumulation from IgG autoantibodies was seen in lyn 1/À mice. Plasma cell accumulation and polyclonal IgM autoreactivity are therefore controlled separately from, and are insufficient for, the production of IgG against lupus-associated autoantigens. Regulators of either of these two checkpoints may be attractive therapeutic targets for lupus.Key words: Autoimmunity . Btk . IL-6 . Lyn . Plasma cell Supporting Information available online IntroductionThe autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens resulting in autoantibody production, immune complex deposition, inflammation, and end organ damage. Aberrant accumulation of antibody-secreting cells, which include among them autoreactive specificities, is a common feature of human SLE [1] and several genetically distinct models of murine lupus, including NZB Â NZW and Lyn-deficient mice [2][3][4][5][6][7][8][9]. This has been attributed to an unusually favorable splenic plasma cell survival niche and impaired plasma cell homing to the bone marrow in the NZB Â NZW model [5,6] Lyn inhibits responses to BCR-crosslinking by phosphorylating the ITIM of several inhibitory receptors [12]. B cells from lyn À/À mice are thus hyperresponsive to BCR stimulation [4,12]. lyn À/À mice also demonstrate numerous defects in myeloid cells, such as increased myelopoiesis [13], increased integrin signaling [14], and increased production of cytokines by activated macrophages, mast cells, and, in some circumstances, dendritic cells [15][16][17]. These cytokines include IL-6, a plasma cell survival factor [18][19][20], the expression of which has been shown to be elevated in both murine and human lupus [21][22][23]. It plays a role in the production of anti-DNA antibodies in murine lupus models both in vitro and in vivo [23,24].A major target of Lyn-dependent inhibitory pathways in B cells is the BCR signaling component Btk [25][26][27]. Btk and Lyn also have opposing effects in macrophages [15,28], dendritic cells [...

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk−/− mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk−/− mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk−/− mice. A transgene expressing a low level of Btk in B cells (Btk lo ) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk lo B cells. anti-DNA IgG was not observed in 56R.Btk lo mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.

Summary The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of SLE, lack an inhibitor of B- and myeloid cell activation. This results in B-cell hyperresponsiveness, plasma cell (PC) accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn−/− mice. We find that IL-21 mRNA is reduced in the spleens of lyn−/−IL-6−/− and lyn−/−Btklo mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for PC accumulation and IgM autoantibodies in lyn−/− mice, it is required for anti-DNA IgG antibodies and some aspects of T cell activation. Surprisingly, GN still develops in lyn−/−IL-21−/− mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA antigens. These studies identify a specific role for IL-21 in the class switching of anti-DNA B-cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn−/− mice.

B-1 cells are important players in the first line of defense against pathogens. According to current models for the origin of B-1 cells, they either represent a separate lineage from conventional B-2 cells or differentiate from conventional B-2 cells via an intermediate, B-1(int), in response to positive selection by antigen. Here we show that Btk, a Tec family kinase that mediates B cell antigen receptor (BCR) signaling, is required at multiple stages of B-1 cell development. VH12 anti-phosphatidylcholine (PtC) IgH transgenic mice provide a model for the induced differentiation of B-1 cells. This transgene selects for PtC-reactive cells and induces them to adopt a B-1 phenotype. Both processes have been shown to depend on Btk. To determine whether this is secondary to a requirement for Btk in the development of mature B-2 cells, we crossed VH12 transgenic mice to mice expressing low levels of Btk. B-2 cell development occurs normally in Btk(lo) mice despite reduced responsiveness to BCR crosslinking. Analysis of VH12.Btk(lo) mice reveals that Btk regulates the B-1(int) to B-1 transition and/or the survival of splenic B-1 cells, in part via a mechanism independent of its role in BCR signaling. We also show that Btk mediates the survival of, and expression of IL-10 by, those B-1 cells that do develop and migrate to the peritoneum. Multiple roles for Btk in B-1 cell development and maintenance may explain the particular sensitivity of this population to mutations in components of Btk signaling pathways.