Non-apoptotic FAS signaling controls mTOR activation and extrafollicular maturation in human B cells.

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

Augmented neutralization of SARS-CoV-2 Omicron variant by boost vaccination and monoclonal antibodies.

Effective vaccines and monoclonal antibodies have been developed against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the appearance of virus variants with higher transmissibility and pathogenicity is a major concern because of their potential to escape vaccines and clinically approved SARS-CoV-2- antibodies. Here, we use flow cytometry-based binding and pseudotyped SARS-CoV-2 neutralization assays to determine the efficacy of boost immunization and therapeutic antibodies to neutralize the dominant Omicron variant. We provide compelling evidence that the third vaccination with BNT162b2 increases the amount of neutralizing serum antibodies against Delta and Omicron variants, albeit to a lower degree when compared to the parental Wuhan strain. Therefore, a third vaccination is warranted to increase titers of protective serum antibodies, especially in the case of the Omicron variant. We also found that most clinically approved and otherwise potent therapeutic antibodies against the Delta variant failed to recognize and neutralize the Omicron variant. In contrast, some antibodies under preclinical development potentially neutralized the Omicron variant. Our studies also support using a flow cytometry-based antibody binding assay to rapidly monitor therapeutic candidates and serum titers against emerging SARS-CoV-2 variants.

Increased risk of chronic fatigue and hair loss following COVID-19 in individuals with hypohidrotic ectodermal dysplasia.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is a group of genodermatoses in which deficient ectodysplasin A signalling leads to maldevelopment of skin appendages, various eccrine glands, and teeth. Individuals with HED often have disrupted epithelial barriers and, therefore, were suspected to be more susceptible to coronavirus infection. METHODS: 56 households with at least one member who had coronavirus disease of 2019 (COVID-19) were enrolled in a longitudinal study to compare the course of illness, immune responses, and long-term consequences of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection in HED patients (n = 15, age 9-52 years) and control subjects of the same age group (n = 149). RESULTS: In 14 HED patients, mild or moderate typical COVID-19 symptoms were observed that lasted for 4-45 days. Fever during the first days sometimes required external cooling measures. The course of COVID-19 was similar to that in control subjects if patients developed antibodies blocking the SARS-CoV-2 spike protein. Five out of six HED patients with completely abrogated ectodysplasin A signalling (83%) suffered from chronic, in two cases very severe fatigue following COVID-19, while only 25% of HED patients with residual activity of this pathway and 21% of control subjects recovering from COVID-19 experienced postinfectious fatigue. Hair loss after COVID-19 was also more frequent among HED patients (64%) than in the control group (13%). CONCLUSIONS: HED appears to be associated with an increased risk of long-term consequences of a SARS-CoV-2 infection. Preventive vaccination against COVID-19 should be recommended for individuals affected by this rare genetic disorder.

Metabolic impairment of non-small cell lung cancers by mitochondrial HSPD1 targeting.

BACKGROUND: The identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures. METHODS: Gene fitness was determined by bioinformatics analysis from available datasets from genetic screenings. HSPD1 expression was evaluated by immunohistochemistry from formalin-fixed paraffin-embedded tissues from NSCLC patients. Real-time proliferation assays with and without cytotoxicity reagents, colony formation assays and cell cycle analyses were used to monitor growth and drug sensitivity of different NSCLC cells in vitro. In vivo growth was monitored with subcutaneous injections in immune-deficient mice. Cell metabolic activity was analyzed through extracellular metabolic flux analysis. Specific knockouts were introduced by CRISPR/Cas9. RESULTS: We show heat shock protein family D member 1 (HSPD1 or HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients' prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on oxidative phosphorylation and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B. CONCLUSIONS: These results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NSCLC.

The role of miR-200b/c in balancing EMT and proliferation revealed by an activity reporter.

Since their discovery, microRNAs (miRNAs) have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs endogenous levels and activity for in vitro and in vivo applications could lead to a better understanding of their biological role in physiology and disease.

miR-148a controls metabolic programming and survival of mature CD19-negative plasma cells in mice.

Long-lived antibody-secreting plasma cells are essential to establish humoral memory against pathogens. While a regulatory transcription factor network has been established in plasma cell differentiation, the regulatory role of miRNAs remains enigmatic. We have recently identified miR-148a as the most abundant miRNA in primary mouse and human plasma cells. To determine whether this plasma cell signature miRNA controls the in vivo development of B cells into long-lived plasma cells, we established mice with genomic, conditional, and inducible deletions of miR-148a. The analysis of miR-148a-deficient mice revealed reduced serum Ig, decreased numbers of newly formed plasmablasts and reduced CD19-negative, CD93-positive long-lived plasma cells. Transcriptome and metabolic analysis revealed an impaired glucose uptake, a reduced oxidative phosphorylation-based energy metabolism, and an altered abundance of homing receptors CXCR3 (increase) and CXCR4 (reduction) in miR-148a-deficient plasma cells. These findings support the role of miR-148a as a positive regulator of the maintenance of long-lived plasma cells.

TFG is required for autophagy flux and to prevent endoplasmic reticulum stress in CH12 B lymphoma cells.

Plasma cells depend on quality control of newly synthesized antibodies in the endoplasmic reticulum (ER) via macroautophagy/autophagy and proteasomal degradation. The cytosolic adaptor protein TFG (Trk-fused gene) regulates ER-Golgi transport, the secretory pathway and proteasome activity in non-immune cells. We show here that TFG is upregulated during lipopolysaccharide- and CpG-induced differentiation of B1 and B2 B cells into plasmablasts, with the highest expression of TFG in mature plasma cells. CRISPR-CAS9-mediated gene disruption of tfg in the B lymphoma cell line CH12 revealed increased apoptosis, which was reverted by BCL2 but even more by ectopic TFG expression. Loss of TFG disrupted ER structure, leading to an expanded ER and increased expression of ER stress genes. When compared to wild-type CH12 cells, tfg KO CH12 cells were more sensitive toward ER stress induced by tunicamycin, monensin and proteasome inhibition or by expression of an ER-bound immunoglobulin (Ig) µ heavy (µH) chain. CH12 tfg KO B cells displayed more total LC3, lower LC3-II turnover and increased numbers and size of autophagosomes. Tandem-fluorescent-LC3 revealed less accumulation of GFP-LC3 in starved and chloroquine-treated CH12 tfg KO B cells. The GFP:RFP ratio of tandem-fluorescent-LC3 was higher in tunicamycin-treated CH12 tfg KO B cells, suggesting less autophagy flux during induced ER stress. Based on these data, we suggest that TFG controls autophagy flux in CH12 B cells and propose that TFG is a survival factor that alleviates ER stress through the support of autophagy flux in activated B cells and mature plasma cells.Abbreviations: Ab, antibody; Ag, antigen; ASC, antibody-secreting cells; ATG, autophagy-related; BCR, B cell receptor; COPII, coat protein complex II; CpG, non-methylated CpG oligonucleotide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FO, follicular; GFP, green fluorescent protein; HC, heavy chain; Ig, immunoglobulin; IRES, internal ribosomal entry site; LC, light chain; MZ, marginal zone; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; TLR, toll-like receptor; UPR, unfolded protein response.

Unraveling the mysteries of plasma cells.

Antibody-secreting plasma cells are the central pillars of humoral immunity. They are generated in a fundamental cellular restructuring process from naive B cells upon contact with antigen. This outstanding process is guided and controlled by a complex transcriptional network accompanied by a fascinating morphological metamorphosis, governed by the combined action of Blimp-1, Xbp-1 and IRF-4. The survival of plasma cells requires the intimate interaction with a specific microenvironment, consisting of stromal cells and cells of hematopoietic origin. Cell-cell contacts, cytokines and availability of metabolites such as glucose and amino acids modulate the survival abilities of plasma cells in their niches. Moreover, plasma cells have been shown to regulate immune responses by releasing cytokines. Furthermore, plasma cells are central players in autoimmune diseases and malignant transformation of plasma cells can result in the generation of multiple myeloma. Hence, the development of sophisticated strategies to deplete autoreactive plasma cells and myeloma cells represents a challenge for current and future research.

Extracellular aggregated alpha synuclein primarily triggers lysosomal dysfunction in neural cells prevented by trehalose.

Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an important role in the progression of alpha synucleinopathies. A critical step for the propagation process is the accumulation of extracellular aSyn within recipient cells. Here, we investigated the trafficking of distinct exogenous aSyn forms and addressed the mechanisms influencing their accumulation in recipient cells. The aggregated aSyn species (oligomers and fibrils) exhibited more pronounced accumulation within recipient cells than aSyn monomers. In particular, internalized extracellular aSyn in the aggregated forms was able to seed the aggregation of endogenous aSyn. Following uptake, aSyn was detected along endosome-to-lysosome and autophagosome-to-lysosome routes. Intriguingly, aggregated aSyn resulted in lysosomal activity impairment, accompanied by the accumulation of dilated lysosomes. Moreover, analysis of autophagy-related protein markers suggested decreased autophagosome clearance. In contrast, the endocytic pathway, proteasome activity, and mitochondrial homeostasis were not substantially affected in recipient cells. Our data suggests that extracellularly added aggregated aSyn primarily impairs lysosomal activity, consequently leading to aSyn accumulation within recipient cells. Importantly, the autophagy inducer trehalose prevented lysosomal alterations and attenuated aSyn accumulation within aSyn-exposed cells. Our study underscores the importance of lysosomes for the propagation of aSyn pathology, thereby proposing these organelles as interventional targets.

Regulation of Energy Metabolism during Early B Lymphocyte Development.

The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body's own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca2+-mediated mitoflashes. Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.

Interleukin-36 receptor mediates the crosstalk between plasma cells and synovial fibroblasts.

The IL-1 family member IL-36α has proinflammatory and pathogenic properties in psoriasis. IL-36α binds to the IL-36 receptor leading to nuclear factor kappa B/mitogen activated protein kinase mediated cytokine release. The IL-36R antagonist prevents recruitment of IL-1 receptor accessory protein and therefore IL-36-dependent cell activation. In inflamed human tissue, we previously could show that resident B cells and plasma cells (PC) express IL-36α. Further, fibroblast-like synoviocytes (FLS) produced proinflammatory cytokines upon IL-36α-stimulation. We hypothesize an IL-36-specific crosstalk between B cells/PCs and FLS permitting a proinflammatory B cell niche. Here, we firstly demonstrated that B cell lines and B cells from healthy donors express IL-36α and stimulation increased IL-36α in B cells and primary plasmablasts/PCs. Moreover, FLS respond specifically to IL-36α by proliferation and production of matrix metalloproteinases via p38/HSP27 signaling. Importantly, IL-36R-deficiency abrogated IL-36α-induced production of inflammatory mediators in FLS and changed the intrinsic FLS-phenotype. Using an in vitro co-culture system, we could show that IL-36R-deficient FLS had a limited capacity to support PC survival compared to wild-type FLS. Hence, we demonstrated an IL-36R-dependent crosstalk between B cells/PCs and FLS. Our data support the concept of initiation and maintenance of a proinflammatory niche by B cells in the joints.

Fra-2 regulates B cell development by enhancing IRF4 and Foxo1 transcription.

The role of AP-1 transcription factors in early B cell development and function is still incompletely characterized. Here we address the role of Fra-2 in B cell differentiation. Deletion of Fra-2 leads to impaired B cell proliferation in the bone marrow. In addition, IL-7-stimulated pro-B cell cultures revealed a reduced differentiation from large pre-B cells to small B cells and immature B cells. Gene profiling and chromatin immunoprecipitation sequencing analyses unraveled a transcriptional reduction of the transcription factors Foxo1, Irf4, Ikaros, and Aiolos in Fra-2-deficient B cells. Moreover, expression of IL7Rα and Rag 1/2, downstream targets of Irf4 and Foxo1, were also reduced in the absence of Fra-2. Pro-B cell proliferation and small pre-B cell differentiation were fully rescued by expression of Foxo1 and Irf4 in Fra-2-deficient pro-B cells. Hence, Fra-2 is a key upstream regulator of Foxo1 and Irf4 expression and influences proliferation and differentiation of B cells at multiple stages.

A new staining protocol for detection of murine antibody-secreting plasma cell subsets by flow cytometry.

We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.

A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1.

B-cell development in the bone marrow comprises proliferative and resting phases in different niches. We asked whether B-cell metabolism relates to these changes. Compared to pro B and small pre B cells, large pre B cells revealed the highest glucose uptake and ROS but not mitochondrial mass, whereas small pre B cells exhibited the lowest mitochondrial membrane potential. Small pre B cells from Rag1-/-;33.C9 µ heavy chain knock-in mice revealed decreased glycolysis (ECAR) and mitochondrial spare capacity compared to pro B cells from Rag1-/- mice. We were interested in the step regulating this metabolic switch from pro to pre B cells and uncovered that Swiprosin-2/EFhd1, a Ca2+-binding protein of the inner mitochondrial membrane involved in Ca2+-induced mitoflashes, is expressed in pro B cells, but downregulated by surface pre B-cell receptor expression. Knockdown and knockout of EFhd1 in 38B9 pro B cells decreased the oxidative phosphorylation/glycolysis (OCR/ECAR) ratio by increasing glycolysis, glycolytic capacity and reserve. Prolonged expression of EFhd1 in EFhd1 transgenic mice beyond the pro B cell stage increased expression of the mitochondrial co-activator PGC-1α in primary pre B cells, but reduced mitochondrial ATP production at the pro to pre B cell transition in IL-7 cultures. Transgenic EFhd1 expression caused a B-cell intrinsic developmental disadvantage for pro and pre B cells. Hence, coordinated expression of EFhd1 in pro B cells and by the pre BCR regulates metabolic changes and pro/pre B-cell development.

JAK1-dependent transphosphorylation of JAK2 limits the antifibrotic effects of selective JAK2 inhibitors on long-term treatment.

OBJECTIVES: Janus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-ß. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts. METHODS: The antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance. RESULTS: The antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis. CONCLUSION: Fibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.

Orphan nuclear receptor NR4A1 regulates transforming growth factor-ß signaling and fibrosis.

Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-ß (TGF-ß) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-ß target genes, thereby limiting pro-fibrotic TGF-ß effects. Even though temporary upregulation of TGF-ß in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-ß signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-ß signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells.

The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell-specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.

Swiprosin-1/EFhd2 limits germinal center responses and humoral type 2 immunity.

Activated B cells are selected for in germinal centers by regulation of their apoptosis. The Ca2+ -binding cytoskeletal adaptor protein Swiprosin-1/EFhd2 (EFhd2) can promote apoptosis in activated B cells. We therefore hypothesized that EFhd2 might limit humoral immunity by repressing both the germinal center reaction and the expected enhancement of immune responses in the absence of EFhd2. Here, we established EFhd2(-/-) mice on a C57BL/6 background, which revealed normal B- and T-cell development, basal Ab levels, and T-cell independent type 1, and T-cell independent type 2 responses. However, T cell-dependent immunization with sheep red blood cells and infection with the helminth Nippostrongylus brasiliensis (N.b) increased production of antibodies of multiple isotypes, as well as germinal center formation in EFhd2(-/-) mice. In addition, serum IgE levels and numbers of IgE+ plasma cells were strongly increased in EFhd2(-/-) mice, both after primary as well as after secondary N.b infection. Finally, mixed bone marrow chimeras unraveled an EFhd2-dependent B cell-intrinsic contribution to increased IgE plasma cell numbers in N.b-infected mice. Hence, we established a role for EFhd2 as a negative regulator of germinal center-dependent humoral type 2 immunity, with implications for the generation of IgE.

Inhibition of bone remodeling in young mice by bisphosphonate displaces the plasma cell niche into the spleen.

The bone marrow provides niches for early B cell differentiation and long-lived plasma cells. Therefore, it has been hypothesized that perturbing bone homeostasis might impact B cell function and Ab production. This hypothesis is highly relevant for patients receiving long-term treatment with antiresorptive drugs. We therefore analyzed the humoral immune response of mice chronically treated with ibandronate, a commonly used bisphosphonate. We confirmed the increased bone mass caused by inhibition of osteoclast activity and also the strongly reduced bone formation because of decreased osteoblast numbers in response to ibandronate. Thus, bisphosphonate drastically inhibited bone remodeling. When ibandronate was injected into mice after a primary immunization to mimic common antiosteoporotic treatments, the generation of the various B cell populations, the response to booster immunization, and the generation of plasma cells were surprisingly normal. Mice also responded normally to immunization when ibandronate was applied to naive mice. However, there, ibandronate shunted the homing of bone marrow plasma cells. Interestingly, ibandronate reduced the numbers of megakaryocytes, a known component of the bone marrow plasma cell niche. In line with normal Ab responses, increased plasma cell populations associated with increased megakaryocyte numbers were then observed in the spleens of the ibandronate-treated mice. Thus, although inhibition of bone remodeling disturbed the bone marrow plasma cell niche, a compensatory niche may have been created by relocating the megakaryocytes into the spleen, thereby allowing normal B cell responses. Therefore, megakaryocytes may act as a key regulator of plasma cell niche plasticity.