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The plasma cell proliferative index (PCPI), determined by a slide technique or by flow cytometry, detects cells in the S phase of the cell cycle and is a useful prognostic tool in patients with plasma cell disorders such as multiple myeloma and amyloidosis. We conducted a retrospective review analyzing the prognostic effect of PCPI in 306 patients with smoldering multiple myeloma (SMM). Seventy-nine (26%) patients had an elevated PCPI (>0.5). An elevated PCPI predicted an inferior time to progression (median, 3.0 vs 7.1 years for those with a low PCPI; P = .0004). Within 24 months, the progression rate was significantly higher for patients with an elevated PCPI (49% vs. 20%; P < .0001). PCPI is a valuable tool in risk stratifying patients with SMM and identifies patients with earlier progression who may benefit from closer follow-up and consideration of early intervention trials., (© 2018 by The American Society of Hematology.)

Even though more than 30 years have passed since the eradication of smallpox, high titers of smallpox-specific antibodies are still detected in the blood of subjects vaccinated in childhood. In fact, smallpox-specific antibody levels are maintained in serum for more than 70 years. The generation of life-long immunity against infectious diseases such as smallpox and measles has been thoroughly documented. Although the mechanisms behind high persisting antibody titers in the absence of the causative agent are still unclear, long lived plasma cells (LLPCs) play an important role. Most of the current knowledge on LLPCs is based on experiments performed in mouse models, although the amount of data derived from human studies is increasing. As the results from mouse models are often directly extrapolated to humans, it is important to keep in mind that there are differences. These are not only the obvious such as the life span but there are also anatomical differences, for instance the adiposity of the bone marrow (BM) where LLPCs reside. Whether these differences have an effect on the function of the immune system, and in particular on LLPCs, are still unknown. In this review, we will briefly discuss current knowledge of LLPCs, comparing mice and humans.

In the transition from B cells to Ab-secreting cells (ASCs) many genes are induced, such as ELL2, Irf4, Prdm1, Xbp1, whereas other mRNAs do not change in abundance. Nonetheless, using splicing array technology and mouse splenic B cells plus or minus LPS, we found that induced and "uninduced" genes can show large differences in splicing patterns between the cell stages, which could influence ASC development. We found that ∼55% of these splicing changes depend on ELL2, a transcription elongation factor that influences expression levels and splicing patterns of ASC signature genes, genes in the cell-cycle and N-glycan biosynthesis and processing pathways, and the secretory versus membrane forms of the IgH mRNA. Some of these changes occur when ELL2 binds directly to the genes encoding those mRNAs, whereas some of the changes are indirect. To attempt to account for the changes that occur in RNA splicing before or without ELL2 induction, we examined the amount of the small nuclear RNA molecules and found that they were significantly decreased within 18 h of LPS stimulation and stayed low until 72 h. Correlating with this, at 18 h after LPS, endoplasmic reticulum stress and Ire1 phosphorylation are induced. Inhibiting the regulated Ire1-dependent mRNA decay with 4u8C correlates with the reduction in small nuclear RNA and changes in the normal splicing patterns at 18 h. Thus, we conclude that the RNA splicing patterns in ASCs are shaped early by endoplasmic reticulum stress and Ire1 phosphorylation and later by ELL2 induction., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Antigen-dependent engagement of germinal center (GC) B cell receptors (BCRs) promotes antigen internalization and presentation for follicular helper T cells. However, whether BCR signaling is critical or synergistic with T cell help for GC B cell selection or differentiation is unclear. Here, by adapting an experimental approach that enables independent delivery of BCR-crosslinking antigen or T cell help to GC B cells in vivo, we showed that T cell help was sufficient to induce GC B cell expansion and plasmablast formation. However, although BCR crosslinking could not by itself promote GC B cell selection or differentiation, it could synergize with T cell help to enhance the GC and plasmablast responses when T cell help was limiting. These findings indicate that GC B cells can integrate variable inputs from T cell help and BCR signaling in vivo for an optimal process of selection and differentiation, critical for potent long-term humoral immunity., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

B cells undergo epigenetic remodeling as they differentiate into Ab-secreting cells (ASC). LSD1 is a histone demethylase known to decommission active enhancers and cooperate with the ASC master regulatory transcription factor Blimp-1. The contribution of LSD1 to ASC formation is poorly understood. In this study, we show that LSD1 is necessary for proliferation and differentiation of mouse naive B cells (nB) into plasmablasts (PB). Following LPS inoculation, LSD1-deficient hosts exhibited a 2-fold reduction of splenic PB and serum IgM. LSD1-deficient PB exhibited derepression and superinduction of genes involved in immune system processes; a subset of these being direct Blimp-1 target-repressed genes. Cell cycle genes were globally downregulated without LSD1, which corresponded to a decrease in the proliferative capacity of LSD1-deficient activated B cells. PB lacking LSD1 displayed increased histone H3 lysine 4 monomethylation and chromatin accessibility at nB active enhancers and the binding sites of transcription factors Blimp-1, PU.1, and IRF4 that mapped to LSD1-repressed genes. Together, these data show that LSD1 is required for normal in vivo PB formation, distinguish LSD1 as a transcriptional rheostat and epigenetic modifier of B cell differentiation, and identify LSD1 as a factor responsible for decommissioning nB active enhancers., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1 -/- Prdm1 +/gfp mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1 -/- plasmablasts show a reduced activation of β1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

TACI signals activate B cell proliferation, isotype switch and antibody production in both normal immunity and autoimmune states. In contrast to murine TACI, the human TACI gene undergoes alternative splicing to produce short and long isoforms (TACI-S and TACI-L). In previous studies, we showed that transduction of the short, but not long isoform, into murine B cells or human pre-B cells lacking TACI, caused them to become transcriptional and morphologically identical to plasma cells. These data suggest that the expression of different isoforms in humans provides unique controls on B cell maturation. In these studies we show that TACI-S and TACI-L form complexes in a ligand-independent manner, not dependent on a single extracellular domain. Both TACI isoforms are detectable in the endosomal cellular compartment where they co-localize with MyD88, TRAF6, and the activated 65 kDa form of TLR9, depending on a conserved intracellular TACI sequence. In contrast to TACI-L expressing cells, or cells bearing both isoforms, TACI-S binds ligands BAFF and APRIL with substantially greater affinity and promotes enhanced NF-kB activation. Using isoform-specific monoclonal antibodies, we show that while TACI-L is predominant as a surface receptor surface on human B cells, significantly more TACI-S is noted in the intracellular compartment and also in marginal zone, isotype switched and plasmablast in resting B cells. TACI-S is increased in tonsillar B cells and also in the intracellular compartment of activated peripheral B cells. These data shows that alternative splicing of the human TACI gene leads to two isoforms both of which intersect with MyD88 and TRAF6 and form complexes with TLR9, but the two isoforms have different ligand binding capacities, subcellular locations and activation capabilities.

Aims: The number of precursor and mature lymphoid cells and plasma cells in normal bone marrow trephine (BMT) biopsies throughout the human lifespan is unknown. Reference ranges have been established from aspirated marrow, but due to haemodilution errors, these do not accurately reflect the native marrow milieu. We aimed to define age-specific, normal reference ranges for lymphoid and plasma cells in BMT biopsy specimens using a combined immunophenotyping and digital enumeration approach., Methods: Morphologically normal BMT biopsy specimens (n=483) were obtained from patients aged 1 month to 90 years of age. Immunohistochemistry was performed to identify lymphoid progenitors , T-lymphocytes (CD3), B-lymphocytes (CD20) and plasma cells (CD138 and MUM1). Positive cells were counted using digital enumeration software, and the percent positivity for each antigen was determined per case. Mean values were generated for specific age groups, and age-defined reference ranges were determined for each antigen using normalised data., Results: A mean of 16 609 cells (range: 7210-34 097) were counted per biopsy. Infant marrows showed a predominance of immature lymphoid progenitors and B cells. With increasing age, an increase in mean T cell and plasma cell numbers were observed. The results showed the same trends to flow cytometry references for aspirate material although the absolute values differed., Conclusions: Combined immunohistochemistry and automated enumeration gives an accurate, reproducible number of antigen-positive cells and has generated normal reference ranges for these cell types in BMT biopsies. The method and ranges we have established have the potential to be applied in routine clinical practice., Competing Interests: Competing interests: None to declare., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The unfolded protein response (UPR) and activation of XBP1 is necessary for high secretory efficiency and functional differentiation of antibody secreting cells (ASCs). The UPR additionally includes a branch in which membrane-bound transcription factors, exemplified by ATF6, undergo intramembrane-proteolysis by the sequential action of site-1 (MBTPS1/S1P) and site-2 proteases (MBTPS2/S2P) and release of the cytoplasmic domain as an active transcription factor. Such regulation is shared with a family of CREB3-related transcription factors and sterol regulatory element-binding proteins (SREBPs). Of these, we identify that the CREB3 family member CREB3L2 is strongly induced and activated during the transition from B-cell to plasma cell state. Inhibition of site-1 protease leads to a profound reduction in plasmablast number linked to induction of autophagy. Plasmablasts generated in the presence of site-1 protease inhibitor segregated into CD38 high and CD38 low populations, the latter characterized by a marked reduction in the capacity to secrete IgG. Site-1 protease inhibition is accompanied by a distinctive change in gene expression associated with amino acid, steroid and fatty acid synthesis pathways. These results demonstrate that transcriptional control of metabolic programs necessary for secretory activity can be targeted via site-1 protease inhibition during ASC differentiation.

Long-lived plasma cells (PCs) not only provide protective humoral immunity, they are also an essential component of the autoreactive immunologic memory that may drive chronic immune responses in systemic autoimmunity, such as systemic lupus erythematosus (SLE). The therapeutic relevance of their targeting has been demonstrated in preclinical models and severe, treatment-refractory cases of autoimmune diseases using the proteasome inhibitor bortezomib. Herein, we describe in detail the dynamic serologic changes and effects on immune effector cells in eight SLE patients receiving a median two cycles of 1.3 mg/m 2 intravenous bortezomib. Upon proteasome inhibition, immunoglobulin levels gradually declined by ∼30%, associated with a significant reduction of autoantibodies, and serum complement whereas B-cell activation factor levels increased. While proteasome inhibition was associated with a significant depletion of short- and long-lived PCs in peripheral blood and bone marrow by ∼50%, including those with a distinctly mature CD19 - phenotype, their precursor B cells and T cells largely remained unaffected, resulting in a rapid repopulation of short-lived PCs after bortezomib withdrawal, accompanied by increasing autoantibody levels. Collectively, these findings identify proteasome inhibitors as a promising treatment option for refractory SLE, but also indicate that PC depletion needs to be combined with targeted B-cell therapies for sustained responses in systemic autoimmunity., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To what extent immune responses against the gut flora are compartmentalized within mucosal tissues in homeostatic conditions remains a much-debated issue. We describe here, based on an inducible AID fate-mapping mouse model, that systemic memory B cell subsets, including mainly IgM + B cells in spleen, together with IgA + plasma cells in spleen and bone marrow, are generated in mice in the absence of deliberate immunization. While the IgA component appears dependent on the gut flora, IgM memory B cells are still generated in germ-free mice, albeit to a reduced extent. Clonal relationships and renewal kinetics after anti-CD20 treatment reveal that this long-lasting splenic population is mainly sustained by output of B cell clones persisting in mucosal germinal centers. IgM-secreting hybridomas established from splenic IgM memory B cells showed reactivity against various bacterial isolates and endogenous retroviruses. Ongoing activation of B cells in gut-associated lymphoid tissues thus generates a diversified systemic compartment showing long-lasting clonal persistence and protective capacity against systemic bacterial infections., (© 2018 Le Gallou et al.)

Migration of human plasmablast to the bone marrow is essential for the final differentiation of plasma cells and maintenance of effective humoral immunity. This migration is controlled by CXCL12/CXCR4-mediated activation of the protein kinase AKT. Herein, we show that the CXCL12-induced migration of human plasmablasts is dependent on glucose oxidation. Glucose depletion markedly inhibited plasmablast migration by 67%, and the glucose analog 2-deoxyglucose (2-DG) reduced the migration by 53%; conversely, glutamine depletion did not reduce the migration. CXCL12 boosted the oxygen consumption rate (OCR), and 2-DG treatment significantly reduced the levels of all measured tricarboxylic acid (TCA) cycle intermediates. AKT inhibitors blocked the CXCL12-mediated increase of OCR. CXCL12 enhanced the pyruvate dehydrogenase (PDH) activity by 13.5-fold in an AKT-dependent manner to promote mitochondrial oxidative phosphorylation. The knockdown and inhibition of PDH confirmed its indispensable role in CXCL12-induced migration. Cellular ATP levels fell by 91% upon exposure to 2-DG, and the mitochondrial ATP synthase inhibitor oligomycin inhibited CXCL12-induced migration by 85%. Low ATP levels inhibited the CXCL12-induced activation of AKT and phosphorylation of myosin light chains by 42%, which are required for cell migration. Thus, we have identified a mechanism that controls glucose oxidation via AKT signaling and PDH activation, which supports the migration of plasmablasts. This mechanism can provide insights into the proper development of long-lived plasma cells and is, therefore, essential for optimal humoral immunity. To our knowledge, this study is the first to investigate metabolic mechanisms underlying human plasmablast migration toward CXCL12.

Antibody Secreting Cells (ASCs) are a fundamental component of humoral immunity, however, deregulated or excessive antibody production contributes to the pathology of autoimmune diseases, while transformation of ASCs results in the malignancy Multiple Myeloma (MM). Despite substantial recent improvements in treating these conditions, there is as yet no widely used ASC-specific therapeutic approach, highlighting a critical need to identify novel methods of targeting normal and malignant ASCs. Surface molecules specifically expressed by the target cell population represent ideal candidates for a monoclonal antibody-based therapy. By interrogating the ASC gene signature that we previously defined we identified three surface proteins, Plpp5, Clptm1l and Itm2c, which represent potential targets for novel MM treatments. Plpp5 , Clptm1l and Itm2c are highly and selectively expressed by mouse and human ASCs as well as MM cells. To investigate the function of these proteins within the humoral immune system we have generated three novel mouse strains, each carrying a loss-of-function mutation in either Plpp5 , Clptm1l or Itm2c . Through analysis of these novel strains, we have shown that Plpp5, Clptm1l and Itm2c are dispensable for the development, maturation and differentiation of B-lymphocytes, and for the production of antibodies by ASCs. As adult mice lacking either protein showed no apparent disease phenotypes, it is likely that targeting these molecules on ASCs will have minimal on-target adverse effects.

Antibody-secreting plasma cells (PCs) arise rapidly during adaptive immunity to control infections. The early PCs are retained within the reactive lymphoid organ where their localization and homeostasis rely on extrinsic factors, presumably produced by local niche cells. While myeloid cells have been proposed to form those niches, the contribution by colocalizing stromal cells has remained unclear. Here, we characterized a subset of fibroblastic reticular cells (FRCs) that forms a dense meshwork throughout medullary cords of lymph nodes (LNs) where PCs reside. This medullary FRC type is shown to be anatomically, phenotypically, and functionally distinct from T zone FRCs, both in mice and humans. By using static and dynamic imaging approaches, we provide evidence that medullary FRCs are the main cell type in contact with PCs guiding them in their migration. Medullary FRCs also represent a major local source of the PC survival factors IL-6, BAFF, and CXCL12, besides also producing APRIL. In vitro, medullary FRCs alone or in combination with macrophages promote PC survival while other LN cell types do not have this property. Thus, we propose that this FRC subset, together with medullary macrophages, forms PC survival niches within the LN medulla, and thereby helps in promoting the rapid development of humoral immunity, which is critical in limiting early pathogen spread., Competing Interests: The authors declare no conflict of interest.

B cells have a critical role in the initiation and acceleration of autoimmune diseases, especially those mediated by autoantibodies. In the peripheral lymphoid system, mature B cells are activated by self or/and foreign antigens and signals from helper T cells for differentiating into either memory B cells or antibody-producing plasma cells. Accumulating evidence has shown that epigenetic regulations modulate somatic hypermutation and class switch DNA recombination during B-cell activation and differentiation. Any abnormalities in these complex regulatory processes may contribute to aberrant antibody production, resulting in autoimmune pathogenesis such as systemic lupus erythematosus. Newly generated knowledge from advanced modern technologies such as next-generation sequencing, single-cell sequencing and DNA methylation sequencing has enabled us to better understand B-cell biology and its role in autoimmune development. Thus this review aims to summarize current research progress in epigenetic modifications contributing to B-cell activation and differentiation, especially under autoimmune conditions such as lupus, rheumatoid arthritis and type 1 diabetes.

PKCβ-null (Prkcb -/- ) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCβ failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb -/- B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCβ as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transitioning from a metabolically quiescent naive B cell to an antibody-secreting plasmablast requires division-dependent cellular differentiation. Though cell division demands significant ATP and metabolites, the metabolic processes used for ATP synthesis during plasmablast formation are not well described. Here, the metabolic requirements for plasmablast formation were determined. Following T-independent stimulation with lipopolysaccharide, B cells increased expression of the oxidative phosphorylation machinery in a stepwise manner. Such activated B cells have increased capacity to perform oxidative phosphorylation but showed dependency on glycolysis. Plasmablasts displayed higher oxidative metabolism to support antibody secretion, as inhibiting oxidative ATP production resulted in decreased antibody titers. Differentiation by Blimp1 was required for this increase in oxidative metabolism, as Blimp1-deficient cells proliferate but do not upregulate oxidative phosphorylation. Together, these findings identify a shift in metabolic pathways as B cells differentiate, as well as the requirement for increased metabolic potential to support antibody production., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immunoglobulin G4-related disease (IgG4-RD) is an uncommon disorder that demonstrates characteristic clinicopathologic features including sclerosing lesions with storiform fibrosis, increased IgG4+ plasma cells with an increased IgG4+/IgG+ plasma cell ratio, obliterative phlebitis, and often an increased serum IgG4 level. This review summarizes the characteristic histopathologic and clinical features of IgG4-RD with detailed discussion of the histopathologic characteristics of the most commonly involved anatomic sites. We also present recent advances in our understanding of the pathophysiologic mechanisms of IgG4-RD and discuss updates on the treatment, prognosis, and outcomes of this rare disease, including discussion of the possible association between IgG4-RD and malignancy., (© 2018 APMIS. Published by John Wiley & Sons Ltd.)

Plasma cells (PCs) acquiring long lifespans in the bone marrow (BM) play a pivotal role in the humoral arm of immunological memory. The PCs reside in a special BM niche and produce antibodies against past-encountered pathogens or vaccine components for a long time. In BM, cysteine-X-cysteine (CXC) chemokine receptor type 4 (CXCR4)-expressing PCs and myeloid cells such as dendritic cells are attracted to and held by CXC chemokine ligand 12 (CXCR12)-secreting stromal cells, where survival of the PCs is supported by soluble factors such as IL-6 and APRIL (a proliferation-inducing ligand) produced by neighboring myeloid cells. Although these stromal cells are also supposed to be involved in the support of the survival and antibody production, the full molecular mechanism has not been clarified yet. Here, we show that BM PDGFRα+Sca-1+-enriched mesenchymal stem cells (MSCs), which can contribute as stromal cells for hematopoietic stem cells, also support in vitro survival of and antibody production by BM PCs. IL-6 produced by MSCs was found to be involved in the support. Immunohistochemistry of BM sections suggested a co-localization of a minor population of PCs with PDGFRα+Sca-1+ MSCs in the BM. We also found that the sort-purified MSC preparation was composed of multiple cell groups with different gene expression profiles, as found on single-cell RNA sequencing, to which multiple roles in the in vitro PC support could be attributed.

B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cellular division and is linked to DNA hypomethylation. Conversely, little is known about how de novo deposition of DNA methylation affects B cell fate and function. Here we show that genetic deletion of the de novo DNA methyltransferases Dnmt3a and Dnmt3b (Dnmt3-deficient) in mouse B cells results in normal B cell development and maturation, but increased cell activation and expansion of the germinal center B cell and plasma cell populations upon immunization. Gene expression is mostly unaltered in naive and germinal center B cells, but dysregulated in Dnmt3-deficient plasma cells. Differences in gene expression are proximal to Dnmt3-dependent DNA methylation and chromatin changes, both of which coincide with E2A and PU.1-IRF composite-binding motifs. Thus, de novo DNA methylation limits B cell activation, represses the plasma cell chromatin state, and regulates plasma cell differentiation.

Idiopathic pulmonary arterial hypertension (IPAH) is a devastating pulmonary vascular disease in which autoimmune and inflammatory phenomena are implicated. B cells and autoantibodies have been associated with IPAH and identified as potential therapeutic targets. However, the specific populations of B cells involved and their roles in disease pathogenesis are not clearly defined. We aimed to assess the levels of activated B cells (plasmablasts) in IPAH, and to characterize recombinant antibodies derived from these plasmablasts. Blood plasmablasts are elevated in IPAH, remain elevated over time, and produce IgA autoantibodies. Single-cell sequencing of plasmablasts in IPAH revealed repertoires of affinity-matured antibodies with small clonal expansions, consistent with an ongoing autoimmune response. Recombinant antibodies representative of these clonal lineages bound known autoantigen targets and displayed an unexpectedly high degree of polyreactivity. Representative IPAH plasmablast recombinant antibodies stimulated human umbilical vein endothelial cells to produce cytokines and overexpress the adhesion molecule ICAM-1. Together, our results demonstrate an ongoing adaptive autoimmune response involving IgA plasmablasts that produce anti-endothelial cell autoantibodies in IPAH. These antibodies stimulate endothelial cell production of cytokines and adhesion molecules, which may contribute to disease pathogenesis. These findings suggest a role for mucosally-driven autoimmunity and autoimmune injury in the pathogenesis of IPAH., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although the aetiology of systemic lupus erythematosus (SLE) is unclear, dysregulated B cell responses have been implicated. Here we show that an unusual CD11c hi T-bet + B cell subset, with a unique expression profile including chemokine receptors consistent with migration to target tissues, is expanded in SLE patients, present in nephrotic kidney, enriched for autoreactive specificities and correlates with defined clinical manifestations. IL-21 can potently induce CD11c hi T-bet + B cells and promote the differentiation of these cells into Ig-secreting autoreactive plasma cells. While murine studies have identified a role for T-bet-expressing B cells in autoimmunity, this study describes and exemplifies the importance of CD11c hi T-bet + B cells in human SLE.

Eosinophils were reported to serve as an essential component of the plasma cell niche within the bone marrow. As the potential contribution of eosinophils to humoral immunity has remained incompletely understood, we aimed to further characterize their role during antibody responses and to additionally investigate their role in autoimmune disease. Contrary to our expectations and the currently prevailing paradigm, we found that eosinophils are fully dispensable for the survival of murine bone marrow plasma cells and accordingly do not contribute to antibody production and autoantibody-mediated disease. Littermate wild type and eosinophil-deficient ΔdblGATA-1 animals showed similar numbers and frequencies of plasma cells and did not differ in steady state levels of immunoglobulins or their ability to raise antigen-specific antibody responses. Eosinophils were likewise dispensable for autoantibody production or autoantibody-induced disease in a mouse model of systemic lupus erythematosus. Our findings thus argue against a role of eosinophils during the maintenance of the plasma cell pool and challenge the hitherto postulated concept of an eosinophil-sustained bone marrow niche., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lasting antibody responses are maintained by long-lived plasma cells, which are thought to lodge in the BM in specialized survival niches. Eosinophils have been reported to function as a critical component of the BM survival niche where they are thought to provide pro-survival signals to nearby plasma cells. Recent study shows that many BM plasma cells are recently generated and chiefly short-lived cells, raising the possibility that rare plasma cell-eosinophil interactions are a rate-limiting step needed to establish lasting humoral immunity. To address these issues, we examined the impact of eosinophil depletion on short- and long-lived BM plasma cells in the context of antibody responses induced by both T-cell dependent and T-cell independent antigens. Surprisingly, our results failed to support a role for eosinophils in either plasma cell generation or survival. These studies included examination of plasma cell frequencies in mice lacking eosinophils either after antibody-mediated depletion, or due to mutation of the GATA1 locus., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The transcription factor (TF) interferon regulatory factor-4 (IRF4) promotes both germinal center (GC) reactions and plasma cell (PC) differentiation by binding to alternative DNA motifs including AP-1-IRF composite elements, Ets-IRF composite elements (EICEs), and interferon sequence response elements (ISREs). Although all of these motifs mediate transcriptional activation by IRF4, it is still unknown how some of the IRF4 target genes are downregulated upon PC differentiation. Here, we revealed a molecular mechanism of IRF4-mediated gene downregulation during PC differentiation. By combining IRF4 chromatin immunoprecipitation sequence and gene expression analysis, we identified zinc finger-IRF composite elements (ZICEs) in IRF4 binding regions aligned with genes whose expression was downregulated in PCs. The zinc finger TFs Ikaros and Aiolos were identified as IRF4 binding partners in PCs, and Ikaros but not Aiolos was essential for IRF4 binding to the ZICE sequence and for PC differentiation. The Ebf1 gene, which positively controls B-cell activation and GC reactions, was identified as one of the Ikaros/IRF4 target genes. Importantly, while the ZICE embeds the ISRE motif, IRF4 bound the ZICE motif as heterodimers with Ikaros for repression of target genes, which include Ebf1 In contrast, if the zinc finger motif is juxtaposed to the EICE motif, the Ikaros/PU.1/IRF4 complex functioned to activate target gene expression. Our findings revealed a novel mode of IRF4 activity upon PC differentiation where upon forming an Ikaros/IRF4 DNA-bound complex, a subset of genes is repressed., (© 2018 by The American Society of Hematology.)

Competing Interests: There are no conflicts of interest

Memory B cells (MBCs) and plasma cells (PCs) constitute the two cellular outputs of germinal center (GC) responses that together facilitate long-term humoral immunity. Although expression of the transcription factor BLIMP-1 identifies cells undergoing PC differentiation, no such marker exists for cells committed to the MBC lineage. Here, we report that the chemokine receptor CCR6 uniquely marks MBC precursors in both mouse and human GCs. CCR6 + GC B cells were highly enriched within the GC light zone (LZ), were the most quiescent of all GC B cells, exhibited a cell-surface phenotype and gene expression signature indicative of an MBC transition, and possessed the augmented response characteristics of MBCs. MBC precursors within the GC LZ predominantly possessed a low affinity for antigen but also included cells from within the high-affinity pool. These data indicate a fundamental dichotomy between the processes that drive MBC and PC differentiation during GC responses., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pre-existing serum antibodies play an important role in vaccine-mediated protection against infection but the underlying mechanisms of immune memory are unclear. Clinical studies indicate that antigen-specific antibody responses can be maintained for many years, leading to theories that reactivation/differentiation of memory B cells into plasma cells is required to sustain long-term antibody production. Here, we present a decade-long study in which we demonstrate site-specific survival of bone marrow-derived plasma cells and durable antibody responses to multiple virus and vaccine antigens in rhesus macaques for years after sustained memory B cell depletion. Moreover, BrdU + cells with plasma cell morphology can be detected for 10 years after vaccination/BrdU administration, indicating that plasma cells may persist for a prolonged period of time in the absence of cell division. On the basis of these results, long-lived plasma cells represent a key cell population responsible for long-term antibody production and serological memory.

Plasma cell differentiation is a tightly regulated process that requires appropriate T cell helps to reach the induction threshold. To further understand mechanisms by which T cell inputs regulate B cell fate decision, we investigate the minimal IL-2 stimulation for triggering human plasma cell differentiation in vitro. Here we show that the timed repression of BACH2 through IL-2-mediated ERK/ELK1 signalling pathway directs plasma cell lineage commitment. Enforced BACH2 repression in activated B cells unlocks the plasma cell transcriptional program and induces their differentiation into immunoglobulin M-secreting cells. RNA-seq and ChIP-seq results further identify BACH2 target genes involved in this process. An active regulatory region within the BACH2 super-enhancer, under ELK1 control and differentially regulated upon B-cell activation and cellular divisions, helps integrate IL-2 signal. Our study thus provides insights into the temporal regulation of BACH2 and its targets for controlling the differentiation of human naive B cells.

Humoral immunity requires B cells to respond to multiple stimuli, including antigen, membrane and soluble ligands, and microbial products. Ets family transcription factors regulate many aspects of haematopoiesis, although their functions in humoral immunity are difficult to decipher as a result of redundancy between the family members. Here we show that mice lacking both PU.1 and SpiB in mature B cells do not generate germinal centers and high-affinity antibody after protein immunization. PU.1 and SpiB double-deficient B cells have a survival defect after engagement of CD40 or Toll-like receptors (TLR), despite paradoxically enhanced plasma cell differentiation. PU.1 and SpiB regulate the expression of many components of the B cell receptor signaling pathway and the receptors for CD40L, BAFF and TLR ligands. Thus, PU.1 and SpiB enable B cells to appropriately respond to environmental cues.

Background: Alloantibodies to red blood cell (RBC) antigens can cause significant hemolytic events. Prior studies have demonstrated that inflammatory stimuli in animal models and inflammatory states in humans, including autoimmunity and viremia, promote alloimmunization. However, molecular mechanisms underlying these findings are poorly understood. Given that Type 1 interferons (IFN-α/β) regulate antiviral immunity and autoimmune pathology, the hypothesis that IFN-α/β regulates RBC alloimmunization was tested in a murine model., Study Design and Methods: Leukoreduced murine RBCs expressing the human KEL glycoprotein were transfused into control mice (WT), mice lacking the unique IFN-α/β receptor (IFNAR1 -/- ), or bone marrow chimeric mice lacking IFNAR1 on specific cell populations. Anti-KEL IgG production, expressed as mean fluorescence intensity (MFI), and B-cell differentiation were examined., Results: Transfused WT mice produced anti-KEL IgG alloantibodies (peak response MFI, 50.4). However, the alloimmune response of IFNAR1 -/- mice was almost completely abrogated (MFI, 4.2; p < 0.05). The response of bone marrow chimeric mice lacking IFNAR1 expression in all hematopoietic cells or specifically in B cells was also diminished (MFI, 3.8 and 5.4, respectively, compared to control chimeras, MFI, 65.6; p < 0.01). Accordingly, transfusion-induced differentiation of IFNAR1 -/- B cells into germinal center B cells and plasma cells was significantly reduced, compared to WT B cells., Conclusions: This study demonstrates that B cells require signaling from IFN-α/β to produce alloantibodies to the human KEL glycoprotein in mice. These findings provide a potential mechanistic basis for inflammation-induced alloimmunization. If these findings extend to human studies, patients with IFN-α/β-associated conditions may have an elevated risk of alloimmunization and benefit from personalized transfusion protocols., (© 2017 AABB.)

Monoclonal antibodies have found wide applications in the treatment of cancer, as well as of autoimmune, infectious, and other diseases. Several dozen new antibodies are currently undergoing different stages of clinical trials, and some of them will soon be added to the list of immunotherapeutic drugs. Most of these antibodies have been generated using hybridoma technology or a phage display. In recent years, new methods of obtaining human monoclonal antibodies have been actively developing. These methods rely on sequencing immunoglobulin genes from B lymphocytes, as well as on the creation of antibody-secreting stable B-cell lines. The term next-generation antibody-discovery platforms has already been established in the literature to refer to these approaches. Our review focuses on describing the results obtained by these methods.

Objectives: This study sought to investigate the genome-wide transcriptional effects of a combination of disease modifying anti-rheumatic drugs (tDMARD; methotrexate, sulfasalazine and hydroxychloroquine) in synovial tissues obtained from early rheumatoid arthritis (RA) patients. While combination DMARD strategies have been investigated for clinical efficacy, very little data exists on the potential molecular mechanism of action. We hypothesized that tDMARD would impact multiple biological pathways, but the specific pathways were unknown., Methods: Paired synovial biopsy samples from early RA patients before and after 6 months of tDMARD therapy were collected by arthroscopy (n = 19). These biopsies as well as those from subjects with normal synovium (n = 28) were profiled by total RNA sequencing., Results: Large differences in gene expression between RA and control biopsies (over 5000 genes) were identified. Despite clinical efficacy, the expression of a restricted set of less than 300 genes was reversed after 6 months of treatment. Many genes remained elevated, even in patients who achieved low disease activity. Interestingly, tDMARD downregulated genes included those involved in T cell activation and signaling and plasmablast/plasma cell differentiation and function., Conclusions: We have identified transcriptomic signatures that characterize synovial tissue from RA patients with early disease. Analysis after 6 months of tDMARD treatment highlight consistent alterations in expression of genes related to T cell activation and plasmablast/plasma cell differentiation. These results provide novel insight into the biology of early RA and the mechanism of tDMARD action and may help identify novel drug targets to improve rates of treatment-induced disease remission.

The name plasma cell was introduced by the anatomist Heinrich H. von Hartz-Waldeyer in 1875. Plasma cells derive from small B lymphocytes after their activation. A fully mature plasma cell lacks surface immunoglobulin expression. Its form is round or oval, with characteristic basophilic cytoplasm and an eccentric nucleus that contains coarse heterochromatin. Antigen activation of mature B cells leads initially to germinal center development, the transient generation of plasmablasts that secrete antibody while still dividing, and short-lived extrafollicular plasma cells that secrete antigen-specific germ line-encoded antibodies. Plasma cells are characterized by the co-expression of CD138 and CD38, which allows their identification in flow cytometry in bone marrow, peripheral blood, or cell suspensions from tissues. The identification of plasma cells as antibody producers was a key discovery that paved the way for the development of monoclonal antibodies., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background/aim: In the colorectum, lymphoid follicles hyperplasia (LH) is sometimes observed as small, round, yellowish-white nodules. The novel image-enhanced endoscopy system named blue laser imaging (BLI) provides enhanced the contrast of surface vessels using lasers for light illumination. We investigated the endoscopic features of LH observed by using BLI endoscopy and its association with chronic bowel symptoms., Patients/methods: 300 participants undergoing colonoscopy for various indications were enrolled. Entire colorectum was observed by using BLI-bright mode with non-magnification view. LH was defined as well demarcated white nodules. Elevated LH with erythema was distinguished as LH severe., Results: LHs were observed more clearly by using BLI-bright mode compared to conventional white light colonoscopy and were also histologically confirmed as intense infiltration of lymphocytes or plasmacytes. LH was observed in 134 subjects (44.6%) and 67 (22.3%) were LH severe. LH was associated younger age (Odds ratio (OR) = 1.05, 95%Confidence Interval (95%CI) = 1.03-1.07, P<0.0001) and chronic bowel symptoms including constipation, hard stools, diarrhea and loose stools (all LH: OR = 4.03, 95%CI = 2.36-6.89, P<0.0001, LH severe: OR = 5.31, 95%CI = 2.64-10.71, P<0.0001). LH severe was closely associated with both constipation associated symptoms (OR = 3.94, 95%CI = 1.79-8.66, P = 0.0007) and diarrhea associated symptoms (OR = 5.22, 95%CI = 2.09-13.05, P = 0.0004). In particular, LH severe in the ascending colon was strongly associated with bowel symptoms (P<0.0001)., Conclusion: LH, visualized by using BLI endoscopy was associated with bowel symptom, raising the possibility of pathogenic role of this endoscopic finding in the functional lower gastrointestinal disorders.

Being the sole source of antibody, plasmablasts and plasma cells are essential for protective immunity. Due to their relative rarity, heterogeneity and the loss of many canonical B-cell markers, antibody-secreting cells (ASCs) have often been problematic to identify and further characterize. In the mouse, the combination of the expression of CD138 and BLIMP-1, has led to many insights into ASC biology, although this approach requires the use of a GFP reporter strain. In the current issue of the European Journal of Immunology, two independent studies by Wilmore et al. and Pracht et al. provide alternative approaches to identify all murine ASCs using antibodies against the cell surface proteins, Sca-1 and TACI, respectively. Here we will discuss the advantages of these new approaches to identify ASCs in the context of our emerging knowledge of the cell surface phenotype and gene expression program of various ASC subsets in the murine and human systems., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Increasing evidence indicates that alternative processing of mRNA, including alternative splicing, 3' alternative polyadenylation, and regulation of mRNA stability/translation, represents a major mechanism contributing to protein diversification. For example, in alternative polyadenylation, the 3' end of the immunoglobulin heavy chain mRNA is processed during B cell differentiation, and this processing involves RNA-binding proteins. hnRNPLL (heterogeneous nuclear ribonucleoprotein L-like protein) is an RNA-binding protein expressed in terminally differentiated lymphocytes, such as memory T cells and plasma cells. hnRNPLL regulates various processes of RNA metabolism, including alternative pre-mRNA splicing and RNA stability. In plasma cells, hnRNPLL also regulates the transition from the membrane isoform of the immunoglobulin heavy-chain (mIgH) to the secreted isoform (sIgH), but the precise mechanism remains to be identified. In this study, we report that hnRNPLL specifically associates with cytoplasmic PABPC1 (poly(A)-binding protein 1) in both T cells and plasma cells. We found that although PABPC1 is not required for the alternative splicing of CD45, a primary target of hnRNPLL in lymphocytes, PABPC1 does promote the binding of hnRNPLL to the immunoglobulin mRNA and regulates switching from mIgH to sIgH in plasma cells. Given the recently identified role of PABPC1 in mRNA alternative polyadenylation, our findings suggest that PABPC1 recruits hnRNPLL to the 3'-end of RNA and regulates the transition from membrane Ig to secreted Ig through mRNA alternative polyadenylation. In conclusion, our study has revealed a mechanism that regulates immunoglobulin secretion in B cells via cooperation between a plasma cell-specific RBP (hnRNPLL) and a universally expressed RBP (PABPC1)., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Plasma cells (PCs) are a major source of alloantibody in transplant patients and are resistant to current therapy. Because receptor-ligand interactions in stromal microenvironments play important roles in the localization, development, and survival of normal PCs, we hypothesized that interfering with CXCR4/CXCL12 interactions with plerixafor might cause PC depletion and enhance the efficacy of the proteasome inhibitor bortezomib. PCs in mouse spleen, bone marrow, and peripheral blood demonstrated CXCR4 expression. We then treated with plerixafor in doses ranging from 240 μg/kg in a single dose to a 1-mg/kg daily dose for 10 days. CXCR4/CXCL12 blockade with plerixafor resulted in increased mobilization of PCs into the peripheral blood. Splenectomy completely abrogated this effect, suggesting that all plerixafor-mobilized cells were from the spleen. The total number of PCs in the spleen and marrow remained constant despite treatment with plerixafor. Bortezomib caused a reduction in PCs, but adding plerixafor did not increase killing. We conclude that CXCR4/CXCL12 interactions are important for the retention of a subpopulation of PCs in the spleen, but this interaction has minimal effect on PCs in the marrow. The lack of enhancement of bortezomib-mediated depletion suggests that factors other than CXCR4/CXCL12 interactions are responsible for drug resistance., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Follicular T helper (T FH ) cells orchestrate the germinal center (GC) reaction locally. Local mechanisms regulating their dynamics and helper functions are not well defined. Here we found that GC-expressed ephrin B1 (EFNB1) repulsively inhibited T cell to B cell adhesion and GC T FH retention by signaling through T FH -expressed EPHB6 receptor. At the same time, EFNB1 promoted interleukin-21 production from GC T FH cells by signaling predominantly through EPHB4. Consequently, EFNB1-null GCs were associated with defective production of plasma cells despite harboring excessive T FH cells. In a competitive GC reaction, EFNB1-deficient B cells more efficiently interacted with T FH cells and produced more bone-marrow plasma cells, likely as a result of gaining more contact-dependent help. Our results reveal a contact-dependent repulsive guidance system that controls GC T FH dynamics and effector functions locally., (Copyright © 2017, American Association for the Advancement of Science.)

Antibody secreting cells (ASCs) are critical effector cells and long-lived sentinels for immune memory. ASCs are highly dependent on exogenous soluble factors such as interleukin-6 (IL-6) and APRIL, to prevent their cell death. We have found that the canonical surface marker of ASCs, CD138 (syndecan-1), which is upregulated during ASC maturation, is required in a cell-intrinsic manner to mount an effective long-term humoral immune response following immunization. Surface expression of CD138 increased heparan sulfate levels on ASCs, which are known to bind pro-survival cytokines, leading to increased survival in a cell-intrinsic manner in vivo. In IL-6 and APRIL-deficient hosts, ASCs underwent extensive apoptosis independently of CD138 expression. We propose a model in which CD138 expression on fully mature ASCs provides a selective survival advantage over less mature, newly minted ASCs, by enhancing pro-survival cytokine signaling., (© 2017 by The American Society of Hematology.)

Humoral immune responses are tailored to the invading pathogen through regulation of key transcription factors and their networks. This is critical to establishing effective antibody-mediated responses, yet it is unknown how B cells integrate pathogen-induced signals to drive or suppress transcriptional programs specialized for each class of pathogen. Here, we detail the key role of the transcription factor c-Myb in regulating the T-bet-mediated anti-viral program. Deletion of c-Myb in mature B cells significantly increased serum IgG2c and CXCR3 expression by upregulating T-bet, normally suppressed during Th2-cell-mediated responses. Enhanced expression of T-bet resulted in aberrant plasma cell differentiation within the germinal center, mediated by CXCR3 expression. These findings identify a dual role for c-Myb in limiting inappropriate effector responses while coordinating plasma cell differentiation with germinal center egress. Identifying such intrinsic regulators of specialized antibody responses can assist in vaccine design and therapeutic intervention in B-cell-mediated immune disorders., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The necessity for bone marrow aspiration and the lack of highly sensitive assays to detect residual disease present challenges for effective management of multiple myeloma (MM), a plasma cell cancer. We show that a microfluidic cell capture based on CD138 antigen, which is highly expressed on plasma cells, permits quantitation of rare circulating plasma cells (CPCs) in blood and subsequent fluorescence-based assays. The microfluidic device is based on a herringbone channel design, and exhibits an estimated cell capture efficiency of ~40-70%, permitting detection of <10 CPCs/mL using 1-mL sample volumes, which is difficult using existing techniques. In bone marrow samples, the microfluidic-based plasma cell counts exhibited excellent correlation with flow cytometry analysis. In peripheral blood samples, the device detected a baseline of 2-5 CD138 + cells/mL in healthy donor blood, with significantly higher numbers in blood samples of MM patients in remission (20-24 CD138 + cells/mL), and yet higher numbers in MM patients exhibiting disease (45-184 CD138 + cells/mL). Analysis of CPCs isolated using the device was consistent with serum immunoglobulin assays that are commonly used in MM diagnostics. These results indicate the potential of CD138-based microfluidic CPC capture as a useful 'liquid biopsy' that may complement or partially replace bone marrow aspiration.

Secondary lymphoid organs are organized into distinct zones, governed by different types of mesenchymal stromal cells. These stromal cell subsets are critical for the generation of protective humoral immunity because they direct the migration of, and interaction between, multiple immune cell types to form the germinal centre. The germinal centre response generates long-lived antibody-secreting plasma cells and memory B cells which can provide long-term protection against re-infection. Stromal cell subsets mediate this response through control of immune cell trafficking, activation, localization and antigen access within the secondary lymphoid organ. Further, distinct populations of stromal cells underpin the delicate spatial organization of immune cells within the germinal centre. Because of this, the interactions between immune cells and stromal cells in secondary lymphoid organs are fundamental to the germinal centre response. Herein we review how this unique relationship leads to effective germinal centre responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Germinal centers (GCs) form in secondary lymphoid tissues in response to antigenic challenge and are the site of somatic hypermutation, generating GC B cells with increasing affinity for the inciting agent that are positively selected over time. However, it is not until GC B cells differentiate into memory B cells and plasma cells and egress from the GC back into the circulation that effective long-lived humoral immunity is conferred upon the host. Here we review what is known about the signals that initiate the transition from a GC B cell into the memory B cell and plasma cell compartments and the downstream transcriptional regulation of these processes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Memory B cell generation and antibody production result from a differentiation process that begins when the surface BCR on naïve B cells binds an antigen. How the choice between these fates is tempo-spatially regulated is still obscure, but recent advances have reinforced the concept that the combination of B cell-intrinsic heterogeneity and -extrinsic heterogeneity provided by cells such as T cells is a key determinant. As molecular regulators, the transcription factors IRF4 and Bach2, which participate in these fate choices, have been emerging., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Interleukin (IL) 17A in chronic inflammation is also produced by innate immune cells as neutrophils. Mice with chronic humoral response induced by venom of Thalassophryne nattereri (VTn) proved to be a good tool for evaluating the impact of IL-17A on the development of long-lived plasma cells in the inflamed peritoneal cavity. Here, we report that VTn induces IL-17A production by neutrophils accumulating in the peritoneal cavity and triggers the extrusion of IL-17A along with neutrophil extracellular traps (NETs). Neutrophil depletion reduced the number of IL17A-producing cells in VTn-immunized mice and blocked the differentiation of long-lived plasma cells. Specific antibody production and survival of long-lived plasma cells was ablated in VTn-immunized mice deficient in CD4, while CD28 signaling had the opposite effect on differentiation of long-lived plasma cells. Further, maturation of long-lived plasma cells in inflamed peritoneal cavity was IL-1R1 and COX-2 dependent. Finally, when both the Raf-MEK-ERK pathway and the IL-17A or IL-1R1 activities were blocked, neutrophils were unable to promote the differentiation of memory B cells into long-lived plasma cells, confirming the essential role of neutrophils and IL-17A along with NETs in an IL-1/IL-1R-dependent manner as the novel helping partner for plasma cell differentiation in chronically inflamed tissues.