B cell-activating factor (BAFF) from dendritic cells, monocytes and neutrophils is required for B cell maturation and autoantibody production in SLE-like autoimmune disease.

Purpose and methods: B cell-activating factor (BAFF) contributes to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Although several anti-BAFF Abs and derivatives have been developed for the treatment of SLE, the specific sources of BAFF that sustain autoantibody (auto-Ab) producing cells have not been definitively identified. Using BAFF-RFP reporter mice, we identified major changes in BAFF-producing cells in two mouse spontaneous lupus models (Tlr7 Tg mice and Sle1), and in a pristane-induced lupus (PIL) model. Results: First, we confirmed that similar to their wildtype Tlr7 Tg and Sle1 mice counterparts, BAFF-RFP Tlr7 Tg mice and BAFF-RFP Sle1 mice had increased BAFF serum levels, which correlated with increases in plasma cells and auto-Ab production. Next, using the RFP reporter, we defined which cells had dysregulated BAFF production. BAFF-producing neutrophils (Nphs), monocytes (MOs), cDCs, T cells and B cells were all expanded in the spleens of BAFF-RFP Tlr7 Tg mice and BAFF-RFP Sle1 mice compared to controls. Furthermore, Ly6Chi inflammatory MOs and T cells had significantly increased BAFF expression per cell in both spontaneous lupus models, while CD8- DCs up-regulated BAFF expression only in the Tlr7 Tg mice. Similarly, pristane injection of BAFF-RFP mice induced increases in serum BAFF levels, auto-Abs, and the expansion of BAFF-producing Nphs, MOs, and DCs in both the spleen and peritoneal cavity. BAFF expression in MOs and DCs, in contrast to BAFF from Nphs, was required to maintain homeostatic and pristane-induced systemic BAFF levels and to sustain mature B cell pools in spleens and BMs. Although acting through different mechanisms, Nph, MO and DC sources of BAFF were each required for the development of auto-Abs in PIL mice. Conclusions: Our findings underscore the importance of considering the relative roles of specific myeloid BAFF sources and B cell niches when developing treatments for SLE and other BAFF-associated autoimmune diseases.

Immune complex-driven neutrophil activation and BAFF release: a link to B cell responses in SLE.

OBJECTIVE: The role of neutrophils in driving pathogenic B cell responses in SLE is not fully understood. In this study, we explored the link between immune complex (IC)-driven neutrophil activation, the release of B cell pro-survival factor BAFF and B cell activation using SLE clinical samples. METHODS: BAFF levels were analysed in serum samples from patients with SLE (n=60) and healthy controls (HCs, n=20) by ELISA and correlated with markers of neutrophil activation and circulating IC levels. Neutrophils were stimulated with RNP/IgG ICs and neutrophil activation, the release of BAFF, and neutrophil-mediated B cell responses were studied in vitro. RESULTS: Levels of BAFF in patients with SLE were associated with markers of disease activity, including anti-dsDNA antibody titres (r=0.33, p<0.05), serum C3 levels (r=-0.57, p<0.001) and levels of circulating ICs (r=0.39, p<0.05). Stimulation of neutrophils from healthy individuals with RNP-ICs in vitro induced the release of BAFF (p<0.05), concomitant with formation of neutrophil extracellular traps (NETs) (p<0.05). In culture, neutrophils promoted B cell survival (p<0.05), proliferation (p<0.05) and CD27hiCD38hi plasmablast differentiation. CONCLUSIONS: Our results support a new mechanism by which ICs, on NET formation, induce the release of B cell pro-survival factor BAFF by neutrophils. Furthermore, neutrophils directly promoted B cell activation and cell differentiation. Targeting neutrophil-B cell interactions can be further explored as an approach for inhibiting pathogenic B cell responses in SLE.

B cell activating factor (BAFF) from neutrophils and dendritic cells is required for protective B cell responses against Salmonella typhimurium infection.

Mice lacking B cells are more susceptible to S. typhimurium infection. How B cells contribute to protective immunity against Salmonella and what signals drive their activation are still unclear. Neutrophils (Nphs), monocytes (MOs), and dendritic cells (DCs) are involved in early immune responses to control the initial replication of S. typhimurium. These cells can produce B cell activating factor (BAFF) required for mature B cell survival and may help regulate B cell responses during Salmonella infection. Using BAFF reporter mice (BAFF-RFP+/-), we discovered that an i.p. infection with a virulent strain of S. typhimurium increased BAFF expression in splenic conventional DCs (cDC) and inflammatory Ly6Chi MOs/DCs four days post-infection. S. typhimurium infection induced the release of BAFF from Nphs, a decrease of BAFF-RFP expression and expansion of BAFF-RFP+ Nphs in the spleen and peritoneal cavity. After S. typhimurium infection, serum BAFF levels and immature and mature B cell subsets and plasma cells increased substantially. Conditional knockout (cKO) mice lacking BAFF in either Nphs or cDCs compared to control Bafffl/fl mice had reduced up-regulation of systemic BAFF levels and reduced expansion of mature and germinal center B cell subsets after infection. Importantly, the cKO mice lacking BAFF from either Nphs or cDCs had impaired induction of Salmonella-specific IgM Abs, and were more susceptible to S. typhimurium infection. Thus, Nphs and cDCs are major cellular sources of BAFF driving B cell responses, required for mounting optimal protective immunity against lethal Salmonella infection.

Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients.

BACKGROUND: CD40-CD40L is a key co-stimulatory pathway for B cell activation. As such, its blockade can inhibit pathogenic B cell responses in autoimmune diseases, such as Sjogren's syndrome (SjS) and systemic lupus erythematosus (SLE). In this study, we examined the in vitro effects of KPL-404, a humanized anti-CD40 monoclonal antibody (Ab), on primary human B cells derived from either healthy donors (HD) or autoimmune patients and compared them to the effects of G28-5, a partially antagonistic anti-CD40 antibody. METHODS: PBMCs from HD or SjS and SLE patients were cultured in high-density cell cultures in the presence of IgG4 isotype control or anti-CD40 Abs KPL-404 or G28-5. Cells were stimulated with anti-CD3/CD28 cross-linking reagent ImmunoCult (IC) to induce CD40L-CD40-mediated B cell responses. B cell proliferation and activation, measured by dilution of proliferation tracker dye and the upregulation of CD69 and CD86, respectively, were assessed by flow cytometry. Anti-CD40 Ab cell-internalization was examined by imaging flow cytometry. Cytokine release in the PBMC cultures was quantified by bead-based multiplex assay. RESULTS: KPL-404 binds to CD40 expressed on different subsets of B cells without inducing cell depletion, or B cell proliferation and activation in in vitro culture. Under the same conditions, G28-5 promoted proliferation of and increased CD69 expression on otherwise unstimulated B cells. KPL-404 efficiently blocked the CD40L-CD40-mediated activation of B cells from HD at concentrations between 1 and 10 µg/ml. Treatment with KPL-404 alone did not promote cytokine production and blocked the production of IFNß in healthy PBMC cultures. KPL-404 efficiently blocked CD40L-CD40-mediated activation of B cells from patients with SjS and SLE, without affecting their anti-IgM responses or affecting their cytokine production. Consistent with the differences of their effects on B cell responses, KPL-404 was not internalized by cells, whereas G28-5 showed partial internalization upon CD40 binding. CONCLUSIONS: Anti-CD40 mAb KPL-404 showed purely antagonistic effects on B cells and total PBMCs. KPL-404 inhibited CD40L-CD40-mediated B cell activation in PBMC cultures from both healthy controls and autoimmune patients. These data support the therapeutic potential of CD40 targeting by KPL-404 Ab for inhibiting B cell responses in SjS and SLE.

BAFF Produced by Neutrophils and Dendritic Cells Is Regulated Differently and Has Distinct Roles in Antibody Responses and Protective Immunity against West Nile Virus.

B cell activating factor (BAFF) is essential for B cells to develop and respond to Ags. Dysregulation of BAFF contributes to the development of some autoimmune diseases and malignancies. Little is known about when, where, and how BAFF is produced in vivo and about which BAFF-producing cells contribute to B cell responses. To better understand BAFF functions, we created BAFF reporter (BAFF-RFP) mice and Baff floxed (Bafffl/fl ) mice. Splenic and bone marrow neutrophils (Nphs) from BAFF-RFP mice expressed the highest constitutive levels of BAFF; other myeloid subsets, including conventional dendritic cells (cDCs) and monocyte (MO) subsets, expressed lower levels. Treatment of BAFF-RFP mice with polyinosinic:polycytidylic acid increased BAFF expression in splenic Ly6Chi inflammatory MOs, CD11bhi activated NK subset, and in bone marrow myeloid precursors. Postinfection with West Nile virus (WNV), BAFF increased in CD8- cDCs and Nphs, and BAFF+ CD11bhi NK cells expanded in draining lymph nodes. The cell- and tissue-specific increases in BAFF expression were dependent on type I IFN signaling. MAVS also was required or contributed to BAFF expression in dendritic cell and MO subsets, respectively. Mice with deletion of Baff in either cDCs or Nphs had reduced Ab responses after NP-Ficoll immunization; thus, BAFF produced by both cDCs and Nphs contributes to T cell-independent Ab responses. Conversely, mice with a cDC Baff deficiency had increased mortality after WNV infection and decreased WNV-specific IgG and neutralizing Ab responses. BAFF produced by Nphs and cDCs is regulated differently and has key roles in Ab responses and protective immunity.

The Plasticity of Newly Formed B Cells.

Newly formed B cells (NF-B cells) that emerge from the bone marrow to the periphery have often been referred to as immature or transitional B cells. However, NF-B cells have several striking characteristics, including a distinct BCR repertoire, high expression of AID, high sensitivity to PAMPs, and the ability to produce cytokines. A number of findings do not support their designation as immature because NF-B cells have the potential to become Ab-producing cells and to undergo class-switch recombination. In this review, we provide a fresh perspective on NF-B cell functions and describe some of the signals driving their activation. We summarize growing evidence supporting a role for NF-B cells in protection against infections and as a potential source of autoantibody-producing cells in autoimmune diseases such as systemic lupus erythematosus.

High TLR7 Expression Drives the Expansion of CD19+CD24hiCD38hi Transitional B Cells and Autoantibody Production in SLE Patients.

Signaling through Toll-like receptor 7 (TLR7) drives the production of type I IFN and promotes the activation of autoreactive B cells and is implicated in the pathogenesis of systemic lupus erythematosus (SLE). While TLR7 has been extensively studied in murine lupus, much less is known about its role in the pathogenesis of human SLE. Genetic studies support a link between the TLR7 rs3853839 C/G polymorphism, which affects TLR7 mRNA turnover, and SLE susceptibility; however, the effects of this polymorphism on B cells have not been studied. Here we determined how changes in TLR7 expression affect peripheral B cells and auto-Ab production in SLE patients. High TLR7 expression in SLE patients driven by TLR7 rs3853839 C/G polymorphism was associated with more active disease and upregulation of IFN-responsive genes. TLR7hi SLE patients showed an increase in peripheral B cells. Most notably, the percentage and numbers of CD19+CD24++CD38++ newly-formed transitional (TR) B cells were increased in TLR7hi SLE patients as compared to HCs and TLR7norm/lo SLE patients. Using auto-Ab arrays, we found an increase and enrichment of auto-Ab specificities in the TLR7hi SLE group, including the production of anti-RNA/RNP-Abs. Upon in vitro TLR7 ligand stimulation, TR B cells isolated from TLR7hi but not TLR7norm/lo SLE patients produced anti-nuclear auto-Abs (ANA). Exposure of TR B cells isolated from cord blood to IFNα induced the expression of TLR7 and enabled their activation in response to TLR7 ligation in vitro. Our study shows that overexpression of TLR7 in SLE patients drives the expansion of TR B cells. High TLR7 signaling in TR B cells promotes auto-Ab production, supporting a possible pathogenic role of TR B cells in human SLE.

Sources of Pathogenic Nucleic Acids in Systemic Lupus Erythematosus.

A hallmark of systemic lupus erythematosus (SLE), and several related autoimmune diseases, is the presence of autoantibodies against nucleic acids and nucleic acid-binding proteins, as well as elevated type I interferons (IFNs), which appear to be instrumental in disease pathogenesis. Here we discuss the sources and proposed mechanisms by which a range of cellular RNA and DNA species can become pathogenic and trigger the nucleic acid sensors that drive type I interferon production. Potentially SLE-promoting DNA may originate from pieces of chromatin, from mitochondria, or from reverse-transcribed cellular RNA, while pathogenic RNA may arise from mis-localized, mis-processed, ancient retroviral, or transposable element-derived transcripts. These nucleic acids may leak out from dying cells to be internalized and reacted to by immune cells or they may be generated and remain to be sensed intracellularly in immune or non-immune cells. The presence of aberrant DNA or RNA is normally counteracted by effective counter-mechanisms, the loss of which result in a serious type I IFN-driven disease called Aicardi-Goutières Syndrome. However, in SLE it remains unclear which mechanisms are most critical in precipitating disease: aberrant RNA or DNA, overly sensitive sensor mechanisms, or faulty counter-acting defenses. We propose that the clinical heterogeneity of SLE may be reflected, in part, by heterogeneity in which pathogenic nucleic acid molecules are present and which sensors and pathways they trigger in individual patients. Elucidation of these events may result in the recognition of distinct "endotypes" of SLE, each with its distinct therapeutic choices.

CD22: A Regulator of Innate and Adaptive B Cell Responses and Autoimmunity.

CD22 (Siglec 2) is a receptor predominantly restricted to B cells. It was initially characterized over 30 years ago and named "CD22" in 1984 at the 2nd International workshop in Boston (1). Several excellent reviews have detailed CD22 functions, CD22-regulated signaling pathways and B cell subsets regulated by CD22 or Siglec G (2-4). This review is an attempt to highlight recent and possibly forgotten findings. We also describe the role of CD22 in autoimmunity and the great potential for CD22-based immunotherapeutics for the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE).

Regulation of B-lineage cells by caspase 6.

The caspase (Casp) family of proteases regulate both lymphocyte apoptosis and activation. Here, we show that Casp6 regulates early B-cell development. One-week-old Casp6 knockout (Casp6 KO) mice have significantly more splenic B-cell subsets than wild-type (WT) mice. Adult Casp6 KO mice have normal levels of total splenic B cells but have increased numbers of B1a B cells and CD43+ "transitional" or splenic red pulp (RP) B cells. These results suggested that Casp6 may function to control B-cell numbers under nonhomeostatic conditions and during B-cell development. Consistent with this model, reconstitution of B cells was dysregulated in Casp6 KO mice after sublethal irradiation. Furthermore, bone marrow pro-B, pre-B and immature B-cell numbers were significantly higher in 1-week-old Casp6 KO mice than in 1-week-old WT mice. Casp6 KO pro-B cells proliferated more in response to IL-7 than WT pro-B cells, suggesting that Casp6 regulates early B-cell responses to IL-7. Indeed, adult and aged Casp6 KO mice had elevated numbers of IL-7αR+ Sca1+ precursors of common lymphoid progenitors, suggesting Casp6 may help regulate progenitors of B cells and early B-lineage cells. Casp6 regulates B-cell programs both during early development and after antigen stimulation in the periphery.

Targeting CD22 with the monoclonal antibody epratuzumab modulates human B-cell maturation and cytokine production in response to Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) signaling.

BACKGROUND: Abnormal B-cell activation is implicated in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). The B-cell surface molecule CD22, which regulates activation through the B-cell receptor (BCR), is a potential target for inhibiting pathogenic B cells; however, the regulatory functions of CD22 remain poorly understood. In this study, we determined how targeting of CD22 with epratuzumab (Emab), a humanized anti-CD22 IgG1 monoclonal antibody, affects the activation of human B-cell subsets in response to Toll-like receptor 7 (TLR7) and BCR engagement. METHODS: B-cell subsets were isolated from human tonsils and stimulated with F(ab')2 anti-human IgM and/or the TLR7 agonist R848 in the presence of Emab or a human IgG1 isotype control. Changes in mRNA levels of genes associated with B-cell activation and differentiation were analyzed by quantitative PCR. Cytokine production was measured by ELISA. Cell proliferation, survival, and differentiation were assessed by flow cytometry. RESULTS: Pretreatment of phenotypically naïve CD19+CD10-CD27- cells with Emab led to a significant increase in IL-10 expression, and in some but not all patient samples to a reduction of IL-6 production in response to TLR7 stimulation alone or in combination with anti-IgM. Emab selectively inhibited the expression of PRDM1, the gene encoding B-lymphocyte-induced maturation protein 1 (Blimp-1) in activated CD10-CD27- B cells. CD10-CD27-IgD- cells were highly responsive to stimulation through TLR7 as evidenced by the appearance of blasting CD27hiCD38hi cells. Emab significantly inhibited the activation and differentiation of CD10-CD27-IgD- B cells into plasma cells. CONCLUSIONS: Emab can both regulate cytokine expression and block Blimp1-dependent B-cell differentiation, although the effects of Emab may depend on the stage of B-cell development or activation. In addition, Emab inhibits the activation of CD27-IgD- tonsillar cells, which correspond to so-called double-negative memory B cells, known to be increased in SLE patients with more active disease. These data may be relevant to the therapeutic effect of Emab in vivo via modulation of the production of pro-inflammatory and anti-inflammatory cytokines by B cells. Because Blimp-1 is required by B cells to mature into antibody-producing cells, inhibition of Blimp1 may reduce autoantibody production.

Targeting antigens through blood dendritic cell antigen 2 on plasmacytoid dendritic cells promotes immunologic tolerance.

The C-type lectin receptor blood dendritic cell Ag 2 (BDCA2) is expressed exclusively on human plasmacytoid DCs (pDCs) and plays a role in Ag capture, internalization, and presentation to T cells. We used transgenic mice that express human BDCA2 and anti-BDCA2 mAbs to deliver Ags directly to BDCA2 on pDCs in vivo. Targeting Ag to pDCs in this manner resulted in significant suppression of Ag-specific CD4(+) T cell and Ab responses upon secondary exposure to Ag in the presence of adjuvant. Suppression of Ab responses required both a decrease in effector CD4(+) T cells and preservation of Foxp3(+) regulatory T cells (Tregs). Reduction in Treg numbers following Ag delivery to BDCA2 restored both CD4(+) T cell activation and Ab responses, demonstrating that Tregs were required for the observed tolerance. Our results demonstrate that Ag delivery to pDCs through BDCA2 is an effective method to induce immunological tolerance, which may be useful for treating autoimmune diseases or to inhibit unwanted Ab responses.

Controlling immune responses by targeting antigens to dendritic cell subsets and B cells.

Delivering antigens in vivo by coupling them to mAbs specific for unique receptors on antigen-presenting cells (APCs) is a promising approach for modulating immune responses. Antigen delivery to receptors found on myeloid dendritic cell (DC) subsets, plasmacytoid DCs and B cells has shown them all to be viable targets to stimulate either the cellular or humoral arms of the immune system. It is now evident that antigen-targeting approaches can also be used to invoke antigen-specific inhibition of immune responses. The outcome of activation versus inhibition is determined by a combination of factors that include the choice of APC, the receptor that is targeted, whether to include an adjuvant and, if so, which adjuvant to employ. In addition to their use as a means to modulate immune responses, antigen-targeting systems are also a useful method to investigate the function of DC subsets and the early mechanistic events that underlie the initiation of both cellular and humoral immune responses. In this review, we focus on the literature surrounding the control of B-cell responses when antigen is delivered to various APC subsets.

Overexpression of TLR7 promotes cell-intrinsic expansion and autoantibody production by transitional T1 B cells.

Toll-like receptor (TLR), a ligand for single-stranded RNA, has been implicated in the development of pathogenic anti-RNA autoantibodies both in systemic lupus erythematous (SLE) patients and in murine models of lupus. It is still unclear, however, where and how TLR7-mediated interactions affect the development of autoreactive B cells. We found that overexpression of TLR7 in transgenic mice (TLR7.1Tg) leads to marked alterations of transitional (T1) B cells, associated with their expansion and proliferation within the splenic red pulp (RP). This phenotype was intrinsic to the T1 subset of B cells and occurred independently of type 1 IFN signals. Overexpression of RNase in TLR7.1Tg mice significantly limited the expansion and proliferation of T1 cells, indicating that endogenous RNA complexes are driving their activation. TLR7.1Tg T1 cells were hyper-responsive to anti-IgM and TLR7 ligand stimulation in vitro and produced high concentrations of class-switched IgG2b and IgG2c, including anti-RNA antibodies. Our results demonstrate that initial TLR7 stimulation of B cells occurs at the T1 stage of differentiation in the splenic RP and suggest that dysregulation of TLR7 expression in T1 cells can result in production of autoantibodies.

Spontaneous loss of tolerance of autoreactive B cells in Act1-deficient rheumatoid factor transgenic mice.

Self-reactive B cells in BALB/c AM14 transgenic (Tg) rheumatoid factor mice are not subject to central or peripheral tolerization. Instead, they remain at a stage of "clonal ignorance"; that is, they do not proliferate and differentiate into Ab-forming cells. However, the immunoregulatory mechanisms that prevent autoantibody production in these mice remain unclear. In this study, we show that crossing AM14 Tg mice to a mouse strain deficient in Act1, a molecule involved in the regulation of BAFF-R and CD40-signaling in B cells, results in spontaneous activation of AM14 Tg B cells and production of AM14-specific Abs. Three- to 5-mo-old AM14 Tg Act1(-/-) mice showed significant expansion of AM14 Tg B cells, including a 2- to 3-fold increase in the spleen and cervical lymph nodes compared with AM14 Tg Act1(+/+) mice. Furthermore, in the presence of endogenous self-Ag (IgH(a) congenic background), AM14 Tg Act1(-/-) B cells were spontaneously activated and differentiated into Ab-forming cells. In contrast with previous studies using AM14 Tg MLR.Fas(lpr) mice, we found that a significant number of AM14 Tg cells AM14 Tg Act1(-/-) mice displayed phenotypic characteristics of germinal center B cells. Anti-CD40L treatment significantly limited the expansion and activation of AM14 Tg Act1(-/-) B cells, suggesting that CD40L-mediated signals are required for the retention of these cells. Our results support the important role of Act1 in the regulation of self-reactive B cells and reveal how Act1 functions to prevent the production of autoantibodies.

Extrafollicular B cell activation by marginal zone dendritic cells drives T cell-dependent antibody responses.

Dendritic cells (DCs) are best known for their ability to activate naive T cells, and emerging evidence suggests that distinct DC subsets induce specialized T cell responses. However, little is known concerning the role of DC subsets in the initiation of B cell responses. We report that antigen (Ag) delivery to DC-inhibitory receptor 2 (DCIR2) found on marginal zone (MZ)-associated CD8α(-) DCs in mice leads to robust class-switched antibody (Ab) responses to a T cell-dependent (TD) Ag. DCIR2(+) DCs induced rapid up-regulation of multiple B cell activation markers and changes in chemokine receptor expression, resulting in accumulation of Ag-specific B cells within extrafollicular splenic bridging channels as early as 24 h after immunization. Ag-specific B cells primed by DCIR2(+) DCs were remarkably efficient at driving naive CD4 T cell proliferation, yet DCIR2-induced responses failed to form germinal centers or undergo affinity maturation of serum Ab unless toll-like receptor (TLR) 7 or TLR9 agonists were included at the time of immunization. These results demonstrate DCIR2(+) DCs have a unique capacity to initiate extrafollicular B cell responses to TD Ag, and thus define a novel division of labor among splenic DC subsets for B cell activation during humoral immune responses.

Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease.

Act1 is a negative regulator of B-cell activation factor of the TNF family (BAFF) and CD40L-induced signaling. BALB/C mice lacking Act1 develop systemic autoimmunity resembling systemic lupus erythematosus (SLE) and Sjögren's syndrome (SjS). SLE and SjS are characterized by anti-nuclear IgG autoantibody (ANA-IgG) production and inflammation of peripheral tissues. As autoantibody production can occur in a T-cell dependent or T-cell independent manner, we investigated the role of T-cell help during Act1-mediated autoimmunity. Act1-deficiency was bred onto C57Bl/6 (B6.Act1(-/-) ) mice and B6.TCRß(-/-) TCRδ(-/-) Act1(-/-) (TKO) mice were generated. While TCRß/δ-sufficient B6.Act1(-/-) mice developed splenomegaly and lymphadenopathy, hypergammaglobulinemia, elevated levels of ANA-IgG, and kidney pathology, TKO mice failed to develop any such signs of disease. Neither B6.Act1(-/-) nor TKO mice developed SjS-like disease, suggesting that epigenetic interactions on the BALB/C background are responsible for this phenotype in BALB/C.Act1(-/-) mice. Interestingly, BAFF-driven transitional B-cell abnormalities, previously reported in BALB/C.Act1(-/-) mice, were intact in B6.Act1(-/-) mice and largely independent of T cells. In conclusion, T cells are necessary for the development of SLE-like disease in B6.Act1(-/-) mice, but not BAFF-driven transitional B-cell differentiation.

B-cell selection and the development of autoantibodies.

The clearest evidence that B cells play an important role in human autoimmunity is that immunotherapies that deplete B cells are very effective treatments for many autoimmune diseases. All people, healthy or ill, have autoreactive B cells, but not at the same frequency. A number of genes influence the level of these autoreactive B cells and whether they are eliminated or not during development at a central checkpoint in the bone marrow (BM) or at a later checkpoint in peripheral lymphoid tissues. These genes include those encoding proteins that regulate signaling through the B-cell receptor complex such as Btk and PTPN22, proteins that regulate innate signaling via Toll-like receptors (TLRs) such as MyD88 and interleukin-1 receptor-associated kinase 4, as well as the gene encoding the activation-induced deaminase (AID) essential for B cells to undergo class switch recombination and somatic hypermutation. Recent studies have revealed that TLR signaling elements and AID function not only in peripheral B cells to help mediate effective antibody responses to foreign antigens, but also in the BM to help remove autoreactive B-lineage cells at a very early point in B-cell development. Newly arising B cells that leave the BM and enter the blood and splenic red pulp can express both AID and TLR signaling elements like TLR7, and thus are fully equipped to respond rapidly to antigens (including autoantigens), to isotype class switch, and to undergo somatic hypermutation. These red pulp B cells may thus be an important source of autoantibody-producing cells arising particularly in extrafollicular sites, and indeed may be as significant a source of autoantibody-producing cells as B cells arising from germinal centers.

Protein phosphatase 2A and neutral sphingomyelinase 2 regulate IRAK-1 protein ubiquitination and degradation in response to interleukin-1beta.

The IL-1ß signaling cascade is initiated by the phosphorylation of IL-1ß receptor-associated kinase-1 (IRAK-1), followed by its ubiquitination and degradation. This paper investigates the regulation of IRAK-1 degradation in primary hepatocytes and in HEK cells overexpressing the IL-1ß receptor. We provide evidence that protein phosphatase 2A (PP2A) is a negative regulator of the phosphorylation, Lys(48)-linked ubiquitination, and degradation of IRAK-1. PP2A catalytic activity increased within 30 min of stimulation with IL-1ß. siRNA against PP2A catalytic subunit (PP2Ac) or treatment with pharmacological inhibitor, okadaic acid, enhanced IRAK-1 Lys(48)-linked ubiquitination and degradation. Direct interaction between PP2Ac and IRAK-1 was observed, suggesting that IRAK-1 might be a PP2A substrate. The mechanisms of PP2A activation by IL-1ß involved neutral sphingomyelinase-2 (NSMase-2) and an accumulation of ceramide. Overexpression of NSMase-2 delayed IRAK-1 degradation in a PP2A-dependent manner, whereas NSMase-2 silencing had the opposite effect. The addition of sphingomyelinase, ceramide, or a proteasome inhibitor all led to retention of IRAK-1 at the cell membrane and to increased JNK phosphorylation. This study suggests that NSMase-2- and PP2A-dependent regulation of IRAK-1 degradation is a novel mechanism to fine tune the magnitude of IL-1ß response.