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Transphosphorylation by Src family kinases is required for the activation of Bruton's tyrosine kinase (Btk). Differences in the phenotypes of Btk-/- and lyn-/- mice suggest that these kinases may also have independent or opposing functions. B cell development and function were examined in Btk-/-lyn-/- mice to better understand the functional interaction of Btk and Lyn in vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk-/-lyn-/- mice. However, Btk-/-lyn-/- animals had a more severe immunodeficiency than Btk-/- mice. B cell numbers and response to T cell-dependent antigens were reduced. Btk and Lyn therefore play independent or partially redundant roles in the maintenance and function of peripheral B cells. Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn-/- mice. A transgene expressing Btk at approximately 25% of endogenous levels (Btklo) was crossed onto Btk-/- and Btk-/-lyn-/- backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.

We investigated the role of Bruton's tyrosine kinase (Btk) in FcepsilonRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcepsilonRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcepsilonRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcepsilonRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed by the improvement in the ability of btk mutant mast cells to degranulate and to secrete cytokines after the retroviral transfer of wild-type btk cDNA, but not of vector or kinase-dead btk cDNA. Retroviral transfer of Emt (= Itk/Tsk), Btk's closest relative, also partially improved the ability of btk mutant mast cells to secrete mediators. Taken together, these results demonstrate an important role for Btk in the full expression of FcepsilonRI signal transduction in mast cells.

Background: The use of cuffed endotracheal tubes (ETTs) has become the standard of care in pediatric practice. The rationale for the use of a cuffed ETT is to minimize pressure around the cricoid while providing an effective airway seal. However, safe care requires that the cuff lie distal to the cricoid ring following endotracheal intubation. The current study demonstrates the capability of computed tomography (CT) imaging in identifying the position of the cuff of the ETT in intubated patients., Methods: The study included patients ranging in age from 1 month to 10 years who underwent neck and chest CT imaging that required general anesthesia and endotracheal intubation. The location of the ETT and of the cuff within the airway was determined from axial CT images at three levels (proximal, middle, and distal). Anatomical orientations were tabulated, and percent chances of each orientation were determined for the ETT and the cuff., Results: The study cohort included 42 patients ranging in age from 1 to 114 months. An ETT with a polyvinylchloride cuff was used in 24 patients, and an ETT with a polyurethane cuff was used in 18 patients. The ETT was located near the posterior wall of the trachea in approximately 24-38% of patients, being most likely to be centrally located at the proximal end and at its mid-portion. The middle part of the cuff was most likely to be positioned in the mid-portion of the trachea but tended to skew anteriorly at both the proximal and distal ends., Conclusion: This is the first study using CT imaging to identify the uniformity of cuff inflation within the trachea in children. With commonly used cuffed ETTs, cuff inflation and the final position of ETT cuff within the tracheal lumen were not uniform. Future investigations are needed to determine the reasons for this asymmetry and its clinical implications., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Saudi Journal of Anesthesia.)

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

The conventional type 1 dendritic cells (cDC1) play a pivotal role in protective immunity against pathogens and cancer. However, their low frequency in the blood and tissues limits their use in immune therapy. We have recently described a method to vaccinate against neoantigens that are induced in tumor cells by targeted delivery of a TAP siRNA to dendritic cells using a TLR9 binding CpG oligonucleotide. Since TLR9 is also expressed in immune suppressive myeloid populations TLR9 targeting could reduce the effectiveness of this approach. Here, we describe a modular multivalent antibody platform to target the TAP siRNA to resident Clec9a expressing cDC1 and show that it leads to selective and sustained TAP downregulation in cDC1 and inhibits tumor growth in mice more effectively than CpG targeted siRNA. To induce DC maturation an agonistic CD40 antibody was administered to the siRNA treated mice. To obviate the need for a second drug formulation and reduce the risk of toxicity, we exploited the multivalent nature of this targeting platform to co-deliver the TAP siRNA and a DC maturation agent, a CpG containing oligonucleotide, to cDC1 in vivo and show that it was more effective than Clec9a targeting of TAP siRNA in combination with CD40 antibody. This study describes a way to manipulate the function of cDC1 cells in vivo using a broadly applicable antibody-based targeting platform to deliver multiple biological agents to specific cells in vivo to potentiate (immune) therapy and to probe the biology of specific cell types in their natural settings., (© 2024. The Author(s).)

Bruton's tyrosine kinase (Btk) propagates B cell signaling, and BTK inhibitors are in clinical trials for autoimmune disease. Although autoreactive B cells fail to develop in the absence of Btk, its role in mature cells is unknown. To address this issue, a model of conditional removal ( Btk flox / Cre-ER T2 ) was used to excise Btk from mature transgenic B cells that recognize the pathophysiologic autoantigen insulin. Anti-insulin B cells escape central tolerance and promote autoimmune diabetes, mimicking human autoreactive cells. Lifelong Btk deficiency was previously shown to eliminate 95% of anti-insulin B cells, but in this model, mature anti-insulin B cells survived for weeks after targeted Btk deletion, even when competing with a polyclonal repertoire. BCR-stimulated cells could still signal via Syk, PLCy2, and CD22, but failed to upregulate the antiapoptotic protein Bcl-xL, and proliferation was impaired. Surprisingly, Btk -depleted anti-insulin B cells could still present Ag and activate T cells, a critical function in promoting T cell-mediated islet cell destruction. Thus, pharmacologic targeting of Btk may be most effective by blocking expansion of established autoreactive cells, and preventing emergence of new ones., (Copyright © 2021 by The American Association of Immunologists, Inc.)

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim ( BBim fl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren's Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton's tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity., Competing Interests: Authors GC, LC, and TM are employed by AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wright, Bazile, Clark, Carlesso, Boucher, Kleiman, Mahmoud, Cheng, López-Rodríguez, Satterthwaite, Altman, Greidinger and Khan.)

Hepatocellular carcinoma (HCC), the most prevalent primary liver cancer, is a leading cause of cancer-related death worldwide because of rising incidence and limited therapy. Although treatment with sorafenib or lenvatinib is the standard of care in patients with advanced-stage HCC, the survival benefit from sorafenib is limited due to low response rate and drug resistance. Ibrutinib, an irreversible tyrosine kinase inhibitor (TKI) of the TEC (e.g., BTK) and ErbB (e.g., EGFR) families, is an approved treatment for B-cell malignancies. Here, we demonstrate that ibrutinib inhibits proliferation, spheroid formation, and clonogenic survival of HCC cells, including sorafenib-resistant cells. Mechanistically, ibrutinib inactivated EGFR and its downstream Akt and ERK signaling in HCC cells, and downregulated a set of critical genes involved in cell proliferation, migration, survival, and stemness, and upregulated genes promoting differentiation. Moreover, ibrutinib showed synergy with sorafenib or regorafenib, a sorafenib congener, by inducing apoptosis of HCC cells. In vivo , this TKI combination significantly inhibited HCC growth and prolonged survival of immune-deficient mice bearing human HCCLM3 xenograft tumors and immune-competent mice bearing orthotopic mouse Hepa tumors at a dose that did not exhibit systemic toxicity. In immune-competent mice, the ibrutinib-sorafenib combination reduced the numbers of BTK + immune cells in the tumor microenvironment. Importantly, we found that the BTK + immune cells were also enriched in the tumor microenvironment in a subset of primary human HCCs. Collectively, our findings implicate BTK signaling in hepatocarcinogenesis and support clinical trials of the sorafenib-ibrutinib combination for this deadly disease., (©2019 American Association for Cancer Research.)

Objective: Raynaud's phenomenon (RP) is common in anti-RNP-positive patients with rheumatic diseases but is not itself known to be caused by autoimmunity. The aim of this study was to assess autoantibodies that could mediate this process., Methods: Antibodies derived from patient sera and from murine models of anti-RNP autoimmunity were screened for the ability to induce RP-like tissue ischemia and endothelial cell apoptosis in murine models and in vitro systems., Results: RNP-positive sera from RP patients and murine sera from RNP-positive B cell adoptive transfer recipients induced RP-like tissue ischemia and endothelial cell apoptosis. Proteomic analysis identified cytokeratin 10 (K10) as a candidate autoantigen in RP. Monoclonal anti-K10 antibodies reproduced patterns of ischemic tissue loss and endothelial cell apoptosis; K10 knockout or depletion of anti-K10 activity in serum was protective. Cold exposure enhanced K10 expression and in vivo tissue loss., Conclusion: Anti-K10 antibodies are sufficient to mediate RP-like ischemia in murine models and are implicated in the pathogenesis of RP in patients with anti-RNP autoimmunity., (© 2018, American College of Rheumatology.)

Importance: Squamous cell carcinoma (SCC) is the second most common form of skin cancer, and its incidence is increasing. When surgical management is not an option, finding a safe and efficacious treatment is a challenge. Mounting evidence suggests that the human papillomavirus (HPV) is involved in the pathogenesis of some SCCs., Objective: To assess whether the 9-valent HPV vaccine could be an effective treatment strategy for cutaneous SCC., Design, Setting, and Participants: A woman in her 90s with multiple, inoperable cutaneous basaloid SCCs was successfully treated at a university-based outpatient dermatology clinic with a combination of systemic and intratumoral delivery of the 9-valent HPV vaccine from March 17, 2016, through February 27, 2017, and then followed up through May 21, 2018., Main Outcomes and Measures: Reduction in tumor size and number after a combination of systemic and intratumoral administration of the HPV vaccine., Results: All tumors resolved 11 months after the first intratumoral injection of the vaccine. The patient remained free of tumors at the end of follow-up., Conclusions and Relevance: This is the first report, to our knowledge, of complete regression of a cutaneous malignant tumor after combined systemic and direct intratumoral injection of the 9-valent HPV vaccine. This report suggests that the HPV vaccine may have therapeutic utility for SCCs in patients who are poor surgical candidates, have multiple lesions, or defer surgery.

Approximately 12% of all human cancers worldwide are caused by infections with oncogenic viruses. Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8) is one of the oncogenic viruses responsible for human cancers, including Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL), and the lymphoproliferative disorder multicentric Castleman's disease (MCD). Chronic inflammation mediated by KSHV infection plays a decisive role in the development and survival of these cancers. NF-κB, a family of transcription factors regulating inflammation, cell survival, and proliferation, is persistently activated in KSHV-infected cells. The KSHV latent and lytic expressing oncogenes involved in NF-κB activation are vFLIP/K13 and vGPCR, respectively. However, the mechanisms by which NF-κB is activated by vFLIP and vGPCR are poorly understood. In this study, we have found that a host molecule, Cell Adhesion Molecule 1 (CADM1), is robustly upregulated in KSHV-infected PBMCs and KSHV-associated PEL cells. Further investigation determined that both vFLIP and vGPCR interacted with CADM1. The PDZ binding motif localized at the carboxyl terminus of CADM1 is essential for both vGPCR and vFLIP to maintain chronic NF-κB activation. Membrane lipid raft associated CADM1 interaction with vFLIP is critical for the initiation of IKK kinase complex and NF-κB activation in the PEL cells. In addition, CADM1 played essential roles in the survival of KSHV-associated PEL cells. These data indicate that CADM1 plays key roles in the activation of NF-κB pathways during latent and lytic phases of the KSHV life cycle and the survival of KSHV-infected cells.

Bruton's tyrosine kinase (Btk) is a crucial regulator of B cell signaling and is a therapeutic target for lymphoma and autoimmune disease. BTK-deficient patients suffer from humoral immunodeficiency, as their B cells fail to progress beyond the bone marrow. However, the role of Btk in fully developed, mature peripheral B cells is not well understood. Analysis using BTK inhibitors is complicated by suboptimal inhibition, off-target effects, or failure to eliminate BTK's adaptor function. Therefore a Btk flox /Cre-ER T2 mouse model was developed and used to excise Btk after B cell populations were established. Mice lacking Btk from birth are known to have reduced follicular (FO) compartments, with expanded transitional populations, suggesting a block in development. In adult Btk flox /Cre-ER T2 mice, Btk excision did not reduce FO B cells, which persisted for weeks. Autoimmune-prone B1 cells also survived conditional Btk excision, contrasting their near absence in global Btk -deficient mice. Therefore, Btk supports BCR signaling during selection into the FO and B1 compartments, but is not needed to maintain these cell populations. B1-related natural IgM levels remained normal, contrasting global Btk deficiency, but B cell proliferation and T-independent type II immunization responses were blunted. Thus, B cells have nuanced signaling responses that are differentially regulated by Btk for development, survival, and function. These findings raise the possibility that Btk may also be expendable for survival of mature human B cells, therefore requiring prolonged dosing to be effective, and that success of BTK inhibitors may depend in part on off-target effects., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The host employs both cell-autonomous and system-level responses to limit pathogen replication in the initial stages of infection. Previously, we reported that the eukaryotic initiation factor 2α (eIF2α) kinases heme-regulated inhibitor (HRI) and protein kinase R (PKR) control distinct cellular and immune-related activities in response to diverse bacterial pathogens. Specifically for Listeria monocytogenes , there was reduced translocation of the pathogen to the cytosolic compartment in HRI-deficient cells and consequently reduced loading of pathogen-derived antigens on major histocompatibility complex class I (MHC-I) complexes. Here we show that Hri -/- mice, as well as wild-type mice treated with an HRI inhibitor, are more susceptible to listeriosis. In the first few hours of L. monocytogenes infection, there was much greater pathogen proliferation in the liver of Hri -/- mice than in the liver of Hri +/+ mice. Further, there was a rapid increase of serum interleukin-6 (IL-6) levels in Hri +/+ mice in the first few hours of infection whereas the increase in IL-6 levels in Hri -/- mice was notably delayed. Consistent with these in vivo findings, the rate of listeriolysin O (LLO)-dependent pathogen efflux from infected Hri -/- macrophages and fibroblasts was significantly higher than the rate seen with infected Hri +/+ cells. Treatment of cells with an eIF2α kinase activator enhanced both the HRI-dependent and PKR-dependent infection phenotypes, further indicating the pharmacologically malleability of this signaling pathway. Collectively, these results suggest that HRI mediates the cellular confinement and killing of virulent L. monocytogenes in addition to promoting a system-level cytokine response and that both are required to limit pathogen replication during the first few hours of infection., (Copyright © 2018 American Society for Microbiology.)

Infants are generally highly susceptible to oral pathogens. Intestinal infection and the associated diarrhea are significant global causes of morbidity and mortality in infants. Among the enteric pathogens, enteropathogenic Escherichia coli (EPEC) stands out as showing the highest risk for infection-induced death in infants ≤12 months old. We have developed an experimental model of infant infection with EPEC, using the mouse-specific pathogen Citrobacter rodentium Our murine infant model is similar to EPEC infection in human infants since infant mice are much more susceptible to C. rodentium infection than adult mice; infants infected with 50-fold fewer bacteria than the standard adult dose uniformly succumbed to the infection. Infant infection is characterized by high early and sustained bacterial titers and profound intestinal inflammation associated with extensive necrosis and systemic dissemination of the bacteria. Therefore, it seems likely that infant deaths result from sepsis secondary to intestinal damage. Recently, specialized proresolving mediators (SPM) have been found to exert profound beneficial effects in adult models of infection. Thus, we investigated the actions of two proresolving lipid mediators, resolvin D1 (RvD1) and resolvin D5 (RvD5), on the course of infection in infants. Strikingly, postinfection treatment with RvD1 and RvD5 reduced bacterial loads, mitigated inflammation, and rescued the infants from death. Furthermore, postinfection treatment with RvD1 and RvD5 led to protection from reinfection associated with C. rodentium -specific IgG responses comparable to those in adults. These results indicate that SPM may provide novel therapeutic tools for the treatment of pathological intestinal infections in infants., (Copyright © 2017 American Society for Microbiology.)

B lymphocytes play a role in inhibiting the immune response against certain tumors, but the underlying mechanisms are poorly understood. EMT-6 mammary tumors grow well in wild-type (WT) mice but show reduced growth in B-cell-deficient μ(-/-) BALB/c mice (BCDM). WT mice demonstrate extensive B-cell infiltration into the tumor bed, reduced CD8(+) T cell and CD49(+) NK cell infiltration, and markedly reduced cytolytic T-cell response relative to BCDM. Expression of LAP/TGF-β1, CD80, CD86 and PD-L1 is significantly increased in tumor-infiltrating B cells (TIL-B) relative to splenic B cells. LAP/TGF-β1 expression on TIL-B progressively increased from 5.4±1.7% on day 8 to 43.1±6.1% by day 21 post tumor implantation. Co-culture of EMT-6 tumor cells with Naive-B cells ex vivo generated B cells (EMT6-B) with a similar immunophenotype to TIL-B. Purified TIL-B, or in-vitro-generated EMT6-B suppressed CD4(+), CD8(+) and CD4(+)CD25(-) T-cell proliferation, and Th1 cytokine secretion, and also suppressed purified NK-cell proliferation in response to IL-15, compared to naive splenic B cells. Acquired B regulatory function required direct tumor cell: B-cell contact, and was partially reversed by antibody to TGF-β or PD-L1, leading to tumor rejection in vivo B-cell acquisition of a suppressive phenotype following tumor infiltration may result in profound inhibition of T-cell anti-tumor responses., (© The Japanese Society for Immunology. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1, Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogen Listeria monocytogenes Within a few hours of systemic infection, the massive proliferation of L. monocytogenes in Perforin-2(-/-)mice leads to a rapid appearance of acute disease symptoms. We go on to show in cultured Perforin-2(-/-)cells that the vacuole-to-cytosol transitioning of L. monocytogenesis greatly accelerated. Unexpectedly, we found that in Perforin-2(-/-)macrophages,Listeria-containing vacuoles quickly (≤ 15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation of L. monocytogenes to its replicative niche in the cytosol. This hypothesis was supported by our finding that aL. monocytogenes strain expressing virulence factors at a constitutively high level replicated equally well in Perforin-2(+/+)and Perforin-2(-/-)macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification of Listeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

In aged mice, new B-cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B-cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5(low) B-cell precursors generate new B cells which show increased reactivity to the self-antigen/bacterial antigen phosphorylcholine (PC). Pro-B cells in old bone marrow as well as pro-B cells from young adult λ5-deficient mice are resistant to cytokine-induced apoptosis (TNFα; TGFβ), indicating that low λ5 expression in pro-B cells is sufficient to cause increased survival. Transfer of TNFα-producing 'age-associated B cells' (ABC; CD21/35(-) CD23(-)) or follicular (FO) B cells from aged mice into RAG-2 KO recipients led to preferential loss of λ5(high) pro-B cells, but retention of λ5(low), apoptosis-resistant pro-B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα-producing B cells, results in preferential loss of SLC(high) pro-B cells within the bone marrow. Further B-cell development then occurs via an 'SLC(low)' pathway that not only impairs B-cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Unlabelled: Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1., Importance: Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Splenic transitional B-cells (T1 and T2) are selected to avoid self-reactivity and to safeguard against autoimmunity, then differentiate into mature follicular (FO-I and FO-II) and marginal zone (MZ) subsets. Transcriptomic analysis by RNA-seq of the five B-cell subsets revealed T1 cell signature genes included RAG suggesting a potential for receptor revision. T1 to T2 B-cell differentiation was marked by a switch from Myb to Myc, increased expression of the PI3K adapter DAP10 and MHC class II. FO-II may be an intermediate in FO-I differentiation and may also become MZ B-cells as suggested by principle component analysis. MZ B-cells possessed the most distinct transcriptome including down-regulation of CD45 phosphatase-associated protein (CD45-AP/PTPRC-AP), as well as upregulation of IL-9R and innate molecules TLR3, TLR7, and bactericidal Perforin-2 (MPEG1). Among the endosomal TLRs, stimulation via TLR3 further enhanced Perforin-2 expression exclusively in MZ B-cells. Using gene-deleted and overexpressing transgenic mice we show that IL-9/IL-9R interaction resulted in rapid activation of STAT1, 3, and 5, primarily in MZ B-cells. Importantly, CD45-AP mutant mice had reduced transitional and increased mature MZ and FO B-cells, suggesting that it prevents premature entry of transitional B-cells to the mature B-cell pool or their survival and proliferation. Together, these findings suggest, developmental plasticity among splenic B-cell subsets, potential for receptor revision in peripheral tolerance whereas enhanced metabolism coincides with T2 to mature B-cell differentiation. Further, unique core transcriptional signatures in MZ B-cells may control their innate features.

Autoreactive B lymphocytes are essential for the development of T cell-mediated type 1 diabetes (T1D). Cytoplasmic Bruton's tyrosine kinase (BTK) is a key component of B cell signaling, and its deletion in T1D-prone NOD mice significantly reduces diabetes. However, the role of BTK in the survival and function of autoreactive B cells is not clear. To evaluate the contributions of BTK, we used mice in which B cells express an anti-insulin BCR (125Tg) and promote T1D, despite being anergic. Crossing Btk deficiency onto 125Tg mice reveals that, in contrast to immature B cells, mature anti-insulin B cells are exquisitely dependent upon BTK, because their numbers are reduced by 95%. BTK kinase domain inhibition reproduces this effect in mature anti-insulin B cells, with less impact at transitional stages. The increased dependence of anti-insulin B cells on BTK became particularly evident in an Igκ locus site-directed model, in which 50% of B cells edit their BCRs to noninsulin specificities; Btk deficiency preferentially depletes insulin binders from the follicular and marginal zone B cell subsets. The persistent few Btk-deficient anti-insulin B cells remain competent to internalize Ag and invade pancreatic islets. As such, loss of BTK does not significantly reduce diabetes incidence in 125Tg/NOD mice as it does in NOD mice with a normal B cell repertoire. Thus, BTK targeting may not impair autoreactive anti-insulin B cell function, yet it may provide protection in an endogenous repertoire by decreasing the relative availability of mature autoreactive B cells.

The nuclear factor kappa B (NF-κB) plays vital role in the immune system by regulating innate and adaptive immunity, development and survival of lymphocytes, and lymphoid organogenesis. All known NF-κB activators converge on the IkappaB kinase (IKK) complex to activate the canonical and non-canonical NF-κB pathways. The IKK complex contains two catalytic subunits (IKKα and IKKβ) and a regulatory subunit NEMO/IKKγ that regulates the canonical NF-κB pathway, whereas IKKα regulates the non-canonical pathway. The process of IKKα activation and its role in the regulation of canonical NF-κB activation remain elusive. The canonical pathway is rapidly activated and produces a potent inflammatory response to bacterial and viral infections as well as different types of stress; however, uncontrolled NF-κB activation can lead to autoimmune diseases and cancers. Therefore, to keep the inflammatory response in check, elaborate negative regulatory mechanisms operate to terminate NF-κB activation at multiple levels by de novo synthesis of NF-κB inhibitory proteins, and orchestration of protein ubiquitination and deubiquitination. The NF-κB target genes, IκBα and A20, play critical roles in termination of the active canonical NF-κB pathway. In this review, we discuss our recent findings describing a novel function for IKKα in nucleating the ubiquitin-editing enzyme A20 complex, a major negative regulator of canonical NF-κB signaling. Consistently with an inhibitory function of IKKα, it is targeted by the human T-cell leukemia virus 1 (HTLV-1) oncoprotein, Tax, to prevent assembly of the A20 complex to maintain persistent NF-κB activation that promotes transformation and survival of virus-transformed cells.

B-lymphocytes are integral to host defense against microbial pathogens and are associated with many autoimmune diseases. The B-cell receptor implements B-cell self-tolerance based on the antigen specificity, and B-cell-activating factor receptor (BAFF-R) imposes homeostatic control. While shaping the repertoire, the immune tolerance process also culls mature B cells into distinct populations. The activation response of B cells is tailored to the type of pathogen attack and is facilitated by T-cell help via CD40/CD40L interaction and/or innate cell help via toll-like receptors in conjunction with BAFF receptors and ligands. Activated effector B cells not only produce antibodies, but also produce a variety of cytokines to enhance and suppress the immune response. Not surprisingly, B cells play multiple roles in both humoral and cellular immune responses during infection and autoimmune pathogenesis. Here, we discuss how gene expression and signaling networks regulate peripheral B-cell tolerance, B-cell effector functions and emerging therapies targeting B-cell signaling in autoimmune diseases.

Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria. For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.

Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimulation induced Bcl10 association with IκB kinase β, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-x(L), a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells.

T follicular cells help B cells to drive germinal center formation. In this issue of Immunity, Ballesteros-Tato et al. (2012) demonstrate that high amounts of interleukin-2 inhibit production of this critical T effector subset., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion., Methodology and Principal Findings: Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T(H)1 (IgG2a) but not T(H)2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses., Conclusions: We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Type 1 diabetes results from T cell-mediated destruction of insulin-producing beta cells. Although elimination of B lymphocytes has proven successful at preventing disease, modulation of B cell function as a means to prevent type 1 diabetes has not been investigated. The development, fate, and function of B lymphocytes depend upon BCR signaling, which is mediated in part by Bruton's tyrosine kinase (BTK). When introduced into NOD mice, btk deficiency only modestly reduces B cell numbers, but dramatically protects against diabetes. In NOD, btk deficiency mirrors changes in B cell subsets seen in other strains, but also improves B cell-related tolerance, as indicated by failure to generate insulin autoantibodies. Introduction of an anti-insulin BCR H chain transgene restores diabetes in btk-deficient NOD mice, indicating that btk-deficient B cells are functionally capable of promoting autoimmune diabetes if they have a critical autoimmune specificity. This suggests that the disease-protective effect of btk deficiency may reflect a lack of autoreactive specificities in the B cell repertoire. Thus, signaling via BTK can be modulated to improve B cell tolerance, and prevent T cell-mediated autoimmune diabetes.

Generation of mature B lymphocytes from early (T1) and late transitional (T2) precursors requires cooperative signaling through BCR and B cell-activating factor receptor 3 (BR3). Recent studies have shown that BCR signaling positively regulates NF-kappaB2, suggesting BCR regulation of BR3 signaling. To investigate the significance of signal integration from BCR and BR3 in B cell development and function, we crossed Btk-deficient mice (btk(-/-)), which are developmentally blocked between the T2 and the mature follicular B cell stage as a result of a partial defect in BCR signaling, and A/WySnJ mice, which possess a mutant BR3 defective in propagating intracellular signals that results in a severely reduced peripheral B cell compartment, although all B cell subsets are present in relatively normal ratios. A/WySnJ x btk(-/-) mice display a B cell-autonomous defect, resulting in a developmental block at an earlier stage (T1) than either mutation alone, leading to the loss of mature splenic follicular and marginal zone B cells, as well as the loss of peritoneal B1 and B2 cell populations. The competence of the double mutant T1 B cells to respond to TLR4 and CD40 survival and activation signals is further attenuated compared with single mutations as evidenced by severely reduced humoral immune responses in vivo and proliferation in response to anti-IgM, LPS, and anti-CD40 stimulation in vitro. Thus, BCR and BR3 independently and in concert regulate the survival, differentiation, and function of all B cell populations at and beyond T1, earliest transitional stage.

B lymphocyte homeostasis depends on tonic and induced BCR signaling and receptors sensitive to trophic factors, such as B cell-activating factor receptor (BAFF-R or BR3) during development and maintenance. This review will discuss growing evidence suggesting that the signaling mechanisms that maintain B cell survival and metabolic fitness during selection at transitional stages and survival after maturation rely on cross-talk between BCR and BR3 signaling. Recent findings have also begun to unravel the molecular mechanisms underlying this crosstalk. In this review I also propose a model for regulating the amplitude of BCR signaling by a signal amplification loop downstream of the BCR involving Btk and NF-kappaB that may facilitate BCR-dependent B cell survival as well as its functional coupling to BR3 for the growth and survival of B lymphocytes.

Objective: Patients with chronic kidney disease (CKD) have the highest risk for atherosclerotic cardiovascular disease (CVD). Current interventions have been insufficiently effective in lessening excess incidence and mortality from CVD in CKD patients versus other high-risk groups. The mechanisms underlying the heightened risk remain obscure but may relate to differences in CKD-induced atherogenesis, including perturbation of macrophage cholesterol trafficking., Methods and Results: We examined the impact of renal dysfunction on macrophage cholesterol homeostasis in the apoE(-/-) mouse model of atherosclerosis. Renal impairment induced by uninephrectomy dramatically increased macrophage cholesterol content, linked to striking impairment of macrophage cholesterol efflux. This blunted efflux was associated with downregulation of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) and activation of the nuclear factor-kappa B (NF-kappaB). Treatment with the angiotensin receptor blocker (ARB) losartan decreased NF-kappaB and restored cholesterol efflux., Conclusions: Our findings show that mild renal dysfunction perturbs macrophage lipid homeostasis by inhibiting cholesterol efflux, mediated by decreased ABCA1 transporter and activation of NF-kappaB, and that ARB can restore cholesterol efflux.

Signaling from the BCR and B cell activating factor receptor (BAFF-R or BR3) differentially regulates apoptosis within early transitional (T1) and late transitional (T2; CD21(int)-T2) B cells during selection processes to generate mature B lymphocytes. However, molecular mechanisms underlying the differential sensitivity of transitional B cells to apoptosis remain unclear. In this study, we demonstrate that BCR signaling induced more long-term c-Rel activation in T2 and mature than in T1 B cells leading to increased expression of anti-apoptotic genes as well as prosurvival BAFF-R and its downstream substrate p100 (NF-kappaB2). Sustained c-Rel activation required de novo c-Rel gene transcription and translation via Btk-dependent mechanisms. Like T1 cells, mature B cells from Btk- and c-Rel-deficient mice also failed to activate these genes. These findings suggest that the gain of survival potential within transitional B cells is dependent on the ability to produce a long-term c-Rel response, which plays a critical role in T2 B cell survival and differentiation in vivo by inducing anti-apoptotic genes, BAFF-R and NF-kappaB2, an essential component for BAFF-R survival signaling. Thus, acquisition of resistance to apoptosis during transitional B cell maturation is achieved by integration of BCR and BAFF-R signals.

DC recognize microbial components through an array of receptors known as PRR. PRR initiate intracellular signals, which engender DC with the capacity to stimulate T-cell responses. Dectin-1 is a PRR that recognizes beta-glucan, a major constituent of many fungi's outer cell wall. Here we show that Dectin-1 activates DC through phospholipase (PLC)gamma2 signaling. PLCgamma2-deficient DC were unable to expand antigen-specific T cells and induce T(H)1 and T(H)17 differentiation in response to beta-glucan. Mechanistically, PLCgamma2-deficiency impaired the capacity of DC to secrete polarizing cytokines following exposure to beta-glucan. Dectin-1 required PLCgamma2 to activate MAPK, AP-1 and NF-kappaB, which induce cytokine gene expression. Moreover, PLCgamma2 controlled Dectin-1-mediated NFAT activation and induction of NFAT-dependent genes such as IL-2, cyclooxigenase-2 and Egr transcription factors. We conclude that PLCgamma2 is a crucial signaling mediator that modifies DC gene expression program to activate DC responses to beta-glucan-containing pathogens.

Prefoldin is a hexameric chaperone that facilitates posttranslational folding of actins and other cytoskeletal proteins by the Tcp1-containing ring complex chaperonin, TriC. The present study characterized mice with a null mutation in Pfdn1, which encodes the first subunit of the Prefoldin complex. Pfdn1-deficient mice displayed phenotypes characteristic of defects in cytoskeletal function, including manifestations of ciliary dyskinesia, neuronal loss, and defects in B and T cell development and function. B and T cell maturation was markedly impaired at relatively early stages, namely at the transitions from pre-pro-B to pre-B cells in the bone marrow and from CD4-CD8- double-negative to CD4+CD8+ double-positive T cells in the thymus. In addition, mature B and T lymphocytes displayed cell activation defects upon Ag receptor cross-linking accompanied by impaired Ag receptor capping in B cells. These phenotypes illustrate the importance of cytoskeletal function in immune cell development and activation.

B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5x10(6) purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1x10(6 )primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population.

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.

A non-toxic, nanoparticulate polyelectrolyte complex (PEC) drug delivery system was formulated to maintain suitable physicochemical properties at physiological pH. Toxicity, binding, and internalization were evaluated in relevant microvascular endothelial cells. PEC were non-toxic, as indicated by cell proliferation studies and propidium iodide staining. Inhibitor studies revealed that PEC were bound, in part, via heparan sulfate proteoglycans and internalized through macropinocytosis. A novel, flow cytometric, Scatchard protocol was established and showed that PEC, in the absence of surface modification, bind cells non-specifically with positive cooperativity, as seen by graphical transformations.

Through their differential interactions with B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), the three BLyS family receptors play central roles in B cell survival and differentiation. Recent evidence indicates BLyS receptor levels shift following BCR ligation, suggesting that activation cues can alter overall BLyS receptor profiles and thus ligand sensitivity. In this study, we show that TLR stimuli also alter BLyS receptor expression, but in contrast to BCR ligation, TLR9 and TLR4 signals, preferentially increase transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) expression. Although both of these TLRs act through MyD88-dependent mechanisms to increase TACI expression, they differ in terms of their downstream mediators and the B cell subset affected. Surprisingly, only TLR4 relies on c-Rel and p50 to augment TACI expression, whereas TLR9 does not. Furthermore, although all follicular and marginal zone B cells up-regulate TACI in response to TLR9 stimulation, only marginal zone B cells and a subset of follicular B cells respond to TLR4. Finally, we find that both BLyS and APRIL enhance viability among quiescent and BCR-stimulated B cells. However, although BLyS enhances viability among TLR stimulated B cells, APRIL does not, suggesting that TACI but not BLyS receptor 3 may share survival promoting pathways with TLRs.

Functional peripheral mature follicular B (FoB) lymphocytes are thought to develop from immature transitional cells in a BCR-dependent manner. We have previously shown that BCR cross-linking in vitro results in death of early transitional (T1) B cells, whereas late transitional (T2) B cells survive and display phenotypic characteristics of mature FoB cells. We now demonstrate that diacylglycerol (DAG), a lipid second messenger implicated in cell survival and differentiation, is produced preferentially in T2 compared with T1 B cells upon BCR cross-linking. Consistently, inositol 1,4,5-triphosphate is also produced preferentially in T2 compared with T1 B cells. Unexpectedly, the initial calcium peak appears similar in both T1 and T2 B cells, whereas sustained calcium levels are higher in T1 B cells. Pretreatment with 2-aminoethoxydiphenylborate, an inhibitor of inositol 1,4,5-triphosphate receptor-mediated calcium release, and verapamil, an inhibitor of L-type calcium channels, preferentially affects T1 B cells, suggesting that distinct mechanisms regulate calcium mobilization in each of the two transitional B cell subsets. Finally, BCR-mediated DAG production is dependent upon Bruton's tyrosine kinase and phospholipase C-gamma2, enzymes required for the development of FoB from T2 B cells. These results suggest that calcium signaling in the absence of DAG-mediated signals may lead to T1 B cell tolerance, whereas the combined action of DAG and calcium signaling is necessary for survival and differentiation of T2 into mature FoB lymphocytes.

Helicobacter pylori infection of the human stomach causes chronic gastritis that can lead to gastric cancer. Because activated lymphocytes persist in the gastric mucosa, and because a high multiplicity of infection (MOI) of H. pylori is needed to induce apoptosis in vitro, we speculated that resistance of lymphocytes to apoptosis is an important feature of the immune response to H. pylori. Freshly isolated mouse splenocytes underwent substantial spontaneous apoptosis and displayed a biphasic response to H. pylori, in which low MOI (1-10) markedly inhibited apoptosis, whereas high MOI (> or =75) potentiated apoptosis. Low MOI reduced mitochondrial membrane depolarization, caspase-3 and caspase-9 activation, and cytochrome c release and increased Bcl-2 levels. Low MOI also induced cellular proliferation. When cells were subjected to fluorescence-activated cell sorting after coculture with H. pylori, CD19+ B cells were found to be protected from apoptosis and undergoing proliferation at low MOI, whereas CD3+ T cells did not exhibit this pattern. The protective effect of low MOI on apoptosis persisted even when B cells were isolated before activation. Immunophenotyping showed that all B-cell subsets examined were protected from apoptosis at low MOI. Additionally, gastric infection with H. pylori resulted in protection of splenic B cells from spontaneous apoptosis. Our results suggest that the low levels of H. pylori infection that occur in vivo are associated with B-cell survival and proliferation, consistent with their potential to evolve into mucosa-associated lymphoid tissue lymphoma.

Phytochemical investigations on the chloroform and ethyl acetate soluble fractions of the roots of Ranunculus repens led to the isolation of methyl 3,4,5-trihydroxybenzoate 1, R(+)- 4-methoxydalbergione 2 and R(+)-dalbergiophenol 3. The structures of these compounds were established through spectral studies including 1D and 2D NMR experiments and by comparison with literature data. These compounds showed potent inhibitory activity against urease.

Transcription factor NF-kappaB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF). In the absence of TNF, NF-kappaB is sequestered in the cytoplasm by inhibitory IkappaB proteins. Phosphorylation of IkappaBby the beta-catalytic subunit of IKK, a multicomponent IkappaB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-kappaB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKbeta, which greatly stimulates IkappaB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IkappaB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121-179 of the IKKgamma regulatory subunit. Third, deletion of the PP2A-binding site in IKKgamma attenuates T loop phosphorylation and catalytic activation of IKKbeta in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK.PP2A complexes is required for the proper induction of IkappaB kinase activity in vivo.

Engagement of the B-cell antigen receptor (BCR) induces the activation of various transcription factors, including NFAT (nuclear factor of activated T-cells) and NF-kappaB (nuclear factor kappaB), which participate in long-term biological responses such as proliferation, survival and differentiation of B-lymphocytes. We addressed the biochemical basis of this process using the DT40 chicken B-cell lymphoma. We discovered that Bruton's tyrosine kinase (BTK) and phospholipase C-gamma2 (PLC-gamma2) are required to activate NFAT and NF-kappaB, and to produce the lipid second messenger diacylglycerol in response to BCR cross-linking. Therefore the functional integrity of the BTK/PLC-gamma2/diacylglycerol signalling axis is crucial for BCR-directed activation of both transcription factors NFAT and NF-kappaB.

BCR engagement initiates intracellular calcium ([Ca2+]i) mobilization which is critical for the activation of multiple transcription factors including NF-kappaB and NFAT. Previously, we showed that Bruton's tyrosine kinase (BTK)-deficient (btk-/-) B cells, which display a modestly reduced calcium response to BCR crosslinking, do not activate NF-kappaB. Here we show that BTK is also essential for the activation of NFAT following BCR engagement. Pharmacological mobilization of [Ca2+]i in BTK-deficient DT40 B cells (DT40.BTK) does not rescue BCR directed activation of NF-kappaB and only partially that of NFAT, suggesting existence of additional BTK-signaling pathways in this process. Therefore, we investigated a requirement for BTK in the production of diacylglycerol (DAG). We found that DT40.BTK B cells do not produce DAG in response to BCR engagement. Pharmacological inhibition of PKC isozymes and Ras revealed that the BCR-induced activation of NF-kappaB requires conventional PKCbeta, whereas that of NFAT may involve non-conventional PKCdelta and Ras pathways. Consistent with an essential role for BTK in the regulation of NFAT, B cells from btk-/- mice display defective expression of CD5, a gene under the control of NFAT. Together, these results suggest that BCR employs distinct BTK-dependent molecular mechanisms to regulate the activation of NF-kappaB versus NFAT.

Mature B-lymphocytes develop sequentially from transitional type 1 (T1) and type 2 (T2) precursors in the spleen. To elucidate the mechanisms that regulate the developmental fate of these distinct B cell subsets, we investigated their biochemical and biological responses following stimulation through the B-cell antigen receptor (BCR). As compared with the T1 subset, T2 cells are more responsive to BCR engagement, as evidenced by their robust induction of activation markers, expression of the prosurvival protein Bcl-x(L), and enhanced proliferation. BCR stimulation of T2 cells leads to the appearance of B cells with mature phenotypic characteristics, whereas T1 cells die. All of these T2 responses are dependent on the BCR signal transducer Bruton's tyrosine kinase, which is dispensable for the T1 to T2 transition. Furthermore, the serine/threonine kinases ERK, p38 MAPK, and Akt are predominantly activated in T2 compared with T1 B cells following BCR cross-linking. We conclude that T1 and T2 B cells respond differentially to BCR engagement via the induction of stage-specific signaling pathways. In turn, these signaling pathways probably govern the development and selection processes that are critical for the formation of the mature B cell compartment.

Disruption of Bruton's tyrosine kinase (BTK) function leads to x-linked immunodeficiency (xid) in mice. BTK-deficient (btk(-/-)) B cells are defective for survival. Prior studies show that BTK is required for the induction of Bcl-x(L) following B cell antigen receptor (BCR) engagement. However, the mechanism underlying Bcl-x(L) induction in response to BCR ligation remains unresolved. We now demonstrate that BTK regulates bcl-x expression by transcriptional control in response to BCR engagement. BTK targets nuclear factor-kappaB (NF-kappaB) to activate the bcl-x promoter via a phospholipase C-gamma2 (PLC-gamma2)-dependent mechanism. Perturbation of the BTK/PLC-gamma2/NF-kappaB signaling axis likely contributes to the defective expression of bcl-x and compromised survival of xid B cells.