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Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2Cre/CreMcl1flox/flox (Mcl1ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1flox/flox (Mcl1ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease.

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show thatgins1andgins2mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the GINS complex as neither Gins1 nor Gins2 seem to be present during these stages.

In mice, marginal zone (MZ) B cells are found principally in the MZ of the spleen and characterized as CD23-negative cells, primarily express polyreactive BCRs, high levels of complement receptor-2 and TLRs. Collagen-induced arthritis (CIA) is a commonly used animal model of human rheumatoid arthritis, considered as a Th1-mediated disease. Although the importance of MZ B cells in the initiation of CIA is well established, their role in remission is unexplored. Besides, playing a central role in Th1 cell development, T-box transcription factor (T-bet) has important functions in B cells. T-bet is regulated by IFN-γ and through the BCR and TLR9, the signals that have an impact on regulatory IL-10 production. In this work, we aimed to analyze the contribution of T-bet to the function of IL-10-positive MZ B cells. We demonstrate that during the remission phase of CIA, MZ B cells express an elevated level of T-bet and confirm the existence of IL-10/T-bet coexpressing cells. Moreover, we show that T-bet-expressing MZ B cells migrate toward CXCR3 ligand and secrete IL-10 by inflammatory stimuli. Our data suggest that T-bet might contribute to the remission of CIA by facilitating the regulatory potential of IL-10-positive MZ B cells.

IL-10 is a suppressive cytokine that has been implicated in the pathophysiology of autoimmune disorders and can be produced by different cell types such as regulatory B-cells. Our previous work showed that under inflammatory condition MZ B-cells differentiated into IL-10 producing cells and contributed to the downregulation of collagen-induced arthritis, while follicular B-cells failed to do so. Based on these observations, we aimed to investigate how inflammatory signals mediated through the BCR, TLR9 and IFN-γ receptors trigger IL-10 production in MZ B-cells but leave FO B-cells unresponsive. We particularly focused on the CREB transcription factor as it is involved in all three signalling cascades and analysed its contribution to IL-10 production. Our results demonstrate that the IL-10 production of MZ B-cells induced by the BCR, TLR9 and IFN-γ receptors is mediated by CREB. We showed that the activation of CREB is prolonged in MZ B-cells while the transcription factor only transiently phosphorylated in FO B-cells. The sustained phosphorylation of CREB is clearly associated with its prolonged binding to molecular partner CBP, whereas inhibition of their association decreased IL-10 production. We assume that sustained activation of CREB is required for IL-10 production by B-cells under inflammatory conditions.

The importance of the BCR and TLR9 in autoimmunity and in the production of auto-antibodies is well established but the underlying molecular mechanism still needs to be determined. Here, we aim to characterize the BCR-TLR9 cross-talk by its effect on T-bet, as T-bet is activated and regulated by both receptors and has an important role in class-switching to pathological IgG2a in mice. Using primary mouse B cells, we demonstrate that T-bet expression is synergistically elevated by the cross-talk between the BCR and TLR9. To test the effect of this synergy on IgG2a-switching, the levels of switched B cells were checked by functional tests. We found that BCR costimulation had no additional effect on TLR9-induced IgG2a expression, however the expression of Rad51 was synergistically increased. To check the biological significance of the synergy, we compared T-bet expression in B cells from healthy and collagen-induced arthritis mice but no differences were found. Taken together, we demonstrate here that signaling cascades driven by the BCR and TLR9 have a newly identified meeting point at T-bet. The two cascades act synergistically on T-bet; however additional signals may be needed to induce prolonged functional responses such as class-switch recombination.

Eszter Szarka1*, Zsuzsa Neer1*, Péter Balogh2, Monika Ádori1, Adrienn Angyal1, József Prechl3, Endre Kiss1,3, Dorottya Kövesdi1, Gabriella Sármay11Department of Immunology, Eötvös Loránd University, 1117 Budapest, 2Department of Immunology and Biotechnology, University of Pécs, Pécs, 3Immunology Research Group of the Hungarian Academy of Science at Eötvös Loránd University, 1117 Budapest, Hungary*These authors contributed equally to this workAbstract: Antibodies specific for bovine type II collagen (CII) and Fcγ receptors play a major role in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA) and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv) fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2). These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro-inflammatory cytokines. Taken together, these results suggest that the in vivo emerging immune complexes formed with autoantigen(s) may trigger the IL-12/23 dependent pathways, escalating the inflammation in RA. Thus blockade of these cytokines may be beneficial to downregulate immune complex-induced inflammation in autoimmune arthritis.Keywords: collagen-induced arthritis, Fc gamma receptor, immune complex, inflammation, targeting&nbsp

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.

Engagement of antigen receptors on immature B cells induces apoptosis, while at the mature stage, it stimulates cell activation and proliferation. The difference in B cell receptor (BCR)-mediated signaling pathways regulating death or survival of B cells is not fully understood. We aimed to characterize the pathway leading to BCR-driven apoptosis. Transitional immature B cells were obtained from the spleen of sublethally irradiated and auto-reconstituted mice. We have detected a short-lived BCR-driven activation of mitogen-activated protein kinases (ERK1/2 and p38 MAPK) and Akt/PKB in transitional immature B cells that correlated with the lack of c-Fos expression, reduced phosphorylation of Akt substrates and a susceptibility for apoptosis. Simultaneous signaling through BCR and CD40 protected immature B cells from apoptosis, however, without inducing Bcl-2 expression. The BCR-induced apoptosis of immature B cells is a result of the collapse of mitochondrial membrane potential and the subsequent activation of caspase-3.

In the absence of phosphorylated Gab2, immature B cells are unable to maintain a prolonged signal transduction, which is necessary for the transcriptional regulation and activation of the cells. Thus, the lack of Gab2 phosphorylation may drive immature B cells to apoptosis.

CpG oligodeoxynucleotides (CpG) are widely studied as promising adjuvants in vaccines against a range of diseases including infection, cancer or allergy. Conjugating antigen to CpG has been shown to potentiate the adjuvant effect via enhancing antigen uptake and danger signaling by the very same cell. In the present study, using biotinylated CpG and streptavidin as a model system, we demonstrate that CpG motif containing free and antigen-conjugated oligonucleotides do not compete in terms of cell activation via TLR9, but do compete for cellular uptake. Antigen-conjugated CpG enhances cellular association and uptake of the antigen by antigen-presenting cells (APC) and T cells. Free CpG efficiently competes with antigen-CpG conjugates in BMDC and T cells, but shows weak or no competition in B cells that have higher TLR9 expression. Vaccination with antigen-conjugated CpG or with a mixture of antigen and CpG elevates the level of antigen-specific antibodies but co-administration of CpG-antigen conjugates and free CpG adversely effects immunogenicity. These observations may help optimize CpG-based vaccine formulation. © 2014 Elsevier B.V.

The generation of high-affinity Abs is essential for immunity and requires collaboration between B and T cells within germinal centers (GCs). By using novel mouse models with a conditional deletion of the p110δ catalytic subunit of the PI3K pathway, we established that p110δ is required in T cells, but not in B cells, for the GC reaction. We found the formation of T follicular helper (TFH) cells to be critically dependent on p110δ in T cells. Furthermore, by deleting phosphatase and tensin homolog deleted on chromosome 10, which opposes p110δ in activated T cells, we found a positive correlation between increased numbers of TFH cells and GC B cells. These results are consistent with the hypothesis that T cell help is the limiting factor in the GC reaction. P110δ was not required for the expression of B cell lymphoma 6, the downregulation of CCR7, or T cell entry into primary follicles. Instead, p110δ was the critical catalytic subunit for ICOS downstream signaling and the production of key TFH cytokines and effector molecules. Our findings support a model in which the magnitude of the GC reaction is controlled by the activity of the PI3K pathway in TFH cells.

B-cell fate during maturation and the germinal center reaction is regulated through the strength and the duration of the B-cell receptor signal. Signaling pathways discriminating between apoptosis and survival in B cells are keys in understanding adaptive immunity. Gab2 is a member of the Gab/Dos adaptor protein family. It has been shown in several model systems that Gab/Dos family members may regulate both the anti-apoptotic PI3-K/Akt and the mitogenic Ras/MAPK pathways, still their role in B-cells have not been investigated in detail. Here we studied the role of Gab2 in B-cell receptor mediated signaling. We have shown that BCR crosslinking induces the marked phosphorylation of Gab2 through both Lyn and Syk kinases. Subsequently Gab2 recruits p85 regulatory subunit of PI3-K, and SHP-2. Our results revealed that Ig-alpha/Ig-beta, signal transducing unit of the B-cell receptor, may function as scaffold recruiting Gab2 to the signalosome. Overexpression of Gab2 in A20 cells demonstrated that Gab2 is a regulator of the PI3-K/Akt but not that of the Ras/MAPK pathway in B cells. Accordingly to the elevated Akt phosphorylation, overexpression of wild-type Gab2 in A20 cells suppressed Fas-mediated apoptosis, and enhanced BCR-mediated rescue from Fas-induced cell death. Although PH-domain has only a stabilizing effect on membrane recruitment of Gab2, it is indispensable in mediating its anti-apoptotic effect.

B-cell activating factor of the TNF family (BAFF) is critical for the survival and maturation of B cells. The molecular mechanisms by which BAFF regulates the survival of developing B cells are becoming better understood. Recent evidence has begun to emerge demonstrating a role for the PI3K/Akt signalling pathway in response to BAFF. However, the importance of the PI3K family for BAFF-signalling and the effects of loss of PI3K function on BAFF responses are still unknown. We therefore investigated the BAFF-mediated responses of B cells deficient for the PI3K catalytic subunit P110delta. We find that the loss of P110delta impairs the BAFF-mediated survival of cultured B cells demonstrating a direct role for this member of the PI3K family in regulating the survival of B cells in response to BAFF. P110delta was required for the growth of B cells in response to BAFF and was critical for the upregulation of the receptor for BAFF following BCR crosslinking. Our findings reveal an important role for p110delta in regulating B-cell responses to BAFF.

Antigens coated with complement fragments coligate the B cell receptor (BCR) with the CD21/CD19 complex which results in synergistic activation of B cells. Previous studies identified PI3K, Vav proteins and PLCgamma as important components of this synergy. We now show that protein kinase D (also known as PKCmu) is also a point of convergence of these signalling pathways. We found that PKD activation upon BCR engagement or coligation of the BCR with CD19 is entirely dependent on PI3K and PLCgamma but differ in the requirement for Vav proteins. Whereas PKD activation is Vav1 and Vav2 dependent in response to BCR cross-linking, PKD activation is sensitive to the lack of Vav1 under synergistic stimulation of BCR and CD19. These findings show that Vav proteins and PI3K regulation of PLCgamma contributes to the activation of PKD in response to BCR and or CD19 cross-linking.

Sphingomyelinase (SMase)-mediated release of ceramide in the plasma membrane of T-lymphocytes induced by different stimuli such as ligation of Fas/CD95, irradiation, stress, inflammation or anticancer drugs primarily involves mitochondrial apoptosis signaling, but under specific conditions non-apoptotic Fas-signaling was also reported. Here we investigated, using a quantitative simulation model with exogenous C2-ceramide (and SMase), the dependence of activation and fate of T-cells on the strength and duration of ceramide accumulation. A murine, influenza virus hemagglutinin-specific T-helper cell (IP12-7) alone or together with interacting antigen presenting B-cells (APC) was used. C2-ceramide induced apoptosis of TH cells above a 'threshold' stimulus (>25 microM in 'strength' or >30 min in duration), while below the threshold C2-ceramide was non-apoptotic, as confirmed by early and late apoptotic markers (PS-translocation, mitochondrial depolarization, caspase-3 activation, DNA-fragmentation). The modest ceramide stimuli strongly suppressed the calcium response and inhibited several downstream signal events (e.g. ERK1/2-, JNK-phosphorylation, CD69 expression or IL-2 production) in TH cells during both anti-CD3 induced and APC-triggered activation. Ceramide moderately affected the Ca2+ -release from internal stores upon antigen-specific engagement of TCR in immunological synapses, while the influx phase was remarkably reduced in both amplitude and rate, suggesting that the major target(s) of ceramide-effects are membrane-proximal. Ceramide inhibited Kv1.3 potassium channels, store operated Ca2+ -entry (SOC) and depolarized the plasma membrane to which contribution of spontaneously formed ceramide channels is possible. The impaired function of these transporters may be coupled to the quantitative, membrane raft-remodeling effect of ceramide and responsible, in a concerted action, for the suppressed activation. Our results suggest that non-apoptotic Fas stimuli, received from previously activated, FasL+ interacting lymphocytes in the lymph nodes, may negatively regulate subsequent antigen-specific T-cell activation and thus modulate the antigen-specific T-cell response.

B lymphocytes respond to bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) and CD180 (previously called RP105). We show here that the responses of B lymphocytes to LPS require the function of the Vav family of guanine nucleotide exchange factors. Vav1-mutant mice generate defective humoral immunoglobulin G (IgG) responses following administration of low doses of LPS but respond normally to higher doses, while mice lacking both Vav1 and Vav2 manifest defective responses even after a high dose of LPS. Vav1/2-mutant B cells fail to divide extensively in vitro in response to LPS or CD180, while deficiency of Vav1 alone impairs CD180-but not LPS-driven proliferation. Likewise, activation of Akt (a PI3K [phosphatidylinositol 3-kinase] target) and phosphorylation of IκBα in response to CD180 or LPS required Vav1 and Vav2, while Vav1 deficiency led to defective responses to CD180. In addition, activation of ERK (extracellular signal regulated kinase) required Vav1 and Vav2 in response to CD180 but was Vav1 and vav2 independent in response to LPS. Induction of CD86 and CD25 by anti-CD180 also required Vav function, as did the induction of the antiapoptotic protein Bcl-xL (B-cell leukemia XL). These data provide evidence for the function for the Vav proteins in regulating the responses of B cells to LPS. (Blood. 2005;106:635-640)

Scientific background A functionally important subset of B cells, known as regulatory B cells (Breg), are specialised to control immune response and immunological diseases such as autoimmunity. Breg cells are best characterised by their inducible production of IL-10 and can appear in different phenotypic forms. Various activation signals, including Toll-like receptors (TLRs), B cell antigen receptor (BCR) or CD40 stimulation can trigger the regulatory transformation of B cells, although it is not clear which of these signals are the most effective and which B cells have the most susceptible phenotype. We have studied the effect of the BCR and TLR9 stimulation on three populations of B cells in mouse spleen (Transitional-2-Marginal Zone Precursors (T2-MZP), Marginal Zone (MZ) and Follicular (FO) B cells) to induce differentiation into Bregs. Materials and methods We set up in vitro cultures using sorted B cells from T2-MZP, MZ or FO pools from the spleen of adult mice and stimulated them via the BCR and/or TLR9. The transformation to Breg was monitored by following IL-10 production using gene expression analysis, IL-10-specific ELISA or intracellular IL-10 staining measured by FACS. Results Based on our experimental set up, we found that the BCR signal alone was not sufficient to induce IL-10 production, while TLR9 stimulation mediated the regulatory transformation of all B cell types with different extent. Comparing the three populations, we found that B cells from the marginal zone responded more efficiently for TLR9 stimulation. We detected the highest level of secreted IL-10 and the highest number of induced IL-10 positive cells among MZ B cells. The results at protein level were confirmed by gene expression analysis, additionally, at mRNA level a time-dependent synergistic crosstalk through the BCR and TLR9 was observed. Conclusion We concluded that MZ B cells were the most inducible B cell population and TLR9 signal alone was efficient to trigger IL-10 production. Using the most sensitive B cell population with the efficient stimulus will help us to study the signalling characteristics of Bregs and their important role in autoimmune diseases. This project was supported by OTKA NK 104846 and by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences.

Ligation of B cell receptors (BCR) on immature B cells may induce apoptosis, while in mature B cells it stimulates cell activation and growth. The signaling pathway regulating the differential functional response, death or survival of the B cell is not fully characterized. We have tested the intracellular signaling requirement of these processes using B cells isolated from the spleen of irradiated auto-reconstituted (transitional immature B cells) and untreated mice (mature B cells), respectively. We compared the BCR induced intracellular [Ca2+] transient, protein tyrosine phosphorylation and ERK phosphorylation, furthermore, the activation of Elk-1 and CREB transcription factors. The BCR induced rise of intracellular [Ca2+] did not significantly differ in the two populations, only a slight difference in the late phase of the response was observed. Immature B cells responded with a maximum tyrosine phosphorylation to a five times lower dose of anti-IgM compared to the mature population. Most importantly, we have found a significant difference in the tyrosine phosphorylation of the Gab family adaptor proteins, Gab1/2. In contrast to mature B cells, crosslinking of BCR on immature B cells did not induce tyrosine phosphorylation of Gab2, thus the Gab2-organized signal amplification complex could not be produced. Furthermore, we detected a significant difference in the kinetics of BCR induced ERK, Elk-1 and CREB phosphorylation. In immature B cells, ERK was transiently phosphorylated, ceasing after 120 min, while in mature cells, ERK phosphorylation was sustained. Elk-1 and CREB activation was also transient in immature B cells, followed the kinetics of ERK phosphorylation. The lack of sustained Erk1/2 activation suppresses the transcription factors necessary for the proliferation signal. Since ERK is regulated by the phosphorylated Gab1/2, these data demonstrate that BCR triggered phosphorylation and signal amplification of Gab1/2 is a critical step in a life or death decision of B cells.

We have compared early signaling events at various stages of B cell differentiation using established mouse cell lines. Clustering of pre-B cell antigen receptor (BCR) or BCR induced the tyrosine phosphorylation of various proteins in all cells, although the phosphorylation pattern differed. In spite of the pre-BCR-induced tyrosine phosphorylation, we could not detect an intracellular Ca(2+) signal in pre-B cells. However, co-clustering of the pre-BCR with CD19 did induce Ca(2+) mobilization. In contrast to the immature and mature B cells, where the B cell linker protein (BLNK) went through inducible tyrosine phosphorylation upon BCR clustering, we observed a constitutive tyrosine phosphorylation of BLNK in pre-B cell lines. Both BLNK and phospholipase C (PLC)gamma were raft associated in unstimulated pre-B cells, and this could not be enhanced by pre-BCR engagement, suggesting a ligand-independent PLC gamma-mediated signaling. Further results indicate that the cell lines representing the immature stage are more sensitive to BCR-, CD19- and type II receptors binding the Fc part of IgG (Fc gamma RIIb)-mediated signals than mature B cells.

ur previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag- specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag- specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.