Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders.

The meninges contain adaptive immune cells that provide immunosurveillance of the central nervous system (CNS). These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial-meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells, which may help maintain immune privilege within the CNS.

Imaging of Bone Marrow Plasma Cells and of Their Niches.

Decade-long survival of plasma cells in the bone marrow has long been a puzzling matter. To understand how plasma cells are maintained and supported by survival-niches to account for lifelong antibody production demands new intravital imaging techniques that are able to follow up a single cell and their interaction with other cell types in situ. We achieved to successfully establish longitudinal imaging of the bone marrow (LIMB) that is based on an implantable endoscopic device. In this chapter, basic approaches on how to investigate plasma cell-stroma interaction and surgical implantation procedures are introduced.

RNA-sequencing data-driven dissection of human plasma cell differentiation reveals new potential transcription regulators.

Plasma cells (PCs) play an important role in the adaptive immune system through a continuous production of antibodies. We have demonstrated that PC differentiation can be modeled in vitro using complex multistep culture systems reproducing sequential differentiation process occurring in vivo. Here we present a comprehensive, temporal program of gene expression data encompassing human PC differentiation (PCD) using RNA sequencing (RNA-seq). Our results reveal 6374 differentially expressed genes classified into four temporal gene expression patterns. A stringent pathway enrichment analysis of these gene clusters highlights known pathways but also pathways largely unknown in PCD, including the heme biosynthesis and the glutathione conjugation pathways. Additionally, our analysis revealed numerous novel transcriptional networks with significant stage-specific overexpression and potential importance in PCD, including BATF2, BHLHA15/MIST1, EZH2, WHSC1/MMSET, and BLM. We have experimentally validated a potent role for BLM in regulating cell survival and proliferation during human PCD. Taken together, this RNA-seq analysis of PCD temporal stages helped identify coexpressed gene modules with associated up/downregulated transcription regulator genes that could represent major regulatory nodes for human PC maturation. These data constitute a unique resource of human PCD gene expression programs in support of future studies for understanding the underlying mechanisms that control PCD.

CD36 and LC3B initiated autophagy in B cells regulates the humoral immune response.

Scavenger receptors are pattern recognition receptors that recognize both foreign and self-ligands, and initiate different mechanisms of cellular activation, often as co-receptors. The function of scavenger receptor CD36 in the immune system has mostly been studied in macrophages but it is also highly expressed by innate type B cells where its function is less explored. Here we report that CD36 is involved in macro-autophagy/autophagy in B cells, and in its absence, the humoral immune response is impaired. We found that CD36-deficient B cells exhibit a significantly reduced plasma cell formation, proliferation, mitochondrial mobilization and oxidative phosphorylation. These changes were accompanied by impaired initiation of autophagy, and we found that CD36 regulated autophagy and colocalized with autophagosome membrane protein MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). When we investigated T-cell-dependent immune responses, we found that mice with CD36 deficiency, specifically in B cells, exhibited attenuated germinal center responses, class switching, and antibody production as well as autophagosome formation. These findings establish a critical role for CD36 in B cell responses and may also contribute to our understanding of CD36-mediated autophagy in other cells as well as in B cell lymphomas that have been shown to express the receptor.Abbreviations: AICDA/AID: activation-induced cytidine deaminase; ATG5: autophagy related 5; ATP: adenosine triphosphate; BCR: B-cell receptor; CPG: unmethylated cytosine-guanosine; CQ: chloroquine; DC: dendritic cells; FOB: follicular B cells; GC: germinal center; Ig: immunoglobulin; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MZB: marginal zone B cells; NP-CGG: 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin; OCR: oxygen consumption rate; oxLDL: oxidized low-density lipoprotein; PC: plasma cells; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; SRBC: sheep red blood cells; Tfh: follicular helper T cells; TLR: toll-like receptor.

IRF4 Activity Is Required in Established Plasma Cells to Regulate Gene Transcription and Mitochondrial Homeostasis.

The transcription factor interferon regulatory factor 4 (IRF4) is critical for the development, maintenance, and function of plasma cells. The mechanism by which IRF4 exerts its action in mature plasma cells has been elusive due to the death of all such cells upon IRF4 loss. While we identify apoptosis as a critical pathway for the death of plasma cells caused by IRF4 loss, we also determine that IRF4 did not regulate the intrinsic apoptotic pathway directly. By using an inducible IRF4 deletion system in the presence of the overexpression of anti-apoptotic BCL2, we identify genes whose expression is coordinated by IRF4 and that in turn specify plasma cell identity and mitochondrial homeostasis.

Virus-Specific Secondary Plasma Cells Produce Elevated Levels of High-Avidity Antibodies but Are Functionally Short Lived.

Most vaccines aim at inducing durable antibody responses and are designed to elicit strong B cell activation and plasma cell (PC) formation. Here we report characteristics of a recently described secondary PC population that rapidly originates from memory B cells (MBCs) upon challenge with virus-like particles (VLPs). Upon secondary antigen challenge, all VLP-specific MBCs proliferated and terminally differentiated to secondary PCs or died, as they could not undergo multiple rounds of re-stimulation. Secondary PCs lived in bone marrow and secondary lymphoid organs and exhibited increased production of antibodies with much higher avidity compared to primary PCs, supplying a swift wave of high avidity antibodies early after antigen recall. Unexpectedly, however, secondary PCs were functionally short-lived and most of them could not be retrieved in lymphoid organs and ceased to produce antibodies. Nevertheless, secondary PCs are an early source of high avidity antibodies and induction of long-lived MBCs with the capacity to rapidly differentiate to secondary PCs may therefore be an underestimated possibility to induce durable protection by vaccination.

Plasma Cells Are Obligate Effectors of Enhanced Myelopoiesis in Aging Bone Marrow.

Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.

Bone Marrow Plasma Cells Modulate Local Myeloid-Lineage Differentiation via IL-10.

Bone marrow plasma cells have been reported to represent a major source of IL-10; however, the impact of plasma cell derived IL-10 in that tissue remains poorly understood. We confirm in this study that even in the absence of acute immune reactions, mature plasma cells represent the dominant IL-10+ cell population in the bone marrow, and identify myeloid-lineage cells as a main local target for plasma cell derived IL-10. Using Vert-X IL-10 transcriptional reporter mice, we found that more than 50% of all IL-10+ cells in bone marrow were CD138+ plasma cells, while other IL-10+ B lineage cells were nearly absent in this organ. Accordingly, IL-10 was found in the supernatants of short-term cultures of FACS-sorted bone marrow plasma cells, confirming IL-10 secretion from these cells. IL-10+ bone marrow plasma cells showed a B220-/CD19-/MHCII low phenotype suggesting that these cells represent a mature differentiation stage. Approximately 5% of bone marrow leucocytes expressed the IL-10 receptor (IL-10R), most of them being CD115+/Ly6C+/CD11c- monocytes. Compared to littermate controls, young B lineage specific IL-10 KO mice showed increased numbers of CD115+ cells but normal populations of other myeloid cell types in bone marrow. However, at 7 months of age B lineage specific IL-10 KO mice exhibited increased populations of CD115+ myeloid and CD11c+ dendritic cells (DCs), and showed reduced F4/80 expression in this tissue; hence, indicating that bone marrow plasma cells modulate the differentiation of local myeloid lineage cells via IL-10, and that this effect increases with age. The effects of B cell/plasma cell derived IL-10 on the differentiation of CD115+, CD11c+, and F4/80+ myeloid cells were confirmed in co-culture experiments. Together, these data support the idea that IL-10 production is not limited to early plasma cell stages in peripheral tissues but is also an important feature of mature plasma cells in the bone marrow. Moreover, we provide evidence that already under homeostatic conditions in the absence of acute immune reactions, bone marrow plasma cells represent a non-redundant source for IL-10 that modulates local myeloid lineage differentiation. This is particularly relevant in older individuals.

Neutrophil-Derived MRP14 Supports Plasma Cell Commitment and Protects Myeloma Cells from Apoptosis.

Neutrophils have recently been proposed as cells with high functional plasticity and are involved in the pathogenesis of infections, malignancy, and autoimmune diseases. However, less is known about the role of neutrophil in humoral response. In this study, we examined the importance of neutrophils and the neutrophil-derived DAMP protein, MRP14, in antibody production. Splenic neutrophils and MRP14 that are present in the splenic peri-MZ region have a close contact with MZ B cells and promote their differentiation into plasma cells. Using neutrophil-depleting mice and an MRP14-blocking compound, we showed that the presence of neutrophil and MRP14 is required for class switch, plasma cell maintenance, and antibody production in the spleen. We found that MRP14 could also be produced by neutrophils in the bone marrow and support the maintenance of bone marrow plasma cells. MRP14 binding could enhance the effect of the BAFF signal and protect primary multiple myeloma cells from doxorubicin-induced apoptosis. Our data demonstrate the effects of neutrophils on neighboring B cells and plasma cells, which provides new insights into the connection between neutrophil and humoral responses.

Increased Osteoblastic Activity Suppressed Proliferation of Multiple Myeloma Plasma Cells.

STUDY DESIGN: In vitro experimental study. OBJECTIVE: To investigate the impact of increased osteoblastic activity on the proliferation and survival of multiple myeloma (MM) plasma cells in vitro SUMMARY OF BACKGROUND DATA.: MM is one of representative hematologic malignancies that cause skeletal-related events (SREs) and dysregulation of bone remodeling is known as a key pathomechanism of disease progression and skeletal-related events. However, decreased proliferation of MM at fracture sites is frequently noted in clinical situations regardless of systemic disease activity. METHODS: Co-culture under various conditions was used to investigate effects of increased osteoblastic activity on survival and proliferation of MM plasma cells. MM plasma cells were cultured in culture media (control) and co-cultured with human mesenchymal stem cells (hMSCs, group I), osteoblasts (OBs) induced from hMSCs (group II) or bone morphogenic protein-2 (BMP-2, group III). Proliferation measured as extracellular signal-regulated kinase (ERK) and immunoglobulin G (Ig G) expression and apoptosis measured as fluorescence-activated cell sorting (FACS) with annexin V method, caspase-3, and stat-3 expression were assessed for cultured MM plasma cells, along with expression of sclerostin. RESULTS: After 72 hours of co-culture, group II and III showed decreased ERK expression compared with controls. Lower Ig G expression was also noted for groups II and III compared with controls. Group I did not show significantly decreased Ig G and ERK expression compared with controls. Expressions of caspase-3 in groups II and III were higher than controls. Co-culture with hMSCs showed decreased caspase-3 expression compared with control. FACS with annexin V showed higher apoptosis in groups II and III. Sclerostin expression was also decreased in osteoblastic conditions compared with the control and hMSCs co-culture condition. CONCLUSION: Collectively, our data suggest that increased osteoblastic conditions may provide not only prevention of SREs but also anti-tumor effects on MM cells in the bone marrow environment. LEVEL OF EVIDENCE: N/A.

Exosome-transmitted LINC00461 promotes multiple myeloma cell proliferation and suppresses apoptosis by modulating microRNA/BCL-2 expression.

BACKGROUND: Multiple myeloma (MM) is a hematologic cancer caused by the abnormal expansion of plasma cells, but the exact mechanism underlying MM development is not completely known. Recently, multiple long noncoding RNAs (lncRNAs) were implicated in the regulation of MM development. METHODS: Samples from patients with MM were collected and detected for LINC00461 expression using real-time polymerase chain reaction (PCR). LINC00461 was knocked down in MM cell lines by short hairpin RNAs (shRNAs) to measure its effect on MM cell proliferation and apoptosis. The function of mesenchymal stromal cell (MSC)-derived exosomes was analyzed using chamber assays. RESULTS: LINC00461 was highly expressed in MM. Knockdown of LINC00461 dramatically reduced MM cell proliferation and induced cell apoptosis. Further study showed that LINC00461 relieved the inhibitory effect of microRNA (miR)-15a/miR-16 on BCL-2. In addition, we observed that MSC-derived exosomes promoted MM cell proliferation through LINC00461. CONCLUSION: Our findings demonstrate that LINC00461, a sponge for miR-15a/16, is highly expressed in MSC-derived exosomes, and enhances MM cell proliferation, which may become an excellent candidate for therapeutic applications.

Stochastically Timed Competition Between Division and Differentiation Fates Regulates the Transition From B Lymphoblast to Plasma Cell.

In response to external stimuli, naïve B cells proliferate and take on a range of fates important for immunity. How their fate is determined is a topic of much recent research, with candidates including asymmetric cell division, lineage priming, stochastic assignment, and microenvironment instruction. Here we manipulate the generation of plasmablasts from B lymphocytes in vitro by varying CD40 stimulation strength to determine its influence on potential sources of fate control. Using long-term live cell imaging, we directly measure times to differentiate, divide, and die of hundreds of pairs of sibling cells. These data reveal that while the allocation of fates is significantly altered by signal strength, the proportion of siblings identified with asymmetric fates is unchanged. In contrast, we find that plasmablast generation is enhanced by slowing times to divide, which is consistent with a hypothesis of competing timed stochastic fate outcomes. We conclude that this mechanistically simple source of alternative fate regulation is important, and that useful quantitative models of signal integration can be developed based on its principles.

LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells.

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

Plasma cell differentiation is controlled by multiple cell division-coupled epigenetic programs.

The genomic loci associated with B cell differentiation that are subject to transcriptional and epigenetic regulation in vivo are not well defined, leaving a gap in our understanding of the development of humoral immune responses. Here, using an in vivo T cell independent B cell differentiation model, we define a cellular division-dependent cis-regulatory element road map using ATAC-seq. Chromatin accessibility changes correlate with gene expression and reveal the reprogramming of transcriptional networks and the genes they regulate at specific cell divisions. A subset of genes in naive B cells display accessible promoters in the absence of transcription and are marked by H3K27me3, an EZH2 catalyzed repressive modification. Such genes encode regulators of cell division and metabolism and include the essential plasma cell transcription factor Blimp-1. Chemical inhibition of EZH2 results in enhanced plasma cell formation, increased expression of the above gene set, and premature expression of Blimp-1 ex vivo. These data provide insights into cell-division coupled epigenetic and transcriptional processes that program plasma cells.

DNMTi/HDACi combined epigenetic targeted treatment induces reprogramming of myeloma cells in the direction of normal plasma cells.

BACKGROUND: Multiple myeloma (MM) is the second most common hematologic malignancy. Aberrant epigenetic modifications have been reported in MM and could be promising therapeutic targets. As response rates are overall limited but deep responses occur, it is important to identify those patients who could indeed benefit from epigenetic-targeted therapy. METHODS: Since HDACi and DNMTi combination have potential therapeutic value in MM, we aimed to build a GEP-based score that could be useful to design future epigenetic-targeted combination trials. In addition, we investigated the changes in GEP upon HDACi/DNMTi treatment. RESULTS: We report a new gene expression-based score to predict MM cell sensitivity to the combination of DNMTi/HDACi. A high Combo score in MM patients identified a group with a worse overall survival but a higher sensitivity of their MM cells to DNMTi/HDACi therapy compared to a low Combo score. In addition, treatment with DNMTi/HDACi downregulated IRF4 and MYC expression and appeared to induce a mature BMPC plasma cell gene expression profile in myeloma cell lines. CONCLUSION: In conclusion, we developed a score for the prediction of primary MM cell sensitivity to DNMTi/HDACi and found that this combination could be beneficial in high-risk patients by targeting proliferation and inducing maturation.

The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production.

A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab')2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2-Rac axis in PC differentiation and IgG antibody responses.