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Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification. The transcription factor IRF8 is essential for classical type 1 dendritic cell (cDC1) development. Murphy and colleagues investigate in detail the molecular control of cDC1 fate specification by systematically unpicking the IRF8 enhancer regions.

Due to cross-antagonism of the B cell factor Pax5 and plasma cell regulator Blimp1, it was hypothesized that plasma cells develop only upon Pax5 repression by Blimp1. Unexpectedly, Liu et al. show that ectopic Pax5 expression in plasma cells does not interfere with their development and function., B cell and plasma cell fates are controlled by different transcriptional networks, as exemplified by the mutually exclusive expression and cross-antagonism of the B cell identity factor Pax5 and the plasma cell regulator Blimp1. It has been postulated that repression of Pax5 by Blimp1 is essential for plasma cell development. Here, we challenged this hypothesis by analyzing the IghPax5/+ mouse, which expressed a Pax5 minigene from the immunoglobulin heavy-chain locus. Despite high Pax5 expression, plasma cells efficiently developed in young IghPax5/+ mice at steady state and upon immunization, while their number moderately declined in older mice. Although Pax5 significantly deregulated the plasma cell expression program, key plasma cell regulators were normally expressed in IghPax5/+ plasma cells. While IgM and IgA secretion by IghPax5/+ plasma cells was normal, IgG secretion was modestly decreased. Hence, Pax5 repression is not essential for robust plasma cell development and antibody secretion, although it is required for optimal IgG production and accumulation of long-lived plasma cells.

Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) and Golgi apparatus of antibody-producing cells, reduced antibody secretion, and aberrant IgG N-glycosylation. The data uncover a key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans., Jagunal homolog 1 (JAGN1) has been identified as a critical regulator of neutrophil biology in mutant mice and rare-disease patients carrying JAGN1 mutations. Here, we report that Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) of antibody-producing cells as well as decreased antibody production and secretion. Consequently, mice lacking Jagn1 in B cells exhibit reduced serum immunoglobulin (Ig) levels at steady state and fail to mount an efficient humoral immune response upon immunization with specific antigens or when challenged with viral infections. We also demonstrate that Jagn1 deficiency in B cells results in aberrant IgG N-glycosylation leading to enhanced Fc receptor binding. Jagn1 deficiency in particular affects fucosylation of IgG subtypes in mice as well as rare-disease patients with loss-of-function mutations in JAGN1. Moreover, we show that ER stress affects antibody glycosylation. Our data uncover a novel and key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans.

The transcription factor Blimp1 is not only an essential regulator of plasma cells, but also a risk factor for the development of autoimmune disease in humans. Here, we demonstrate in the mouse that the Prdm1 (Blimp1) gene was partially activated at the chromatin and transcription level in early B cell development, although mature Prdm1 mRNA did not accumulate due to posttranscriptional regulation. By analyzing a mouse model that facilitated ectopic Blimp1 protein expression throughout B lymphopoiesis, we could demonstrate that Blimp1 impaired B cell development by interfering with the B cell gene expression program, while leading to an increased abundance of plasma cells by promoting premature plasmablast differentiation of immature and mature B cells. With progressing age, these mice developed an autoimmune disease characterized by the presence of autoantibodies and glomerulonephritis. Hence, these data identified ectopic Blimp1 expression as a novel mechanism, through which Blimp1 can act as a risk factor in the development of autoimmune disease.

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41-/-Bhlhe40-/- B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12Vκ4 BCR. Expression of a pre-rearranged VH12Vκ4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis , on the surface of the same B cell or in trans , on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis- ligand binding of CD22 is crucial for the regulation of B-cell Ca 2+ signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca 2+ signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca 2+ responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.

CD22, an inhibitory co-receptor of the B-cell receptor, shows a B-cell-specific expression pattern and is expressed on most B-cell lymphomas. The anti-CD22 antibody Epratuzumab is in clinical trials for B-cell non-Hodgkin lymphoma and systemic lupus erythematosus, but shows a mostly unknown mode of action. We generated a new mouse model that expresses human CD22 instead of murine CD22 (Huki CD22 mice), in which human CD22 can be targeted. Expression of human CD22 on the B cells of Huki CD22 mice does not generally interfere with B-cell development. However, Huki CD22 mice show a reduction of the population of mature recirculating B cells in the bone marrow and reduced transitional and marginal zone B cells in the spleen, phenotypes resembling that of CD22-deficient mice. Similarly, enhanced BCR-induced Ca2+ signalling is observed in Huki CD22 mice, which also mount normal immune responses toward different classes of antigens. Huki CD22 B cells show a normal anti-hCD22 antibody-mediated endocytosis. In conclusion, human CD22 cannot fully substitute for murine CD22 functions, possibly due to the changed intracellular tail of the protein or due to lower expression levels. Huki CD22 mice are a valuable new model for both antibody- and immunotoxin-mediated targeting of human CD22.

CD22 is an inhibitory co-receptor of the B-cell receptor (BCR) on B cells. Since CD22 is ubiquitously expressed in the B-cell lineage and CD22 endocytosis can be triggered efficiently, antibodies and antibody-based immunotoxins against CD22 are used to target B cells both in B-cell lymphomas and leukemias, as well as in autoimmune diseases. CD22 recognizes α2,6-linked sialic acids as endogenous ligands. We have developed new synthetic sialosides as ligands for human CD22. These sialosides bind CD22 on human B cells with high affinity and can efficiently enhance IgM-triggered Ca 2+ signaling. We coupled these sialosides to Pseudomonas exotoxin A to generate a novel CD22 ligand-based immunotoxin. This sialoside-exotoxin-A construct can specifically kill CD22-positive B-cell lymphoma cells. It binds specifically to CD22-positive B-cell lymphoma cells and is dominant over endogenous cis-ligands on the B-cell surface. The sialoside-exotoxin-A construct is efficiently internalized by endocytosis into B-cell lymphoma cell lines. Thus we show the development of a new therapeutic compound for targeting CD22 on human B cells, both for B-cell lymphoma, as well as for B-cell-mediated autoimmune diseases.

Busslinger et al. showed that the transcription factors E2A and E2-2 control the expression of genes required for the development of GC B cells and plasma cells., E2A is an essential regulator of early B cell development. Here, we have demonstrated that E2A together with E2-2 controlled germinal center (GC) B cell and plasma cell development. As shown by the identification of regulated E2A,E2-2 target genes in activated B cells, these E-proteins directly activated genes with important functions in GC B cells and plasma cells by inducing and maintaining DNase I hypersensitive sites. Through binding to multiple enhancers in the Igh 3′ regulatory region and Aicda locus, E-proteins regulated class switch recombination by inducing both Igh germline transcription and AID expression. By regulating 3′ Igk and Igh enhancers and a distal element at the Prdm1 (Blimp1) locus, E-proteins contributed to Igk, Igh, and Prdm1 activation in plasmablasts. Together, these data identified E2A and E2-2 as central regulators of B cell immunity.

CD22 is an inhibitory co-receptor of the B-cell receptor (BCR) on B cells. Since CD22 is ubiquitously expressed in the B-cell lineage and CD22 endocytosis can be triggered efficiently, antibodies and antibody-based immunotoxins against CD22 are used to target B cells both in B-cell lymphomas and leukemias, as well as in autoimmune diseases. CD22 recognizes α2,6-linked sialic acids as endogenous ligands. We have developed new synthetic sialosides as ligands for human CD22. These sialosides bind CD22 on human B cells with high affinity and can efficiently enhance IgM-triggered Ca(2+) signaling. We coupled these sialosides to Pseudomonas exotoxin A to generate a novel CD22 ligand-based immunotoxin. This sialoside-exotoxin-A construct can specifically kill CD22-positive B-cell lymphoma cells. It binds specifically to CD22-positive B-cell lymphoma cells and is dominant over endogenous cis-ligands on the B-cell surface. The sialoside-exotoxin-A construct is efficiently internalized by endocytosis into B-cell lymphoma cell lines. Thus we show the development of a new therapeutic compound for targeting CD22 on human B cells, both for B-cell lymphoma, as well as for B-cell-mediated autoimmune diseases.

Background Epratuzumab is a humanized monoclonal antibody that targets the B cell-specific protein CD22 and is currently in Phase 3 clinical trials in patients (pts) with systemic lupus erythematosus (SLE). Epratuzumab does not deplete B cells and appears to act in an immunomodulatory fashion, eg. by inhibiting activation through the B cell receptor (BCR). 1 It has also been shown to modulate adhesion molecule expression on B cells and their responsiveness to chemokines. 2 Objectives The present study aimed to understand the effect of epratuzumab on B cells in vivo using human CD22 knock-in (Huki) mice, in which the B cells express the human CD22 gene instead of the murine gene. 3 Methods Huki mice received a single intravenous injection (0.5mg) of epratuzumab and, at various time points up to Week (Wk) 12, B cell sub-populations in blood, spleen, bone marrow and lymph nodes were measured, along with B cell activation and homing markers. Ex vivo functional assays were also performed: Ca + flux, apoptosis and CD22 internalization on B cells were measured by flow cytometry and the proliferation of B cells was assessed after in vivo administration of BrdU. Results Epratuzumab-treated mice showed rapid and long-lasting human CD22 internalization on B cells from all organs up to Wk8 and an increase of B cell apoptosis (an increase of cells in sub G1 phase) ex vivo . Furthermore, decreased BCR-activated Ca + -flux was demonstrated after epratuzumab treatment in vitro . However, BrdU incorporation in several B cell subsets was unchanged at early time points and there were no consistent effects demonstrated on the numbers or proportions of such subsets in various organs at any time point up to Wk12. Conclusions Epratuzumab treatment of Huki mice induces functional effects on B cells assessed ex vivo but does not seem to strongly influence B cell development and B cell populations in various organs. These data have implications for understanding the effects of epratuzumab treatment on B cell function in SLE pts. References Sieger N. Arth Rheum 2013;65:770. Daridon C. Arth Res Ther 2010;12:R204. Wohner M. Eur J Immunol 2012;42:3009. Acknowledgements C. Brandl and L. Ozgor contributed equally to the work. The authors acknowledge Costello Medical Consulting for editorial assistance which was funded by UCB Pharma. Disclosure of Interest C. Brandl Grant/research support: UCB Pharma, L. Ozgor Grant/research support: UCB Pharma, M. Wohner: None declared, A. Shock Employee of: UCB Pharma, L. Nitschke Grant/research support: UCB Pharma DOI 10.1136/annrheumdis-2014-eular.1751