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The roles distinct B cell subsets play in clonal expansion, isotype switching, and memory B cell differentiation in response to T cell–independent type 2 Ags (TI-2 Ags) has been understudied. Using sorted B cells from VHB1-8 knock-in mice, we evaluated B-1b, marginal zone, and follicular B cell responses to the TI-2 Ag, NP–Ficoll. All subsets extensively divided in response to NP–Ficoll. Nonetheless, B-1b cells exhibited significantly increased IgG switching and differentiation into Ab-secreting cells (ASC)—a finding that coincided with increased AgR signaling capacity and Blimp1 expression by B-1b cells. All subsets formed memory cells and expressed markers previously identified for T cell–dependent memory B cells, including CD80, PDL2, and CD73, although B-1b cells generated the greatest number of memory cells with higher frequencies of IgG- and CD80-expressing cells. Despite memory formation, secondary immunization 4 wk after primary immunization did not increase NP-specific IgG. However, boosting occurred in B-1b cell–recipient mice when IgG levels declined. CD80+ memory B-1b cells divided, class switched, and differentiated into ASC in response to Ag in vivo, but this was inhibited in the presence of NP-specific IgG. Furthermore, CD80 blockade significantly increased memory B-1b cell division and differentiation to ASC upon Ag restimulation. Collectively, these findings demonstrate B-1b, marginal zone B, and follicular B subsets significantly contribute to the TI-2 Ag–specific memory B cell pool. In particular, we show B-1b cells generate a functional CD80-regulated memory population that can be stimulated to divide and differentiate into ASC upon Ag re-encounter when Ag-specific IgG levels decline.

Diverse B cell responses are important for generating antibody-mediated protection against highly variable pathogens. While some antigens can trigger T-independent B cell proliferation and short-term antibody production, development of long-term humoral immunity requires T-dependent B cell responses. The "two-signal" model of B cell activation has long been invoked to explain alternate B cell recruitment into immune response to foreign antigens vs. induction of tolerance to self-antigens. However, a number of other factors appear to influence the fate of mature B cells responding to antigen in vivo. In this review, we will discuss how various spatiotemporal scenarios of antigen access into secondary lymphoid organs, antigen valency and cellular environment of antigen acquisition by B cells, duration of B cell access to antigen and the timing of T cell help may affect follicular B cell fate, including death, survival, anergy, and recruitment into T-dependent responses. We will also highlight unresolved questions related to B cell activation and tolerance in vivo that may have important implications for vaccine development and autoimmunity.

Germinal centers (GCs) are essential for the production of somatically hypermutated, class-switched Abs that are protective against infection, but they also form in the absence of purposeful immunization or infection, and are termed spontaneous GCs (Spt-GCs). Although Spt-GCs can arise in nonautoimmune-prone mice, aberrant regulation of Spt-GCs in autoimmune-prone mice is strongly associated with the development of autoimmune diseases like systemic lupus erythematosus. The formation of Spt-GCs is crucially driven by TLR7-mediated RNA sensing. However, the impact of MAVS-dependent, Rig-like receptor–mediated RNA sensing on the Spt-GC response remains unknown. In this study, we assessed the Spt-GC response and splenic B cell development in two MAVS-deficient mice with distinct genetic backgrounds. Importantly, we found that MAVS differentially controls Spt-GC responses and B cell development, depending on genetic background. B6/129 mixed background MAVSKO mice had nearly absent Spt-GC responses in the spleen and cervical lymph nodes, which were associated with impaired splenic B cell development, in addition to impaired B cell activation and TLR7 expression. Interestingly, treatment of mice with TLR7 agonist could partially rescue GC responses by overcoming follicular B cell activation deficits. Contrastingly, the absence of MAVS on a B6 background resulted in normal B cell development and Spt-GC formation. Our results highlight important differences in the contribution of MAVS to B cell development and Spt-GC function, depending on the genetic background, warranting greater regard for the impact of genetic background in further studies using these mice for the study of autoimmunity.

Sodium antimonials are one of the major and common drugs used against visceral form leishmaniasis (VL). However, the development of drug resistance makes it difficult to manage this disease. Current work investigates the modulation of splenic B cells during experimental infection with antimony-sensitive and -resistant Leishmania&nbsp, donovani infection. Here we phenotypically characterized splenic B cell subsets in BALB/c mice infected with antimony drug-sensitive and -resistant VL strains using flow-cytometry method. In the splenocytes we noticed increased number of Transitional T3 B cells and B1a B cells in drug-resistant VL strain infection. Besides, we also observed alteration in Follicular B cell population of antimony-resistant strain infected mice. Drug-resistant strain induced secretion of elevated level of IL-10 from B1a B cells and IL-6 from Transitional T3 B cell subsets in the splenocytes. Purified splenic B cells from antimony drug-resistant strain infected mice showed decrease in the Lyn kinase gene expression compared to sensitive strain infected and uninfected mice. The current study provides insight into changes in host splenic B-cell subsets during experimental infection with antimony-sensitive and -resistant L. donovani in murine model.

The maturation of immature B cells and the survival of mature B cells is stringently controlled to maintain a diverse repertoire of antibody specificities while avoiding self-reactivity. At the molecular level this is regulated by signalling from membrane immunoglobulin and the BAFF-receptor which sustain a pro-survival programme of gene expression. Whether and how posttranscriptional mechanisms contribute to B cell maturation and survival remains poorly understood. Here we show that the polypyrimidine tract binding proteins (PTBP) PTBP1 and PTBP3 bind to a large and overlapping set of transcripts in B cells. Both PTBP1 and PTBP3 bind to introns and exons where they are predicted to regulate alternative splicing. Moreover, they also show high-density of binding to 3' untranslated regions suggesting they influence the transcriptome in diverse ways. We show that PTBP1 and PTBP3 are required in B cells beyond the immature cell stage to sustain transitional B cells and the B1, marginal zone and follicular B cell lineages. Therefore, PTBP1 and PTBP3 promote the maturation of quiescent B cells by regulating gene expression at the post-transcriptional level. This article is protected by copyright. All rights reserved.

Background: Chemokine (C-C motif) receptor 2 (CCR2) contributes to autoimmune pathogenesis. However, the effect of CCR2 on B cell signaling and its role in autoimmunity remains unclear. Herein, we investigated the role of CCR2 in the B cell receptor (BCR) signaling pathway and aimed to illustrate its potential molecular mechanisms of action. Methods: To investigate the alterations in B cell signaling and the immune response, we used flow cytometry, western blotting, microscopic techniques, Seahorse assay, and immunofluorescence assay on samples from C57BL/6 mice and germinal CCR2-deletion mice. Results: The absence of CCR2 disturbed follicular B cell development. Furthermore, CCR2 absence was correlated with increased mTORC1-mediated energy metabolism and enhanced early B cell activation, which were induced by the up-regulation of BCR proximal signaling and F-actin accumulation. Mst1 and STAT1 were key factors in up-regulating the B cell activation in CCR2 deficient mice. The disrupted peripheral B cell differentiation and enhanced B cell signaling were associated with the inhibition mTORC1, Mst1, and STAT1. Moreover, loss of CCR2 caused a weakened T cell dependent antigen response, resulting in decreased antibody secreting cells and diminished antigen specific IgM levels. Conclusion: CCR2 is involved in the regulation of BCR signaling pathway by sequentially activating signaling pathways dominated by Mst1, mTORC1, and STAT1. Our study suggests that CCR2 might represent a novel therapeutic targeted for autoimmune diseases.

The function of mitochondrial respiration during B cell fate decisions and differentiation remains equivocal. This study reveals that selection for mitochondrial fitness occurs during B cell activation and is essential for subsequent plasma cell differentiation. By expressing a mutated mitochondrial helicase in transitional B cells, we depleted mitochondrial DNA during B cell maturation, resulting in reduced oxidative phosphorylation. Although no changes in follicular B cell development were evident, germinal centers, class switch recombination to IgG, plasma cell generation and humoral immunity were diminished. Defective oxidative phosphorylation led to aberrant flux of the tricarboxylic acid cycle and lowered the amount of saturated phosphatidic acid. Consequently, MTOR activity and BLIMP-1 induction were curtailed whereas HIF1α, glycolysis and AMPK activity were amplified. Exogenous phosphatidic acid increased mTOR activity in activated B cells. Hence, mitochondrial function is required and selected for in activated B cells for the successful generation of functional plasma cells.

Non-Hodgkin lymphomas comprise a heterogeneous group of malignancies, with a wide scope of clinical, radiological and histological presentations. In this paper, a case is presented of a 59-year-old white male with an infraorbital follicular B-cell lymphoma, which appeared as a painless mass in the left cheek. The lymphoma achieved spontaneous remission five and a half months after his diagnostic incision biopsy. The literature is reviewed, focusing on this rare site of presentation and spontaneous remission. In literature, only four cases have been reported with a follicular B-cell lymphoma of the cheek or infraorbital region, and only 26 cases of spontaneous remission of an extracranial non-Hodgkin lymphoma in the head and neck region have been described. To the authors' best knowledge, this is the first time spontaneous remission of an infraorbital follicular lymphoma could be observed. The nature of the processes inducing spontaneous remission remains obscure. It is important to recognize this phenomenon as this might prevent unnecessary treatment. ispartof: PATHOLOGY & ONCOLOGY RESEARCH vol:27 ispartof: location:Switzerland status: published

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

Transitional B cells must actively undergo selection for self-tolerance before maturing into their resting follicular B cell successors. We found that metabolic quiescence was acquired at the follicular B cell stage in both humans and mice. In follicular B cells expression of genes involved in ribosome biogenesis, aerobic respiration, and mammalian target of rapamycin complex 1 (mTORC1) signaling were reduced when compared to that in transitional B cells. Functional metabolism studies, profiling of whole cell metabolites, and analysis of cell surface proteins in human B cells suggested that this transition was also associated with increased extracellular adenosine salvage. Follicular B cells increased the abundance of the cell surface ectonucleotidase CD73, which coincided with 5’ adenosine monophosphate-activated protein kinase (AMPK) activation and mTORC1 inhibition. Differentiation to the follicular B cell stage in vitro correlated with surface acquisition of CD73 on human transitional B cells and was augmented with the AMPK agonist, AICAR. Finally, individuals with gain-of-function PIK3CD (PI3Kδ) mutations exhibite-JRF and SPd a near absence of circulating follicular B cells. Together, our data suggest that mTORC1 attenuation may be necessary for human follicular B cell development. These data identify a distinct metabolic switch during human B cell development at the transitional to follicular stages, which is characterized by an induction of extracellular adenosine salvage, mTORC1 inhibition, and the acquisition of metabolic quiescence.

Marginal zone B cells (MZB) are a mature B cell subset that rapidly respond to blood-borne pathogens. Although the transcriptional changes that occur throughout MZB development are known, the corresponding epigenetic changes and epigenetic modifying proteins that facilitate these changes are poorly understood. The histone demethylase LSD1 is an epigenetic modifier that promotes plasmablast formation, but its role in B cell development has not been explored. In this study, a role for LSD1 in the development of B cell subsets was examined. B cell–conditional deletion of LSD1 in mice resulted in a decrease in MZB whereas follicular B cells and bone marrow B cell populations were minimally affected. LSD1 repressed genes in MZB that were normally upregulated in the myeloid and follicular B cell lineages. Correspondingly, LSD1 regulated chromatin accessibility at the motifs of transcription factors known to regulate splenic B cell development, including NF-κB motifs. The importance of NF-κB signaling was examined through an ex vivo MZB development assay, which showed that both LSD1-deficient and NF-κB–inhibited transitional B cells failed to undergo full MZB development. Gene expression and chromatin accessibility analyses of in vivo– and ex vivo–generated LSD1-deficient MZB indicated that LSD1 regulated the downstream target genes of noncanonical NF-κB signaling. Additionally LSD1 was found to interact with the noncanonical NF-κB transcription factor p52. Together, these data reveal that the epigenetic modulation of the noncanonical NF-κB signaling pathway by LSD1 is an essential process during the development of MZB.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

The signaling axis formed by the chemokine CXCL12 and its receptor CXCR4 plays an important role in B cell development and activation and is finely regulated by a process termed desensitization. Mutations leading to a truncation of the C-terminus tail of CXCR4 and thus to a defective desensitization have been reported in two diseases, a rare immunodeficiency called the WHIM syndrome and a B cell plasmacytoma called Waldenstrom’s Macroglobulinemia (WM). How CXCR4 desensitization may impact B cell activation in the context of a T-independent extra-follicular response is still unknown. Here using a unique mouse model bearing an orthologous gain of function mutation ofCxcr4we report that Cxcr4 desensitization is an essential gatekeeper controlling B lymphocyte entry into cycle, plasma cell differentiation, migration and maturation upon Myd88-dependent signaling. Altogether, our results support an essential role for Cxcr4 desensitization in limiting the depth and width of the B cell extra-follicular response and PC development.

The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.

Here we report a rare case of concomitant pulmonary chondroid hamartoma and follicular non Hodgkin lymphoma in a 65-year-old woman with a history of 4-month nonspecific symptoms like fever, sweating and weight loss.

The relative potency and quality of mouse B cell response to Toll-like receptors (TLRs) signaling varies significantly depending on the B cell subset and on the TLR member being engaged. Although it has been shown that marginal zone cells respond faster than follicular (FO) splenic B cells to TLR4 stimulus, FO B cells retain full capacity to proliferate and generate plasmablasts and plasma cells (PBs/PCs) with 2–3 days delayed kinetics. It is not clear whether this scenario could be extended to other members of the TLR family. Here, using quantitative cell culture conditions optimized for B cell growth and differentiation, we show that TLR9 signaling by CpG, while promoting vigorous proliferation, completely fails to induce differentiation of FO B cells into PBs/PCs. Little or absent Ig secretion following TLR9 stimulus was accompanied by lack of expression of cell surface markers and canonical transcription factors involved in PB/PC differentiation. Moreover, not only TLR9 did not induce plasmocyte differentiation, but it also strongly inhibited the massive PB/PC differentiation of FO B cells triggered by LPS/TLR4. Our study reveals unexpected opposite roles for TLR4 and TLR9 in the control of plasma cell differentiation program and disagrees with previous conclusions obtained in high-density cultures conditions on the generation of plasmocytes by TRL9 signaling. The potential implications of these findings on the role of TLR9 in controlling self-tolerance, clonal sizes and regulation of humoral responses are discussed.

The Mst1 and 2 cytosolic serine/threonine protein kinases are the mammalian orthologs of the Drosophila Hippo protein. Mst1 has been shown previously to participate in T-cell and B-cell trafficking and the migration of lymphocytes into secondary lymphoid organs in a cell intrinsic manner. We show here that the absence of Mst1 alone only modestly impacts B cell homing to lymph nodes. The absence of both Mst1 and 2 in hematopoietic cells results in relatively normal B cell development in the bone marrow and does not impact migration of immature B cells to the spleen. However, follicular B cells lacking both Mst1 and Mst2 mature in the splenic white pulp but are unable to recirculate to lymph nodes or to the bone marrow. These cells also cannot traffic efficiently to the splenic red pulp. The inability of late transitional and follicular B cells lacking Mst 1 and 2 to migrate to the red pulp explains their failure to differentiate into marginal zone B cell precursors and marginal zone B cells. Mst1 and Mst2 are therefore required for follicular B cells to acquire the ability to recirculate and also to migrate to the splenic red pulp in order to generate marginal zone B cells. In addition B-1 a B cell development is defective in the absence of Mst1.

Summary Systemic rheumatic diseases are characterized by abnormal B cell activation with autoantibody production and hypergammaglobulinaemia. Ro52/SSA, also denoted tripartite motif (TRIM)21, is a major autoantigen in Sjögren's syndrome and systemic lupus erythematosus. Interestingly, TRIM21-deficient mice develop systemic autoimmunity with B cell-driven manifestations such as autoantibodies, hypergammaglobulinaemia and glomerulonephritis following tissue injury. The mechanisms by which TRIM21-deficiency leads to enhanced B cell activation and antibody production are, however, not well understood, and to further elucidate the role of TRIM21 in systemic autoimmunity, we investigated the B cell phenotype and antibody responses of Trim21−/− mice following immunization with thymus-dependent (TD) and thymus-independent (TI) antigens. We found that TRIM21-deficient mice developed significantly higher specific antibody titres than their wild-type counterparts upon B cell receptor (BCR) engagement by TD and TI type II antigens, and this was accompanied by an altered B cell phenotype. Furthermore, BCR cross-linking, but not anti-CD40 stimulation, in vitro resulted in a significantly higher proliferation of Trim21−/− cells. We also observed that splenic follicular B cells were expanded not only in immunized mice but also already in young, unmanipulated Trim21−/− mice, and transcriptomic analysis of these cells revealed an up-regulation of genes associated with B cell differentiation, indicating a role for TRIM21 in their regulation. In conclusion, in this study we describe a link between the rheumatic autoantigen Ro52/TRIM21 and increased antibody production associated with follicular B cell expansion, implicating a potential role for Ro52/TRIM21 in the pathogenesis of systemic autoimmune diseases.

The proapoptotic BH3-only protein BIM (Bcl2l11) plays key roles in the maintenance of multiple hematopoietic cell types. In mice, germline knockout or conditional pan-hematopoietic deletion of Bim results in marked splenomegaly and significantly increased numbers of B cells. However, it has remained unclear whether these abnormalities reflect the loss of cell-intrinsic functions of BIM within the B lymphoid lineage and, if so, which stages in the lifecycle of B cells are most impacted by the loss of BIM. Here, we show that B lymphoid-specific conditional deletion of Bim during early development (i.e., in pro-B cells using Mb1-Cre) or during the final differentiation steps (i.e., in transitional B cells using Cd23-Cre) led to a similar >2-fold expansion of the mature follicular B cell pool. Notably, while the expansion of mature B cells was quantitatively similar in conditional and germline Bim-deficient mice, the splenomegaly was significantly attenuated after B lymphoid-specific compared to global Bim deletion. In vitro, conditional loss of Bim substantially increased the survival of mature B cells that were refractory to activation by lipopolysaccharide. Finally, we also found that conditional deletion of just one Bim allele by Mb1-Cre dramatically accelerated the development of Myc-driven B cell lymphoma, in a manner that was comparable to the effect of germline Bim heterozygosity. These data indicate that, under physiological conditions, BIM regulates B cell homeostasis predominantly by limiting the life span of non-activated mature B cells, and that it can have additional effects on developing B cells under pathological conditions.

Persistent humoral immunity depends on the follicular B cell response and on the generation of somatically mutated high-affinity plasma cells and memory B cells. Upon activation by an antigen, cognately activated follicular B cells and follicular T helper (TFH ) cells initiate germinal centre (GC) reaction during which high-affinity effector cells are generated. The differentiation of activated follicular B cells into plasma cells and memory B cells is guided by complex selection events, both at the cellular and molecular level. The transition of B cell into a plasma cell during the GC response involves alterations in the microenvironment and developmental state of the cell, which are guided by cell-extrinsic signals. The developmental cell fate decisions in response to these signals are coordinated by cell-intrinsic gene regulatory network functioning at epigenetic, transcriptional and post-transcriptional levels.

Composite lymphoma of T-/B-cell type is rare, and follicular lymphoma composite with peripheral T-cell lymphoma (PTCL) has not previously been reported. We report such a case with both neoplastic components displaying a unique zone of distribution. A 75-year-old male patient presented with generalized lymphadenopathy. Sections of axillary lymph node demonstrated potentially 2 clonal processes, PTCL with aberrant CD20 expression and follicular lymphoma. Interestingly, the 2 neoplastic components were confined to their respective classic distribution zones, with PTCL occupying the interfollicular areas and follicular lymphoma residing in follicles. Both populations were detected by flow cytometry, but their immunophenotypes were insufficient to define clonality. Nonetheless, biclonality was demonstrated by lymphoid receptor gene rearrangement analyses. Molecular cytogenetics showed IGH/BCL2 fusion in the follicular lymphoma and amplification of IGH gene or trisomy/tetrasomy 14 in the PTCL. The current case underscores the complexity of composite lymphoma and advocates a multimodal approach to establishing the diagnosis.

// Oshrat Hershkovitz-Rokah 1, 2, 3, * , Polina Geva 1, * , Mali Salmon-Divon 1 , Ofer Shpilberg 2, 3, 4 and Stella Liberman-Aronov 1 1 Department of Molecular Biology, Faculty of Natural Sciences, Ariel University, Ariel, Israel 2 Translational Research Laboratory, Assuta Medical Centers, Tel Aviv, Israel 3 Institude of Hematology, Assuta Medical Centers, Tel Aviv, Israel 4 Pre-Medicine Department, School of Health Sciences, Ariel University, Ariel, Israel * These authors have contributed equally to this work Correspondence to: Stella Liberman-Aronov, email: stellar@ariel.ac.il Keywords: miRNA signature; DLBCL; FL; miRNA-mRNA pairing; B cell lymphoma Received: June 14, 2017 Accepted: December 21, 2017 Published: January 05, 2018 ABSTRACT MicroRNAs (miRs) are short non-coding regulatory RNAs that control gene expression at the post-transcriptional level and play an important role in cancer development and progression, acting either as oncogenes or as tumor suppressors. Identification of aberrantly expressed miRs in patients with hematological malignancies as compared to healthy individuals has suggested that these molecules may serve as novel clinical diagnostic and prognostic biomarkers. We conducted a systematic literature review of articles published between 2007 and 2017 and re-analyzed experimentally-validated human miR expression signatures in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) from various biological sources (tumor tissue, peripheral blood, bone marrow and cell lines). A unique miR expression pattern was observed for each disease. Compared to healthy individuals, 61 miRs were aberrantly expressed in DLBCL and 85 in FL; 20-30% of aberrantly expressed miRs overlapped between the two lymphoma subtypes. Analysis of integrative positive and negative miRNA-mRNA relationships using the Ingenuity Pathway Analysis (IPA) system revealed 970 miR-mRNA pairs for DLBCL and 90 for FL. Through gene ontology analysis, we found potential regulatory pathways that are deregulated in DLBCL and FL due to improper expression of miR target genes. By comparing the expression level of the aberrantly expressed miRs in DLBCL to their expression levels in other malignancies, we identified seven miRs that are aberrantly expressed in DLBCL tumor tissues (miR-15a, miR-16, miR-17, miR-106, miR-21, miR-155 and miR-34a-5p). This specific expression pattern may be a potential diagnostic tool for DLBCL.

Mice lacking secreted IgM (sIgM−/−) antibodies display abnormal splenic B cell development, which results in increased marginal zone and decreased follicular B cell numbers. However, the mechanism by which sIgM exhibit this effect is unknown. Here, we demonstrate that B cells in sIgM−/− mice display increased B cell receptor (BCR) signaling as judged by increased levels of phosphorylated Bruton’s tyrosine kinase (pBtk), phosphorylated Spleen tyrosine kinase (pSyk), and nuclear receptor Nur77. Low dosage treatment with the pBtk inhibitor Ibrutinib reversed the altered B cell development in the spleen of sIgM−/− mice, suggesting that sIgM regulate splenic B cell differentiation by decreasing BCR signaling. Mechanistically, we show that B cells, which express BCRs specific to hen egg lysozyme (HEL) display diminished responsiveness to HEL stimulation in presence of soluble anti-HEL IgM antibodies. Our data identify sIgM as negative regulators of BCR signaling and suggest that they can act as decoy receptors for self-antigens that are recognized by membrane bound BCRs.

Complex interactions govern the interplay of central nervous and immune systems, including the generation, homeostatic maintenance, and activation of B cells. Accordingly, spinal cord injury will likely impact all of these processes. Several laboratories have recently explored this possibility, and their observations in aggregate reveal both acute and chronic consequences that can vary based on the injury location. Acute effects include a transient cessation of bone marrow B lymphopoiesis, with a corresponding drop in the peripheral follicular and transitional B cell subsets, whereas the marginal zone subset is preserved. Despite recovery of B lymphopoiesis by 28 days post injury, follicular B cell numbers remain depressed; this may reflect reduced levels of the homeostatic cytokine BLyS. In general, the ability to mount T dependent antibody responses after injury are intact, as are pre-existing memory B cell pools and antibody levels. In contrast, T-independent responses are chronically compromised. Both glucocorticoid-dependent and -independent processes mediate these effects, but a detailed understanding of the mechanisms involved awaits further study. Nonetheless, these observations in toto strengthen the growing appreciation for bidirectional interactions between the CNS and immune system, highlighting the need for further basic and translational efforts.

Rationale:Itisnowwellestablishedthatimmuneresponsescantake place outside of primary and secondary lymphoid organs. We previously described the presence of tertiary lymphoid structures (TLS) in patients with non–small cell lung cancer (NSCLC) characterized by clusters of mature dendritic cells (DCs) and T cells surrounded by B-cell follicles. We demonstrated that the density of these mature DCs was associated with favorable clinical outcome. Objectives: To study the role of follicular B cells in TLS and the potentiallinkwithalocalhumoralimmuneresponseinpatientswith NSCLC. Methods: The cellular composition of TLS was investigated by immunohistochemistry. Characterization of B-cell subsets was performedby flowcytometry.Aretrospectivestudywasconductedin two independent cohorts of patients. Antibody specificity was analyzed by ELISA. Measurements and Main Results: Consistent with TLS organization, all stages of B-cell differentiation were detectable in most tumors. Germinal center somatic hypermutation and class switch recombination machineries were activated, associated with the generation of plasma cells. Approximately half of the patients showed antibody reactivity against up to 7 out of the 33 tumor antigens tested. A high density of follicular B cells correlated with long-termsurvival,bothinpatientswithearly-stageNSCLCandwith advanced-stage NSCLC treated with chemotherapy. The combination of follicular B cell and mature DC densities allowed the identification of patients with the best clinical outcome. Conclusions: B-cell density represents a new prognostic biomarker for NSCLC patient survival, and makes the link between TLS and a protective B cell–mediated immunity.

Immunometabolism Transitional B cell precursors mature into follicular B cells, which are involved in antibody responses. Farmer et al. discovered a metabolic checkpoint in this developmental process. Compared with transitional B cells, mouse and human follicular B cells were metabolically quiescent and had increased activation of the kinase AMPK and increased levels of the cell surface ectoenzymes CD39 and CD73, which generate extracellular adenosine. Transitional human B cells that expressed CD73 or were exposed to an AMPK agonist preferentially acquired a follicular B cell phenotype. Sci. Signal. 12 , eaaw5573 (2019).

Transgenic over-expression of CD83 on B cells leads to a reduced response to BCR engagement but to an enhanced secretion of IL-10 upon LPS stimulation. In this study, we analyzed the differential influence of CD83 on the stimulation of different B cell subsets via the BCR or TLR4. Neither wild type nor CD83 transgenic (CD83tg) B cells produced any IL-10 in response to BCR stimulation. BCR engagement led to reduced activation of LYN, SYK and ERK1/2 resulting in reduced numbers of proliferating cells in all CD83tg B cell subsets. Moreover, CD83tg follicular (FO) but not marginal zone (MZ) or transitional (TN) B cells showed significantly enhanced cell death. In contrast, LPS stimulation led to normal frequencies of proliferating CD83tg FO, MZ and TN B cells although TLR4 engagement did not rescue FO B cells from apoptosis. Furthermore, LPS stimulation led to high IL-10 production derived from CD83tg MZ B cells that reacted to LPS stimulation with enhanced ERK1/2 activation. Finally, we show that CD83 co-localizes with the BCR complex as well as with the LPS receptor complex suggesting that CD83 interacts with components of both signaling complexes. Taken together, the results of this study show that CD83 already inhibits the initiation of BCR signaling leading to insufficient activation signals in all B cells and reduced survival especially of FO B cells. On the other hand, CD83 supports TLR4-mediated IL-10 release exclusively in MZ B cells. Thus, CD83 differentially modulates FO and MZ B cell responses.

The splenic marginal zone is a unique microenvironment where resident immune cells are exposed to the open blood circulation. Even though it has an important role in responses against blood-borne antigens, lymphocyte migration in the marginal zone has not been intravitally visualized due to challenges associated with achieving adequate imaging depth in this abdominal organ. Here we develop a two-photon microscopy procedure to study marginal zone and follicular B-cell movement in the live mouse spleen. We show that marginal zone B cells are highly motile and exhibit long membrane extensions. Marginal zone B cells shuttle between the marginal zone and follicles with at least one-fifth of the cells exchanging between compartments per hour, a behaviour that explains their ability to deliver antigens rapidly from the open blood circulation to the secluded follicles. Follicular B cells also transit from follicles to the marginal zone, but unlike marginal zone B cells, they fail to undergo integrin-mediated adhesion, become caught in fluid flow and are carried into the red pulp. Follicular B-cell egress via the marginal zone is sphingosine-1-phosphate receptor-1 (S1PR1)-dependent. This study shows that marginal zone B cells migrate continually between marginal zone and follicles and establishes the marginal zone as a site of S1PR1-dependent B-cell exit from follicles. The results also show how adhesive differences of similar cells critically influence their behaviour in the same microenvironment.

The cytokine IL-12 induces IFN-γ production by T and NK cells. In preclinical models, it contributes to antitumor immunity. However, in clinical testing, it has shown limited benefit in patients with any one of a variety of malignancies. Moreover, in a clinical trial testing a combination of IL-12 and rituximab in patients with follicular B cell non-Hodgkin lymphoma (FL), those treated with IL-12 showed a lower response rate, suggesting that IL-12 actually plays a detrimental role. Here, we investigated whether the failure of IL-12 treatment for FL was due to T cell exhaustion, a condition characterized by reduced T cell differentiation, proliferation, and function, which has been observed in chronic viral infection. We found that extended exposure to IL-12 induced T cell exhaustion and contributed to the poor prognosis in FL patients. Long-term exposure of freshly isolated human CD4+ T cells to IL-12 in vitro caused T cell dysfunction and induced expression of TIM-3, a T cell immunoglobulin and mucin domain protein with a known role in T cell exhaustion, via an IFN-γ–independent mechanism. TIM-3 was required for the negative effect of IL-12 on T cell function. Importantly, TIM-3 also was highly expressed on intratumoral T cells that displayed marked functional impairment. Our findings identify IL-12– and TIM-3–mediated exhaustion of T cells as a mechanism for poor clinical outcome when IL-12 is administered to FL patients.

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.

Follicular lymphoma exhibits intratumoral infiltration by non-malignant T lymphocytes, including CD4+CD25+ regulatory T (T(reg)) cells. We combined denileukin diftitox with rituximab in previously untreated, advanced-stage follicular lymphoma patients anticipating that denileukin diftitox would deplete CD25+ T(reg) cells while rituximab would deplete malignant B cells. Patients received rituximab 375 mg/m(2) weekly for 4 weeks and denileukin diftitox 18 mcg/kg/day for 5 days every 3 weeks for 4 cycles; neither agent was given as maintenance therapy. Between August 2008 and March 2010, 24 patients were enrolled. One patient died before treatment was given and was not included in the analysis. Eleven of 23 patients (48%; 95% confidence interval (CI): 27-69%) responded; 2 (9%) had complete responses and 9 (39%) had partial responses. The progression-free rate at 2 years was 55% (95%CI: 37-82%). Thirteen patients (57%) experienced grade ≥3 adverse events and one patient (4%) died. In correlative studies, soluble CD25 and the number of CD25+ T cells decreased after treatment; however, there was a compensatory increase in IL-15 and IP-10. We conclude that although the addition of denileukin diftitox to rituximab decreased the number of CD25+ T cells, denileukin diftitox contributed to the toxicity of the combination without an improvement in response rate or time to progression.

Elevated serum levels of the soluble form of IL-2 receptor α (sIL-2Rα) have been correlated with a poor prognosis in a variety of different types of cancers. However, its biologic relevance remains unclear and controversial. In patients with follicular B-cell non-Hodgkin lymphoma (FL), we observed that serum sIL-2Rα levels were elevated compared with controls and that elevated sIL-2Rα levels before treatment were associated with a poor outcome. To explore the mechanism by which sIL-2Rα may contribute to a poor prognosis in FL, we determined the effects of sIL-2Rα on IL-2 signaling and found that the sIL-2Rα–IL-2 complex promoted T-cell differentiation toward to inhibitory Treg cells rather than TH1 or TH17 cells. Shed by activated T cells that express membrane-bound IL-2Rα, sIL-2Rα further enhanced IL-2–mediated phosphorylation of Stat5 thereby significantly up-regulating Foxp3 expression in CD4+ T cells. We found that CD4+ T cells treated with either IL-2 or sIL-2Rα–IL-2 complex, but not with sIL-2Rα alone, inhibited the function of CD8+ T cells. Taken together, these results indicate that sIL-2Rα actually plays an active biologic role in FL by binding IL-2 and promoting IL-2 signaling rather than depleting IL-2 and blocking its function.

Splenic B-2 cells can be divided into two major subsets: follicular (FO) and marginal zone (MZ) B cells. FO and MZ B cells are generated from immature transitional B cells. Few transcription factors have been identified that regulate FO B cell differentiation. The highly related proteins PU.1, Spi-B, and Spi-C are transcription factors of the E26-transformation-specific family and are important for B cell differentiation and function. To determine whether these proteins play a role in the differentiation of FO B cells, we performed a detailed analysis of splenic B cells in mice with inactivating mutations in the genes encoding PU.1 (Sfpi1) or Spi-B (Spib). Sfpi1+/− Spib−/− (PUB) mice had a 9-fold reduction in the frequency of CD23+ FO B cells compared with that of wild-type mice. In contrast, PUB mice had a 2-fold increase in the frequency of MZ B cells that was confirmed by immunofluorescence staining. Expression of Spi-C in Eμ-Spi-C transgenic PUB mice partially rescued frequencies of CD23+ B cells. Gene expression analysis, in vitro reporter assays, and chromatin immunoprecipitation experiments showed that transcription of the Fcer2a gene encoding CD23 is activated by PU.1, Spi-B, and Spi-C. These results demonstrate that FO B cell differentiation is regulated by the E26-transformation-specific transcription factors PU.1, Spi-B, and Spi-C.

BAFF deficiency in mice impairs B cell development beyond the transitional stage 1 in the spleen and thus severely reduces the size of follicular and marginal zone B cell compartments. Moreover, humoral immune responses in these mice are dramatically impaired. We now addressed the question whether the decrease in mature B cell numbers and the reduced humoral immune responses in BAFF-deficient mice could be overcome by the injection of recombinant BAFF. We therefore engineered a recombinant protein containing the human IgG1 Fc moiety fused to receptor-binding domain of human BAFF (Fc-BAFF). At 1 week after the second injection of this fusion protein a complete rescue of the marginal zone B cell compartment and a 50% rescue of the follicular B cell compartment was observed. Moreover these mice mounted a T cell-dependent humoral immune response indistinguishable from wild-type mice. By day 14 upon arrest of Fc-BAFF treatment mature B cell numbers in the blood dropped by 50%, indicating that the life span of mature B cells in the absence of BAFF is 14 days or less. Collectively these findings demonstrate that injection of Fc-BAFF in BAFF-deficient mice results in a temporary rescue of a functional mature B cell compartment.

Talin1 is a key integrin coactivator. We investigated the roles of this cytoskeletal adaptor and its target integrins in B-cell lymphogenesis, differentiation, migration, and function. Using CD19 Cre-mediated depletion of talin1 selectively in B cells, we found that talin1 was not required for B-cell generation in the bone marrow or for the entry of immature B cells to the white pulp of the spleen. Loss of talin1 also did not affect B-cell maturation into follicular B cells but compromised differentiation of marginal zone B cells. Nevertheless, serum IgM and IgG levels remained normal. Ex vivo analysis of talin1-deficient spleen B cells indicated a necessary role for talin1 in LFA-1 and VLA-4 activation stimulated by canonical agonists, but not in B-cell chemotaxis. Consequently, talin1 null B splenocytes could not enter lymph nodes nor return to the bone marrow. Talin1 deficiency in B cells was also impaired in the humoral response to a T cell-dependent antigen. Collectively, these results indicate that talin1 is not required for follicular B-cell maturation in the spleen or homeostatic humoral immunity but is critical for integrin-dependent B lymphocyte emigration to lymph nodes and optimal immunity against T-dependent antigens.