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During acute brain injury and/or sterile inflammation, release of danger-associated molecular patterns (DAMPs) activates pattern recognition receptors (PRRs). Microglial toll-like receptor (TLR)-4 activated by DAMPs potentiates neuroinflammation through inflammasome-induced IL-1β and pathogenic Th17 polarization which critically influences brain injury. TLR4 activation accompanies increased CD40, a cognate costimulatory molecule, involved in microglia-mediated immune responses in the brain. During brain injury, excessive release of extracellular ATP (DAMPs) is involved in promoting the damage. However, the regulatory role of CD40 in microglia during ATP-TLR4-mediated inflammasome activation has never been explored. We report that CD40, in the absence of ATP, synergizes TLR4-induced proinflammatory cytokines but not IL-1β, suggesting that the response is independent of inflammasome. The presence of ATP during TLR4 activation leads to NLRP3 inflammasome activation and caspase-1-mediated IL-1β secretion which was inhibited during CD40 activation, accompanied with inhibition of ERK1/2 and reactive oxygen species (ROS), and elevation in p38 MAPK phosphorylation. Experiments using selective inhibitors prove indispensability of ERK 1/2 and ROS for inflammasome activation. The ATP-TLR4-primed macrophages polarize the immune response toward pathogenic Th17 cells, whereas CD40 activation mediates Th1 response. Exogenous supplementation of IFN-γ (a Th1 cytokine and CD40 inducer) results in decreased IL-1β, suggesting possible feedback loop mechanism of inflammasome inhibition, whereby IFN-γ-mediated increase in CD40 expression and activation suppress neurotoxic inflammasome activation required for Th17 response. Collectively, the findings indicate that CD40 is a novel negative regulator of ATP-TLR4-mediated inflammasome activation in microglia, thus providing a checkpoint to regulate excessive inflammasome activation and Th17 response during DAMP-mediated brain injury.

Objective: To observe the effect of CD40 siRNA on expression of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody of systemic lupus erythematosus (SLE) animal model MRL/Lpr mice and to discuss its therapy on MRL/Lpr mice., Methods: In the study, 16 female MRL/Lpr mice were randomly divided into control group (n=4), empty vector group (n=4), CD40-siRNA1 group (n=4) and CD40-siRNA2 group (n=4). The vectors expressing siRNA against CD40 were injected by tail veil into MRL/Lpr mice, while MRL/Lpr mice in control group and empty vector group were injected with the same dose of PBS and pGFP-V-RS vector respectively. The injection was given six times and every one day. The mice were sacrificed 14 d after injection, and the spleen tissue was weighed. The pGFP-V-RS was labeled by green fluorescent protein (GFP) and the tissue sections were observed whether siRNA expressed in the spleen. The expression levels of IFN-γ, IL-17, IL-4 and anti-dsDNA antibody in the sera were detected by ELISA method on the 1st day before the first time and the 2nd, 5th, 8th, 11th, and 14th days after last injection, and the expression levels of CD40 mRNA in spleen tissue of MRL/Lpr mice were detected by RT-PCR and the expression levels of CD40 protein in spleen tissue of MRL/Lpr mice were detected by immunohistochemistry method., Results: The expression vector of CD40-siRNA could express in the spleen of MRL/Lpr. The spleens in CD40-siRNA1 group [(78.85±5.61) mg] and CD40-siRNA2 group [(80.25±4.07) mg] were lower than those in control [(141.88±7.81) mg] and empty vector group [(153.10±7.60) mg]. The levels of IL-17, IFN-γ and anti-dsDNA antibody were lower and the levels of IL-4 was higher in CD40-siRNA1 group and CD40-siRNA2 group on the 2nd, 5th and 8th days after last injection than on the 1st day before the first time (P<0.05). The levels of IFN-γ in CD40-siRNA1 group were (118.74±10.32) ng/L, (115.24±8.26) ng/L and (113.71±5.02) ng/L in turn, the levels of IFN-γ in CD40-siRNA2 group were (117.83±6.83) ng/L, (114.07±0.97) ng/L and (112.67±9.66) ng/L in turn. The levels of IL-17 in CD40-siRNA1 group were (7.05±0.41) ng/L, (6.34±0.76) ng/L and (5.83±0.43) ng/L in turn, the levels of IL-17 in CD40-siRNA2 group were (7.07±0.22) ng/L, (6.35±0.49) ng/L and (6.12±0.80) ng/L in turn. The levels of anti-dsDNA antibody in CD40-siRNA1 group were (7.51±0.29) ng/L, (6.74±0.45) ng/L and (6.32±0.39) ng/L in turn, the levels of anti-dsDNA antibody in CD40-siRNA2 group were (8.19±0.38) ng/L, (7.14±0.50) ng/L and (6.48±0.29) ng/L in turn. The levels of IL-4 in CD40-siRNA1 group were (26.51±1.81)ng/L (27.80±1.72) ng/L and (28.08±2.21) ng/L in turn, the level of IL-4 in CD40-siRNA2 group were (26.28±2.03) ng/L, (28.15±2.95) ng/L and (28.37±1.71) ng/L in turn. The expression levels of IL-17 and IFN-γ antibody increased gradually and the levels of IL-4 decreased gradually in CD40-siRNA1 group and CD40-siRNA2 group on the 11th and 14th days after last injection, then reached to the levels of control group and empty vector group (P>0.05). Though the levels of anti-dsDNA antibody in CD40-siRNA1 group and CD40-siRNA2 group on the 11th day was higher than on the 8th day, there was more significance than those in control group and empty vector group (P<0.05). There was no significance between the 4 groups on the 14th day. The levels of CD40 mRNA and protein were lower in CD40-siRNA1 group and CD40-siRNA2 group than in control group and empty vector group on the 14th day after last injection (P<0.05)., Conclusion: CD40-siRNA can reduce the concentration of IL-17, IFN-γ and of anti-dsDNA antibody in serum, and at the same time, it can elevate the concentration of IL-4 and suppress CD40 mRNA and protein of spleen in MRL/Lpr. Meanwhile after suppressing CD40 mRNA and protein, it can reduce inflammatory response of the mice and the disease activity of MRL/Lpr, suggesting that CD40-siRNA has therapy effect on SLE.

The CD40-mediated immune response contributes to a wide variety of chronic inflammatory diseases. CD40 antagonists have potential as novel therapies for immune disorders. However, the CD40 pathway has not been well characterized in the rhesus monkey Macaca mulatta, which is a valuable animal model for human immune disease. An 834 bp transcript was cloned from peripheral blood mononuclear cells (PBMCs) of rhesus monkey using specific primers designed according to the predicted sequence of M. mulatta CD40 (mmCD40) in GenBank. Sequence analysis demonstrated that mmCD40 is highly homologous to human CD40 (hCD40), with an amino acid sequence identity of 94%. Genes encoding the extracellular domain of mmCD40 and the Fc fragment of the hIgG1 were inserted into a pPIC9K plasmid to produce mmCD40Ig by Pichia pastoris. Approximately 15-20 mg of the mmCD40Ig protein with ∼90% purity could be recovered from 1 L of culture. The purified mmCD40Ig protein can form dimers and can specifically bind CD40L-positive cells. Additionally, the mmCD40Ig protein can bind hCD40L protein in phosphate buffered saline and form a stable combination in a size-exclusion chromatography assay using a Superdex 200 column. Moreover, mmCD40Ig is as efficient as M. mulatta CTLA4Ig (mmCTLA4Ig) to suppress Con A-stimulated lymphocyte proliferation. Additionally, mmCD40Ig only showed mild immunosuppressive activity in a one-way mixed lymphocyte reaction (MLR) system. These results suggest that mmCD40Ig secreted by P. pastoris was productive and functional, and it could be used as a tool for pathogenesis and therapies for chronic inflammatory diseases in a M. mulatta model., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: B cells play an important role in the pathogenesis of autoimmune diseases. The role of Bruton's tyrosine kinase (Btk) in cytokine-induced human B cell differentiation and class-switch recombination remains incompletely defined. This study analysed the effect of Btk on human activated B cells., Methods: Purified B cells from healthy subjects were stimulated with B cell receptor (BCR) and other stimuli with or without a Btk inhibitor and gene expression was measured. The B cell line BJAB was used to assess Btk-associated signalling cascades. Phosphorylated Btk (p-Btk) in peripheral blood B cells obtained from 10 healthy subjects and 41 patients with RA was measured by flow cytometry and compared with patient backgrounds., Results: IL-21 signalling, in concert with BCR, CD40 and BAFF signals, led to robust expression of differentiation- and class-switch DNA recombination-related genes and IgG production in human B cells, all of which were significantly suppressed by the Btk inhibitor. Although phosphorylation of STAT1 and STAT3 was induced by co-stimulation with IL-21, BCR and CD40, STAT1 phosphorylation in the nucleus, but not in the cytoplasm, was exclusively impaired by Btk blockade. High levels of p-Btk were noted in B cells of RA patients compared with controls and they correlated significantly with titres of RF among RF-positive patients., Conclusion: The findings elucidate a model in which Btk not only plays a fundamental role in the regulation of BCR signalling, but may also mediate crosstalk with cytokine signalling pathways through regulation of IL-21-induced phosphorylation of STAT1 in the nuclei of human B cells. Btk appears to have pathological relevance in RA., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The interaction between the transmembrane glycoprotein surface receptor CD40 expressed by skin epithelial cells (ECs) and its T-cell-expressed ligand CD154 was suggested to exacerbate inflammatory skin diseases. However, the full spectrum of CD40-mediated effects by ECs underlying this observation is unknown. Therefore, changes in gene expression after CD40 ligation of ECs were studied by microarrays. CD40-mediated activation for 2 hours stimulated the expression of a coordinated network of immune-involved genes strongly interconnected by IL8 and TNF, whereas after 24 hours anti-proliferative and anti-apoptotic genes were upregulated. CD40 ligation was associated with the production of chemokines and the attraction of lymphocytes and myeloid cells from peripheral blood mononuclear cells (PBMCs). Thus, CD40-mediated activation of ECs resulted in a highly coordinated response of genes required for the local development and sustainment of adaptive immune responses. The importance of this process was confirmed by a study on the effects of human papilloma virus (HPV) infection to the EC's response to CD40 ligation. HPV infection clearly attenuated the magnitude of the response to CD40 ligation and the EC's capacity to attract PBMCs. The fact that HPV attenuates CD40 signaling in ECs indicates the importance of the CD40-CD154 immune pathway in boosting cellular immunity within epithelia.

We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent "bystander-activated" (CD8(+)CD44high) T cells displaying a CD25(-)NKG2D(+) phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4(+) T cell help for antigen-specific CD8(+) T cell expansion, little is known regarding the role of CD4(+) T cells in antigen-nonspecific bystander-memory CD8(+) T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8(+) T cells upregulated PD-1 in the absence of CD4(+) T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8(+) T cells. Interestingly, compared to CD8(+) T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8(+) response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8(+) T cell expansion, CD4(+) T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8(+) T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.

The role of macrophages and their interactions with T cells during aging is not well understood. We determined if activating elderly-derived macrophages could rescue age-related and tumor-induced T cell dysfunction. Healthy elderly (18-24 months) Balb/c contained significantly more splenic IL-10-secreting M2-macrophages and myeloid-derived suppressor cells than young (6-8 weeks) mice. Exposure to syngeneic mesothelioma or lung carcinoma-conditioned media polarized peritoneal macrophages into suppressive M2-macrophages regardless of age. Tumor-exposed, elderly, but not young-derived, macrophages produced high levels of IL-4 and could not induce T cell IFN-γ production. We attempted to rescue tumor-exposed macrophages with LPS/IFN-γ (M1 stimulus) or IL-2/agonist anti-CD40 antibody. Tumor-exposed, M1-stimulated macrophages retained high CD40 expression, yet TNF-α and IFN-γ production were diminished relative to non-tumor-exposed, M1-stimulated controls. These macrophages induced young and elderly-derived T cell proliferation however, T cells did not secrete IFN-γ. In contrast, tumor-exposed, IL-2/CD40-stimulated macrophages rescued elderly-derived T cell IFN-γ production, suggesting that IL-2/CD40-activated macrophages could rescue T cell immunity in aging hosts.

The two tumour necrosis factor family proteins BAFF (TNFSF13B) and APRIL (TNFSF13) and their receptors [BAFF-R (TNFRSF13C), TACI (TNFRSF13B), BCMA (TNFRSF17)] play a critical role in the survival of normal B cells. The sensitivity of normal B cells to BAFF and APRIL can be modulated by signals regulated by their receptors. This modulation, however, has not been extensively investigated in chronic lymphocytic leukaemia (CLL) cells. We evaluated the expression, regulation and signalling of BAFF and APRIL receptors in normal and in CLL cells upon stimulation through CD40+IL4R and BCR. We further analysed the prognostic value of BAFF and APRIL receptors expression in patients with CLL. BCMA expression was significantly higher on CLL cells than on normal B cells. BCR and CD40+IL4R stimulation promoted an increase in TACI and BCMA expression, cell viability and activation in normal B cells. A similar effect was observed in CLL cells after CD40+IL4R but not BCR stimulation. BCMA expression correlated with unmutated IGHV genes, poor-risk cytogenetics, and short progression-free survival. These findings further characterize the link between CD40+IL4R regulatory signals, BAFF, APRIL and their receptors and the survival of leukaemic cells and clinical features of CLL., (© 2013 John Wiley & Sons Ltd.)

While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4(lo)CD40(+) effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4(lo)CD40(+) T cells. Understanding the dynamics of the CD40 receptor(s) and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones.

Programmed death-1 (PD-1) is an immunoreceptor predominantly expressed on exhausted T cells, which through an interaction with its ligand (PD-L1), controls peripheral tolerance by limiting effector functions of T lymphocytes. qRT-PCR for PD-1, PD-L1 and their splicing forms as well as flow cytometric assessment of surface expression was performed in a cohort of 58 chronic lymphocytic leukemia (CLL) patients. In functional studies, we assessed the influence of the proliferative response of leukemic B-cells induced by IL-4 and CD40L on PD-1 transcripts and expression on the protein level. The median level of PD-1, but not PD-L1, transcripts in CLL patients was higher in comparison to healthy volunteers (HVs, n = 43, p = 0.0057). We confirmed the presence of PD-1 and PD-L1 on the CLL cell surface, and found the expression of PD-1, but not PD-L1, to be higher among CLL patients in comparison to HVs (47.2% vs. 14.8%, p<0.0001). The Kaplan-Meier curves for the time to progression and overall survival in groups with high and low surface expression of PD-1 and PD-L1 revealed no prognostic value in CLL patients. After stimulation with IL-4 and CD40L, protein expression of PD-1 was significantly increased in samples that responded and up-regulated CD38. PD-1, which is aberrantly expressed both at mRNA and cell surface levels in CLL cells might represent a novel immunotolerant molecule involved in the pathomechanism of the disease, and could provide a novel target for future therapies.

Aims/hypothesis: The role of Toll-like receptor 7 (TLR7), a sensor of viral and self RNA, in promoting autoimmune diabetes remains unclear. Our goal was to determine the effect of TLR7 stimulation on the priming and activation of diabetogenic CD8(+) T cells., Methods: We explored the effects of CL097 (TLR7/8 agonist) and immunoregulatory sequence 661 (IRS661, TLR7 inhibitor) on bone marrow-derived dendritic cells (BMDCs), diabetogenic CD8(+) T cell function and autoimmune diabetes onset in NOD and 8.3 NOD T cell receptor transgenic mice (8.3 NOD mice)., Results: TLR7 stimulation of NOD BMDCs increased activation and production of proinflammatory cytokines. In vivo administration of CL097 activated T cells and dendritic cells and increased levels of proinflammatory cytokines and type 1/2 IFNs in NOD mice. In vivo antigen-specific cytotoxicity studies revealed enhanced cytotoxicity against islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, an islet autoantigen) peptide pulsed targets in NOD mice treated with CL097 plus CD40 agonist. This combination treatment accelerated the onset of autoimmune diabetes in 8.3 NOD mice. Likewise, topical treatment of NOD mice with a TLR7 agonist accelerated diabetes onset. Spontaneous disease in 8.3 NOD mice and accelerated disease in CL097+CD40 agonist-treated 8.3 NOD mice were delayed by IRS661 treatment, which is associated with inhibition of the endogenous upregulation of IFN-α levels within the pancreatic lymph nodes., Conclusions/interpretation: TLR7 stimulation accelerates the spontaneous onset of autoimmune diabetes in 8.3 NOD and NOD mice. Conversely, TLR7 inhibition prevents the early events associated with diabetogenesis.

CD40Ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. CD40L induces activation of antigen-presenting cells (APCs) such as DCs, monocytes, B-cells and endothelial cells. However, CD40 activation alone, whilst inducing IL-10 production, is insufficient to induce interleukin (IL)-12p70 release in human APCs suggesting that additional cytokine signals (e.g. GM-CSF, IL-4 or IFN-γ) are required for the induction of a pro-inflammatory cytokine profile. We demonstrate that IFN-γ-induced Janus kinase 1 (JAK1) enhances CD40-induced IL-12p70 release whilst simultaneously inhibiting IL-10 synthesis, resulting in a pro-inflammatory phenotype of CD40L-activated dendritic cells (DCs). JAK2 mediated enhancing effects on IL-12p70 but did not inhibit IL-10 release, whereas Tyk2 mediated inhibitory effects on IL-12p70 release in this system. The mechanism by which complementary IFN-γ/JAK activities affect IL-12p70 production involves STAT1 activation and de novo induction of interferon-responsive factors (IRF)-1 and IRF-8. Simultaneously, JAK1 was unique in inhibiting IL-10 synthesis via STAT1 and IRF-8 with both transcription factors binding to the IL-10 promoter. We demonstrate that CD40- and JAK/STAT/IRF-signalling pathways are strictly complementary for the induction of a pro-inflammatory cytokine profile in human APCs. This suggests that a number of CD40 effects in chronic inflammatory diseases might be weakened by targeting JAK/STAT., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Evolution of apoptosis resistance in both lymphoma and leukemia cells is well documented, and induction of apoptosis in malignant cells is a major goal of cancer therapy. Up-regulation of anti-apoptotic signals is one of the mechanisms whereby resistance to apoptosis emerges. We have previously described the fusion proteins CD40·FasL and CTLA-4·FasL, which are formed from two functional membrane proteins and induce apoptosis of activated T cells. The present study explores the potential use of CD40·FasL and CTLA-4·FasL for the killing of malignant cells of lymphatic origin. Using malignant B and T cell lines that differ in surface expression of costimulatory molecules, we found that CTLA-4·FasL induces effective apoptosis of cells expressing CD95 and activates caspases 3, 8, and 9. Only B7-expressing B cells responded to CTLA-4·FasL with rapid abrogation of cFLIP expression. CD40·FasL effectively killed only the T cells that express high levels of CD40L in addition to CD95. In these cells, CD40·FasL significantly diminished cFLIP expression. Importantly, each of the fusion proteins is more potent than its respective components parts, alone or in combination. Thus, the proteins with their two functional ends deliver a pro-apoptotic signal and, in parallel, inhibit an anti-apoptotic signal, thus optimizing the wanted, death-inducing effect. Therefore, these proteins emerge as promising agents to be used for targeted and specific tumor cell killing.

Autophagy is a homeostatic mechanism of lysosomal degradation. Defective autophagy has been linked to various disorders such as impaired control of pathogens and neurodegeneration. Autophagy is regulated by a complex array of signaling pathways that act upstream of autophagy proteins. Little is known about the role of altered regulatory signaling in disorders associated with defective autophagy. In particular, it is not known if pathogens inhibit autophagy by modulation of upstream regulatory pathways. Cells infected with HIV-1 blocked rapamycin-induced autophagy and CD40-induced autophagic killing of Toxoplasma gondii in bystander (non-HIV-1 infected) macrophage/monocytic cells. Blockade of autophagy was dependent on Src-Akt and STAT3 triggered by HIV-1 Tat and IL-10. Neutralization of the upstream receptors VEGFR, beta-integrin or CXCR4, as well as of HIV-1 Tat or IL-10 restored autophagy in macrophage/monocytic cells exposed to HIV-1-infected cells. Defective autophagic killing of T. gondii was detected in monocyte-derived macrophages from a subset of HIV-1(+) patients. This defect was also reverted by neutralization of Tat or IL-10. These studies revealed that a pathogen can impair autophagy in non-infected cells by activating counter-regulatory pathways. The fact that pharmacologic manipulation of cell signaling restored autophagy in cells exposed to HIV-1-infected cells raises the possibility of therapeutic manipulation of cell signaling to restore autophagy in HIV-1 infection.

B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies. However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10 secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the classical NF-kappaB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-10(-/-) mice showed better clearance of Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity.

Keratinocytes expressing tumor or viral antigens can be eliminated by antigen-primed CD8 cytotoxic T cells. CD4 T-helper cells help induction of CD8 cytotoxic T cells from naive precursors and generation of CD8 T-cell memory. In this study, we show, unexpectedly, that CD4 cells are also required to assist primed CD8 effector T cells in rejection of skin expressing human growth hormone, a neo-self-antigen, in keratinocytes. The requirement for CD4 cells can be substituted by CD40 costimulation. Rejection of skin expressing ovalbumin (OVA), a non-self-antigen, by primed CD8 cytotoxic T cells can in contrast occur without help from antigen-specific CD4 T cells. However, rejection of OVA expressing keratinocytes is helped by antigen-specific CD4 T cells if only low numbers of primed or naive OVA-specific CD8 T cells are available. Effective immunotherapy directed at antigens expressed in squamous cancer may therefore be facilitated by induction of tumor antigen-specific CD4 helper T cells, as well as cytotoxic CD8 T cells.

Follicular lymphoma (FL) cells are malignant counterparts of germinal centre (GC) B cells. Microenvironment of FL B cells has an important role in the progression of FL and might also have an impact on the treatment of FL. CD40 is an important mediator of microenvironmental survival signals in GCs. Here we studied responses of CD40 signalling on TRAIL-, dexamethasone- and doxorubicin-induced apoptosis in three human FL cell lines. In two of the FL cell lines, CD40 protected cells from apoptosis which was entirely dependent on the activation of NF-kappaB. In one of the FL cell lines, CD40 induced apoptosis itself. However, inhibition of NF-kappaB induced apoptosis in all three FL cell lines. Therefore, our results indicate that inhibitors of NF-kappaB or critical downstream anti-apoptotic targets of NF-kappaB instead of blocking CD40 antibodies in combination with TRAIL or other cytotoxic agents should be considered in the treatment of FL in order to prevent the protective effect of the microenvironment.

The Emu-TCL1 transgenic mouse spontaneously develops a CD5(+) B cell lymphoproliferative disorder similar to human chronic lymphocytic leukemia (CLL). Given the ineffectual T cell antitumor responses in this mouse model of CLL, we sought to determine whether combined treatment with anti-CD40 mAb (alphaCD40) and CpG-containing oligodeoxynucleotides (CpG) could exert immunotherapeutic effects. We have previously shown that macrophages activated by sequential ligation of CD40 and TLR9 could become cytotoxic against solid tumor cell lines both in vitro and in vivo. In the current study, we find that alphaCD40 plus CpG-activated macrophages induce tumor B cell apoptosis in vitro and that alphaCD40 plus CpG treatment markedly retards tumor growth in immunodeficient SCID/Beige mice following transplantation of primary tumor B cells. Our results suggest a novel immunotherapeutic strategy for CLL that may be effective even in the face of tumor or chemotherapy-induced T cell immunodeficiency.

A gastric cancer (GC) cell line, AGS, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, and cell death. In this research, we studied the effects of different forms of CD40 stimulation on AGS cells by flow cytometry, Western blotting and siRNA transfection. We found that different forms of CD40 stimulation, either recombinant soluble CD40L (sCD40L, ligation) or agonist anti-CD40 antibody (cross-linking), induced different effects in AGS gastric cancer cells, proliferation or apoptosis. We also showed that VEGF provided a significant contribution to sCD40L-induced proliferation, while agonist anti-CD40 antibody induced GADD45 upregulation and promoted apoptosis.

TLRs are involved in the regulation of immune responses. R-848, a TLR7/8 ligand, has potent anti-viral and anti-tumour properties and has been used as a new immune response modifier for enhancing Th1 immune response. In this study, we found that R-848 significantly inhibited IgE synthesis from murine B cells at the single cell levels by anti-CD40 plus IL-4-stimulated splenocytes, in which R-848 acted on the early stage of B cell differentiation to modulate IgE synthesis. This inhibitory effect of R-848 on IgE synthesis was not isotype specific as it also inhibited IgG1 synthesis. Moreover, R-848 had no significant effect on the production of IgG2a by anti-CD40 plus IL-4-stimulated splenocytes. Further studies showed that R-848 markedly promoted murine B cell activation induced by anti-CD40 plus IL-4 by up-regulating the expression of B cell activation markers CD25, CD69 and co-stimulatory molecule CD80. In contrast, R-848 inhibited the proliferation and division of murine B cells in anti-CD40 plus IL-4-stimulated splenocytes. R-848 promoted the production of IFN-gamma and IL-12 that were partially responsible for its inhibitory effect on IgE production by anti-CD40 plus IL-4 because the addition of anti-IFN-gamma or anti-IL-12 mAbs to the cultures could significantly restore IgE production by splenocytes. Importantly, R-848 had a direct effect on purified B cells to inhibit IgE production induced by anti-CD40 plus IL-4. Taken together, these results demonstrate that R-848 markedly inhibits IgE synthesis, and suggest that R-848 could be used to treat allergic diseases.

The recently discovered nucleotide binding domain-leucine rich repeat (NLR) gene family is conserved from plants to mammals, and several members are associated with human autoinflammatory or immunodeficiency disorders. This family is defined by a central nucleotide binding domain that contains the highly conserved Walker A and Walker B motifs. Although the nucleotide binding domain is a defining feature of this family, it has not been extensively studied in its purified form. In this report, we show that purified Monarch-1/NLRP12, an NLR protein that negatively regulates NF-kappaB signaling, specifically binds ATP and exhibits ATP hydrolysis activity. Intact Walker A/B motifs are required for this activity. These motifs are also required for Monarch-1 to undergo self-oligomerization, Toll-like receptor- or CD40L-activated association with NF-kappaB-inducing kinase (NIK) and interleukin-1 receptor-associated kinase 1 (IRAK-1), degradation of NIK, and inhibition of IRAK-1 phosphorylation. The stable expression of a Walker A/B mutant in THP-1 monocytes results in increased production of proinflammatory cytokines and chemokines to an extent comparable to that in cells in which Monarch-1 is silenced via short hairpin RNA. The results of this study are consistent with a model wherein ATP binding regulates the anti-inflammatory activity of Monarch-1.

CXCL8 is a CXC chemokine that recruits leukocytes to sites of inflammation. Expression of CXCL8 in the CNS has been demonstrated in neuroinflammatory diseases, including human immunodeficiency virus (HIV-1) encephalitis, but the mechanism of secretion of this chemokine is not fully understood. CD40 is a 50-kDa protein on the surface of microglia, and we have previously shown that it is increased in expression in HIV-1-infected brain tissue as well as by interferon-gamma (IFNgamma) in tissue culture. We examined the expression and regulation of CXCL8 in cultured human fetal microglia after ligation of CD40 with soluble trimeric CD40 ligand (sCD40L) as well as the expression of CXCL8 on microglia in HIV encephalitic brain tissue sections. Treatment of cultured microglia with IFNgamma + sCD40L resulted in significant induction of CXCL8. This expression was mediated by activation of the ERK1/2 MAPK pathway, as demonstrated by ELISA and Western blot using a specific inhibitor (U0126). Gel shift analyses demonstrated that NFkappaB and AP-1, but not C/EBPbeta, mediate microglial CXCL8 production. We also found increased colocalization of CXCL8 with CD68/CD40-positive cells in HIV encephalitic brain tissue compared with HIV-infected nonencephalitic and normal tissue. Thus, CD40-CD40L interactions facilitate chemokine expression, leading to the influx of inflammatory cells into the CNS. These events can lead to the pathology that is associated with neuroinflammatory diseases., ((c) 2007 Wiley-Liss, Inc.)

Aim: To explore the optimal in vitro culture condition for producing antibodies to HLA class I molecules by human B lymphocyte cells., Methods: By applying EBV-transformed B lymphoblast cell line (BLCL) from a sensitized kidney transplanted subject as a model, the capacity of Ig and specific HLA class I antibody production of BLCL cells was investigated by ELISA and ELISpot, under three different culture systems, RPMI1640+OPTI mAb, RPMI1640+0.1 mmol/L non-essential amino acid+1 mmol/L sodium pyruvate+40 mg/L apo-transferrin,and serum free medium (SFM) for hybridoma culture, and three different culture conditions, culture medium only, culture medium supplemented with IL-4, IL-10 and alpha-CD40, and culture medium supplemented with IL-4, IL-10 and CD40L., Results: When BLCL cells were cultured in Hybridoma SFM, the IgG and IgM concentrations in culture supernatants were 25.2-28.1 mg/L and 7.14-7.95 mg/L, respectively, which were higher than that of the other two culture systems (P<0.05). Moreover, when Hybridoma SFM was supplemented with IL-4, IL-10 and alpha-CD40 or CD40L, the frequentcies of HLA-I antibody-secreting BLCL cells were 3.06/10(4) BLCL cells and 3.55/10(4) BLCL cells,which were much higher than that of SFM only group (1.77/10(4) BLCL cells)., Conclusion: BLCL cells can produce both higher amount of Ig and specific HLA-I antibodies under Hybridoma SFM supplimented with IL-4, IL-10 and alpha-CD40 or CD40L.

Objective: Intracellular tumor necrosis factor receptor-associated factors (TRAFs) translocation to lipid rafts is a key element in CD40-induced signaling. The purpose of this study was to investigate the influence of anthocyanin on CD40-mediated proinflammatory events in human endothelial cells and the underlying possible molecular mechanism., Methods and Results: Treatment of endothelial cells with anthocyanin prevented from CD40-induced proinflammatory status, measured by production of IL-6, IL-8, and monocyte chemoattractant protein-1 through inhibiting CD40-induced nuclear factor-kappaB (NF-kappaB) activation. TRAF-2 played pivotal role in CD40-NF-kappaB pathway as TRAF-2 small interference RNA (siRNA) diminished CD40-induced NF-kappaB activation and inflammation. TRAF-2 overexpression increased CD40-mediated NF-kappaB activation. Moreover, TRAF-2 almost totally recruited to lipid rafts after stimulation by CD40 ligand and depletion of cholesterol diminished CD40-mediated NF-kappaB activation. Exposure to anthocyanin not only interrupted TRAF-2 recruitment to lipid rafts but also decreased cholesterol content in Triton X-100 insoluble lipid rafts. However, anthocyanin did not influence the interaction between CD40 ligand and CD40 receptor., Conclusions: Our findings suggest that anthocyanin protects from CD40-induced proinflammatory signaling by preventing TRAF-2 translocation to lipid rafts through regulation of cholesterol distribution, which thereby may represent a mechanism that would explain the anti-inflammatory response of anthocyanin.

Germline (GL) transcription is regulated by specific promoters and immunoglobulin heavy chain (IgH) 3' locus enhancers and is necessary for Ig class-switch recombination (CSR). We have generated different transgenic lines containing the GL epsilon promoter, switch (S) epsilon region, and constant (C) epsilon region with or without the DNase I-sensitive regions (HS) 3A-HS1,2 or HS3B-HS4 3' IgH enhancer pairs. The enhancerless construct was expressed in B cells activated by interleukin (IL)-4 and CD40, thus resembling regulation of the endogenous gene. Both enhancer-containing transgenes efficiently increased expression in B cells and were strongly up-regulated by stimuli. In addition, Sepsilon regions of the transgene containing HS3B-HS4 were mutated in activated, sorted B cells. Such mutations are known to precede CSR and are dependent on activation-induced cytidine deaminase (AID). Our findings show that all elements necessary for recruitment of the recombination machinery are present in the transgene containing HS3 and HS4. These enhancers probably provide something more specific than mere increased accessibility of switch regions. We propose that transcription factors binding the enhancers help to target the recombination machinery to the switch regions.

NF-kappaB activation through B cell receptor (BCR) ligation is critical for B cell development, survival and antigen-mediated activation of B cells. CARD domain and MAGUK-domain containing protein-1 (CARMA1), recently identified adaptor molecule, has been shown to play an essential role in BCR-induced NF-kappaB activation. CARMA1-deficient B cells fail to proliferate upon BCR stimulation, leading to defective humoral responses. Surprisingly, CARMA1-deficient B cells are also defective in CD40-induced proliferation. The mechanisms responsible for CD40-induced proliferation defect have not yet been characterized. In this study, we show that signaling cascades activated by CD40 stimulation are largely unaffected in CARMA1-deficient B cells. Instead, we have found that the defective proliferation of CARMA1-deficient B cells is due to two events. First, CARMA1-deficient B cells show defective cell-cycle progression. Secondly, the numbers of marginal zone (MZ) B cells, which are the main responders upon CD40 stimulation, are greatly diminished in CARMA1-deficient mice. Since B cell maturation requires basal signaling through BCR and NF-kappaB activation, we propose that impaired BCR signaling in CARMA1-deficient mice leads to defective maturation of MZ B cell population, which in turn, contributes to impaired proliferation upon CD40 stimulation.

CD40 and its ligand regulate pleiotropic biological responses, including cell proliferation, differentiation, and apoptosis. In many inflammatory lung diseases, tissue damage by environmental or endogenous oxidants plays a major role in disease pathogenesis. As the epithelial barrier is a major target for these oxidants, we postulated that CD40, the expression of which is increased in asthma, plays a role in the regulation of apoptosis of bronchial epithelial cells exposed to oxidants. Using 16HBE 14o- cells exposed to oxidant stress, we found that ligation of CD40 (induced by G28-5 monoclonal antibodies) enhanced cell survival and increased the number of cells in G2/M (interphase between DNA synthesis and mitosis) of the cell cycle. This was associated with NF-kappaB and activator protein-1 activation and increased expression of the inhibitor of apoptosis, c-IAP1. However, oxidant stress-induced apoptosis was found to be caspase- and calpain-independent implicating CD40 ligation as a regulator of caspase-independent cell death. This was confirmed by the demonstration that CD40 ligation prevented mitochondrial release and nuclear translocation of apoptosis inducing factor. In conclusion, we demonstrate a novel role for CD40 as a regulator of epithelial cell survival against oxidant stress. Furthermore, we have identified, for the first time, an endogenous inhibitory pathway of caspase-independent cell death.

CD40 is critically involved in Fas-mediated cholangiocyte apoptosis during liver inflammation, but the underlying signalling events are poorly understood. Our recent work implicated AP-1 in CD40-induced cholangiocyte apoptosis, but suggested involvement of other signalling pathways. Because STAT3 has been implicated in liver regeneration we investigated this signalling pathway during CD40 mediated cholangiocyte apoptosis. Western immunoblotting, electrophoretic mobility gel shift assays, In situ DNA end labelling and caspase-3 activity were used to investigate intracellular signalling and apoptosis in primary human cholangiocytes following CD40 activation. CD40-activation induced caspase-3 dependent cholangiocyte apoptosis and 3-fold increases in JNK/ERK phosphorylation (concomitant with increased AP-1 binding activity) and 4-fold increases in pSTAT3, which were sustained for up to 24 h. Protein levels of c-Jun, c-Fos and pSTAT3 confirmed the upregulation. Phosphorylation of p38 remained unchanged suggesting that this MAP kinase was not involved in CD40 mediated apoptosis. Increased JAK2 phosphorylation accompanied increased STAT3 phosphorylation after CD40 ligation. Cholangiocytes were also shown to express JAK1 and 3 which was phosphorylated following control stimulation with TNFalpha or IL2 respectively but not after CD40 ligation. JNK, ERK and JAK2 inhibitors partially abrogated apoptosis and when used in combination reduced it to basal levels. In conclusion, induction of CD40-mediated cholangiocyte apoptosis requires JAK2-mediated phosphorylation of STAT3 as well as sustained JNK1/2, ERK1/2 activation. This study demonstrates that STAT3 can function as a proapoptotic factor in primary human liver epithelial cells.

Aims: CD40 expression is restricted to Keratinocytes of normal epidermis or stratified squamous epithelium of oral mucosa. Ligation of CD40 inhibits keratinocyte proliferation and apoptosis. The aim of this study was to investigate the functional significance of CD40 in the proliferation, apoptosis, adhesion and migration of human oral keratinocytes in vitro., Methods: The CD40-negative oral keratinocyte line OSC19, its CD40-positive transfected derivative (OSC19T-CD40) and null transfectants (OSC19T-control), with and without stimulation by soluble protein CD40 ligand (sCD40L) or anti-CD40 antibodies were used., Results: OSC19T-CD40 showed significantly (P < 0.001) slower growth than the null transfectants and parent cells. OSC19T-CD40 proliferation was inhibited by ligation with sCD40L and blocking by two anti-CD40 antibodies, but stimulated by a third. Binding of CD40 with ligand or antibody had no effect on keratinocyte apoptosis in any cell line. The capacity of OSC19T-CD40 cells to adhere to CD40L-coated wells was significantly greater (P < 0.001) than that of parent OSC19 and OSC19T-control cells, and the migration rate of OSC19T-CD40 cells was significantly higher than parent OSC19 (P = 0.038 on fibronectin, P = 0.004 on Matrigel) or OSC19T-control (P =0.017 on fibronectin, P = 0.013 on Matrigel) cells., Conclusions: CD40 is an important molecule in keratinocyte homeostasis, and has more than one ligand. The ligand that is bound may be critical in oral epithelial homeostasis, the development of malignancy and the behaviour of the subsequent tumour.

CD40-CD154 interaction is pivotal for resistance against numerous pathogens. However, it is not known if this pathway can also enhance in vivo resistance in gamma interferon (IFN-gamma)-deficient hosts. This is an important question because patients and mice with defects in type 1 cytokine response can control a variety of pathogens. While blockade of endogenous CD154 resulted in a remarkable increase in parasite load in IFN-gamma-/- mice infected with Toxoplasma gondii, in vivo administration of a stimulatory anti-CD40 monoclonal antibody markedly reduced parasite load. This latter effect took place even in T-cell-depleted mice and was accompanied by induction of macrophage toxoplasmacidal activity. CD40 stimulation restricted T. gondii replication independently of STAT1, p47 GTPases, and nitric oxide. In vivo CD40 ligation enhanced tumor necrosis factor alpha (TNF-alpha) production by T. gondii-infected macrophages. In addition, CD40 stimulation required the presence of TNF receptor 2 to reduce parasite load in vivo. These results suggest that CD40-CD154 interaction regulates IFN-gamma-independent mechanisms of host protection through induction of macrophage antimicrobial activity and modulation of TNF-alpha signaling.

Several myeloid leukemia-derived cells have been reported to possess the ability to differentiate into dendritic cells (DC). MUTZ-3, a myeloid leukemia cell line, responds to GM-CSF, IL-4 and TNF-alpha, and acquires a phenotype similar to immature monocyte-derived DC (MoDC). In the present study, MUTZ-3-derived DC (MuDC) showed high level expression of HLA class II molecules, CD80 and CD86, and were able to function as potent antigen presenting cells as previously reported. Interestingly, MuDC maturation was induced by CD40- mediated stimulation, but not by LPS stimulation. We analyzed CCR1, CCR7 and Toll-like receptor (TLR) expressions in MuDC, and measured IL-10 and IL-12 production after maturation stimuli. Although MuDC expressed the mRNA for TLR4, a major component of the LPS receptor system, they did not show an enhanced level of CCR7 or cytokine production after LPS stimulation. In contrast, they responded to CD40 stimulation, which resulted in increased levels of CD83, CD86 and CCR7. Moreover, while LPS- stimulated MoDC could potently stimulate NK cells in a DC-NK cell co-culture, LPS-stimulated MuDC failed to stimulate primary NK cells. Taken together, our findings suggest that, although MuDC express TLR4, unlike TNF-alpha and IL-1beta, LPS does not stimulate MuDC to acquire mature phenotypes, and they may have impaired activity to initiate innate immune response.

Objective: Production of cytokines that support T-cell activation and proliferation and migration to lymph nodes is one of the most important terms of cancer vaccine development. In previous studies we and others used CD40 ligation to obtain higher expression of co-stimulatory and adhesion molecules on leukaemic cells from children with acute lymphoblastic leukaemia (ALL). This time we assess the cytokine and chemokine gene expression profile in CD40-stimulated ALL cells., Material and Methods: Malignant cells from 25 children with BCP-ALL were stimulated (or not) with huCD40LT and rIL-4 for 96 h. Eleven different molecule, cytokine and chemokine mRNAs levels (CCR7, IL-23, TGF-beta-IP, IFN-gamma, IL-10, CD1a, CD40, CD54, CD80, CD83, CD86) were determined using the real-time PCR technique with TaqMan chemistry using ready-to-use low-density arrays for gene expression by Applied Biosystems., Results: 1) Increases in mRNA levels for CD40, CD54 and CD80 after CD40L and IL-4 stimulation were observed, 2) CCR7 mRNA expression was higher after CD40 ligation than before the culture (p = 0.002), 3) IL-10 mRNA expression was higher after the culture with medium than before the culture (p = 0.01)., Conclusions: The results show that leukaemia-derived dendritic cells obtained with CD40 ligation express CCR7 - chemokine is involved in migration to lymph nodes and does not produce higher amounts of IL-10, a potent immunosuppressive cytokine. Our preclinical findings could be used in the design of immunotherapy trials for the treatment of children with ALL.

Background: We previously reported that adenovirus mediated CD40Ig gene therapy (AdCD40Ig) induced long-term acceptance of fully allogeneic rat cardiac allografts, however, the underlying mechanism has not been fully clarified. To address this we have compared the ability of dimeric and monomeric soluble CD40 to prolong allograft survival in vivo and generate regulatory T cells in vitro., Methods: The ability of CD40Ig (soluble dimmer, containing an Fc region) or CD40/Myc/His (soluble monomer, lacking an Fc region) therapy to generate CD4CD25 regulatory T cells in vitro and to prevent rejection of rat cardiac allografts (ACI to LEWIS) was compared. Immunoregulatory capacity of regulatory T cells generated was determined by suppression of alloantigen specific proliferation and cytotoxicity., Results: Dimeric soluble CD40Ig did not inhibit CD4 T cell proliferation but rather promoted IL-2 production and the generation of CD4CD25 T cells, which regulated alloantigen-specific cytotoxic T lymphocyte activity. Treatment with either AdCD40Ig or purified soluble CD40Ig prolonged the survival of rat cardiac allografts. In contrast, although monomeric soluble CD40/Myc/His suppressed IL-12 production in a similar manner to that achieved by CD40Ig, it did not augment IL-2 production. Moreover, while CD40/Myc/His also generated CD4CD25 T cells, they did not exhibit regulatory activity and administration of soluble CD40/Myc/His failed to prolong cardiac allograft survival., Conclusions: These results suggest signaling through CD154 in addition to blocking of CD154-CD40 interaction is important for the immunomodulatory effects of soluble CD40Ig. Taken together, our results provide new insight into the mechanism of immunomodulation by soluble CD40 constructs.

Dendritic cells (DC) have been implicated in the defective function of the immune system during cancer progression. We have demonstrated that patients with cancer have fewer myeloid (CD11c+) and plasmacytoid (CD123(hi)) DC and a concurrent accumulation of CD11c(-)CD123- immature cells expressing HLA-DR (DR(+)IC). Notably, DR(+)IC from cancer patients have a reduced capacity to stimulate allogeneic T-cells. DR(+)IC are also present in healthy donors, albeit in smaller numbers. In this study, we assessed whether DR(+)IC could have an impact on the immune response by comparing their function with DC counterparts. For this purpose, DR(+)IC and DC were purified and tested in the presentation of antigens through major histocompatibility complex (MHC) II and MHC-I molecules. DR(+)IC were less efficient than DC at presenting antigens to T-cells. DR(+)IC induced a limited activation of T-cells, eliciting poor T-helper (Th) 1 and preferentially inducing Th2-biased responses. Importantly, despite DR(+)IC's poor responsiveness to inflammatory factors, in samples from healthy volunteers and breast cancer patients, CD40 ligation induced phenotypic maturation and interleukin 12 secretion, in turn generating more efficient T-cell responses. These data underscore the importance of inefficient antigen presentation as a mechanism for tumor evasion and suggest an approach to improve the efficacy of DC-based immunotherapy for cancer.

Transforming growth factor-beta-activated kinase 1 (TAK1) has been linked to interleukin 1 receptor and tumor necrosis factor receptor signaling. Here we generated mouse strains with conditional expression of a Map3k7 allele encoding part of TAK1. TAK1-deficient embryonic fibroblasts demonstrated loss of responses to interleukin 1beta and tumor necrosis factor. Studies of mice with B cell-specific TAK1 deficiency showed that TAK1 was indispensable for cellular responses to Toll-like receptor ligands, CD40 and B cell receptor crosslinking. In addition, antigen-induced immune responses were considerably impaired in mice with B cell-specific TAK1 deficiency. TAK1-deficient cells failed to activate transcription factor NF-kappaB and mitogen-activated protein kinases in response to interleukin 1beta, tumor necrosis factor and Toll-like receptor ligands. However, TAK1-deficient B cells were able to activate NF-kappaB but not the kinase Jnk in response to B cell receptor stimulation. These results collectively indicate that TAK1 is key in the cellular response to a variety of stimuli.

Key pathological processes in Alzheimer's disease (AD) include the accumulation of amyloid beta peptide (Abeta) which, in excess, triggers pathological cascades including widespread inflammation, partly reflected by chronic microglial activation. It has previously been suggested that CD40/CD40L interaction promotes AD like pathology in transgenic mice. Thus, amyloid burden, gliosis and hyperphosphorylation of tau are all reduced in transgenic models of AD lacking functional CD40L. We therefore hypothesized that cellular events leading to altered APP metabolism, inflammation and increased tau phosphorylation underlying these observations would be regulated at the genomic level. In the present report, we used the Affymetrix (GeneChip) oligonucleotide microarray U133A to gain insight into the global and simultaneous transcriptomic changes in response to microglia activation after CD40/CD40L ligation. As expected, regulation of elements of the NF-kappaB signaling, chemokine and B cell signaling pathways was observed. Taken together, our data also suggest that CD40 ligation in human microglia specifically perturbs many genes associated with APP processing.

Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus CD154 (Ad-CD154, CD40 ligand [CD40L]) gene therapy experienced rapid reductions in leukemia cell counts and lymph node size associated with the induced expression of Fas (CD95). However, CLL cells initially resist CD95-mediated apoptosis within the first 3 days after CD40 ligation in vitro. Thereafter, they become sensitive, which is associated with the CD40-induced expression of the proapoptotic protein B-cell leukemia 2 homology 3 (BH3) interacting domain death agonist (Bid). We hypothesized that the initial resistance to CD95-mediated apoptosis may be due to the high-level expression of X-linked inhibitor of apoptosis protein (XIAP) by CLL cells. Consistent with this, CLL cells from patients 1 day after treatment with autologous Ad-CD154-transduced CLL cells became sensitive to CD95-mediated apoptosis following treatment with a novel XIAP inhibitor, 1540-14. Similarly, 1540-14 specifically enhanced CD95-mediated apoptosis of CLL cells following CD40 ligation in vitro. Immunoblot analyses demonstrated that treatment with 1540-14 allowed CD40-stimulated CLL cells to experience high-level activation of caspases-8 and -3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase following CD95 ligation. This study demonstrates that distal apoptosis regulators contribute to the initial resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that XIAP inhibitors might enhance the effectiveness of immune-based treatment strategies that target CD40, such as CD154 gene therapy.

Microglia are the resident phagocytes of the brain and are an important source of proinflammatory mediators. Human immunodeficiency virus (HIV)-1 infects the central nervous system early in the course of disease, and it is believed that this occurs, in part, through the transmigration of HIV-1-infected cells across the blood-brain barrier. Infected cells release viral proteins, such as Tat and gp120. After microglia interact with these proteins, they become activated and secrete chemokines; up-regulate key surface receptors, such as CD40, and also activate resident cells. This review focuses on the consequences of microglial activation in NeuroAIDS, with an emphasis on chemokine production and CD40 up-regulation after interaction with tat or gp120. The importance of microglial CD40 in two other neurological diseases, Alzheimer's disease and multiple sclerosis, is also discussed., ((c) 2005 Wiley-Liss, Inc.)

CD40, a TNF-R-related cell surface receptor, is shown here to be expressed by glioma cells in vitro and in vivo. Glioma cell lines expressing low levels of CD40 at the cell surface resist cytotoxic effects of CD40L. CD40 gene transfer sensitizes glioma cells to CD40L. Inhibition of protein synthesis potentiates cell death which involves CD40 clustering and caspases 8 and 3 processing. CD40-transfected LN-18 cells acquire resistance to CD95L. In contrast, subtoxic concentrations of CD40L strongly sensitize these cells for TNF-alpha-induced apoptosis. Bispecific CD40xCD95 antibodies specifically kill glioma cells, disclosing the property of endogenous CD40 to facilitate death signalling.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma with poor response to therapy and unfavorable prognosis. Here, we show that retinoic acid (RA) isomers significantly inhibit the proliferation of both primary MCL cultures (n = 7) and established cell lines (Granta 519 and SP-53) as shown by [(3)H]thymidine uptake and carboxyfluorescein diacetate succinimidyl ester labeling coupled with cyclin D1 staining. RA induces cell accumulation in G(0)-G(1) together with a marked up-regulation of p27(Kip1) by inhibiting ubiquitination and proteasome-dependent degradation of the protein. The p21(Cip1) inhibitor was also up-regulated by RA in Granta 519 cells, whereas the expression of cyclin D1 is unaffected. Most of RA-induced p27(Kip1) was bound to cyclin D1/cyclin-dependent kinase 4 complexes, probably contributing to the decreased cyclin-dependent kinase 4 kinase activity and pRb hypophosphorylation observed in RA-treated cells. Experiments with receptor-selective ligands indicate that RA receptor alpha cooperates with retinoid X receptors in mediating RA-dependent MCL cell growth inhibition. Notably, RA isomers, and particularly 9-cis-RA, also inhibited the growth-promoting effect induced in primary MCL cells by CD40 activation alone or in combination with interleukin-4. Immunohistochemical analysis showed that significant numbers of CD40L-expressing lymphoid cells are present in lymph node biopsies of MCL patients. These results therefore further strengthen the possibility that triggering of CD40 by infiltrating CD40L+ cells may continuously promote the growth of MCL cells in vivo. On these grounds, our findings that RA inhibits basal MCL proliferation as well as MCL growth-promoting effects exerted by microenvironmental factors make these compounds highly attractive in terms of potential clinical efficacy in this setting.

The CD40 receptor and the Epstein-Barr virus oncoprotein LMP1 are both members of the TNF-receptor family and share several signaling mediators, including TRAF2 and TRAF3. Depending on the cell lineage and stage of maturation, LMP1 and CD40 can have synergistic, antagonist or unrelated effects. Previous publications have suggested that both TRAF2 and TRAF3 move into lipid rafts upon LMP1 expression or CD40 activation, whereas their proteolysis is only enhanced by CD40. However CD40-induced proteolysis of TRAF2 has only been reported in murine cells, and there are conflicting data regarding translocation of TRAF2 into lipid rafts. We therefore investigated TRAF2 and TRAF3 modifications induced by CD40 and LMP1 signaling in a panel of human cell lines of lymphoid and epithelial origins. Upon CD40 stimulation, a marked redistribution of TRAF2 into the buoyant raft fraction was observed in all cell lines and was often associated with a similar redistribution of TRAF3. In contrast, only TRAF3 was redistributed into the raft fraction upon LMP1 expression. Moreover parallel changes in subcellular distribution of TRAF2 and TRAF3 were recorded by electron microscopy. A significant decrease in TRAF2 and TRAF3 concentrations triggered by CD40 ligation was observed in only 1 cell line and there was no evidence that this decrease was required for the negative feed-back on JNK activation. TRAF2 redistribution into raft-like complexes thus appears as the most significant event distinctive of CD40 and LMP1 signaling. On the other hand, the parallel influence of CD40 and LMP1 on TRAF3 redistribution is consistent with functional similarities between the CD40-TRAF3 and LMP1-TRAF3 axes.

An inflammatory process in association with reactive gliosis has been suggested to play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). One of the key findings is a marked increase in the level of cyclooxygenase-2 (COX-2), a therapeutic target of ALS. We investigated the expression of CD40 in the spinal cord of a transgenic mouse model of ALS (G93A mice), and its relevance to COX-2 upregulation. CD40 was predominantly expressed in neurons in normal spinal cord and upregulated in reactive glial cells in spinal cord injury. In the spinal cord of G93A mice, the expression of CD40 was increased in both reactive microglia and astrocytes, where COX-2 was especially increased. The level of COX-2 was upregulated in microglia and astrocytes by CD40 stimulation in vitro. CD40 stimulation in primary spinal cord cultures caused motor neuron loss that was protected by selective COX-2 inhibitor. These results suggest that CD40, which is upregulated in reactive glial cells in ALS, participates in motor neuron loss via induction of COX-2.

Monoclonal antibodies against CD40, conjugated to antigen, act as potent immunological adjuvants for primary antibody responses. We show here that CD40mAbs can also act as strong adjuvants for memory antibody responses, and for T cell responses as measured by ex vivo T cell proliferation to antigen, and delayed type hypersensitivity. Interferon gamma secretion in response to antigen is also enhanced. Finally, the adjuvant effect of CD40mAbs for secondary antibody responses is transferred with T cells rather than B cells. CD40mAb apparently have potent adjuvant effects on both Th1-like cells, and on T cells able to promote B cell antibody production. It is possible that the adjuvant effects of CD40 are mediated at least in part, indirectly, through enhanced antigen presentation by specific B cells, to T cells.

IL-21 is a cytokine that regulates the activation of T and NK cells and promotes the proliferation of B cells activated via CD40. In this study, we show that rIL-21 strongly induces the production of all IgG isotypes by purified CD19(+) human spleen or peripheral blood B cells stimulated with anti-CD40 mAb. Moreover, it was found to specifically induce the production of IgG(1) and IgG(3) by CD40-activated CD19(+)CD27(-) naive human B cells. Although stimulation of CD19(+) B cells via CD40 alone induced gamma 1 and gamma 3 germline transcripts, as well as the expression of activation-induced cytidine deaminase, only stimulation with both anti-CD40 mAb and rIL-21 resulted in the production of S gamma/S mu switch circular DNA. These results show that IL-21, in addition to promoting growth and differentiation of committed B cells, is a specific switch factor for the production of IgG(1) and IgG(3).

The Wilms' tumor suppressor gene (WT1) has been shown to be overexpressed in acute and chronic leukemias and in a variety of solid human malignancies, including cancers of the breast and lung. In our present study, we investigated the potential role of WT1 gene in human colon cancer. WT1 mRNA and protein expression was analyzed in a panel of human colon cancer cell lines and primary colon carcinomas by RT-PCR and Western blot analysis, respectively. A mutational screen of WT1' zinc-finger region was carried out by sequence analysis. Finally, using peptide-stimulated cytotoxic T cells it was investigated whether WT1-expressing colon tumor cells are a potential target for antigen-specific immunotherapy. Medium to high abundant levels of WT1 mRNA were detected by RT-PCR in 10 of 12 (83%) colon cell lines and by quantitative, real-time RT-PCR in 13 of 15 (87%) primary tumors, whereas only very low levels of expression were found in 2 primary tumors. Interestingly, however, low levels of WT1 mRNA were also detected in all samples derived from normal colon mucosa. When RT-PCR products were examined by sequence analysis, both +KTS and -KTS splice isoforms but no zinc-finger mutations were found, suggesting that the wild-type form of the WT1 gene is expressed. To determine whether the WT1 protein can serve as a target antigen for immunotherapy, 2 HLA-A2.1-restricted WT1 peptides (Db126 and WH187) were used for the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs). The WH187-specific CTLs not only lysed target cells pulsed exogenously with cognate peptide but also WT1-expressing colon tumor cells in a HLA-restricted manner. These findings identify the WT1 protein as an attractive target for the development of antigen-specific immunotherapy in human colon cancer., (Copyright 2004 Wiley-Liss, Inc.)

Preformed CD40/CD40 homodimers were initially observed on human Burkitt lymphoma cell lines, normal B cells, and transitional bladder carcinoma cell lines. However, the nature and the biological relevance of these homodimers have not yet been investigated. In the present study, we demonstrated that Epstein-Barr virus-transformed B cells and CD40-transfected HEK 293 cells constitutively expressed disulfide-linked CD40/CD40 homodimers at low levels. Oligomerization of CD40 leads to a rapid and significant increase in the disulfide-linked CD40/CD40 homodimer formation, a response that could be prevented using a thiol-alkylating agent. Formation of CD40/CD40 homodimers was found to be absolutely required for CD40-mediated activation of phosphatidylinositol 3-kinase, which, in turn regulated B7.2 expression. In contrast, CD40 monomers provided the minimal signal emerging from CD40, activating p38 MAP kinase and inducing homotypic B cell adhesion. CD40/CD40 homodimer formation was totally independent of TRAF1/2/3/5 associations with the threonine at position 254 in the cytoplasmic tail of the CD40 molecules. This finding may be vital to better understanding the molecular mechanisms that govern cell signaling triggered by CD40/CD154 interactions.

The capacity to class switch the IgH chain is critical to the effectiveness of humoral immune responses. We show that in vitro-stimulated splenic B cells from senescent mice are deficient in production of multiple class switch isotypes (IgG1, G2a, G3, and E), class switch recombination (CSR), and induction of the E2A-encoded transcription factor E47. E47 has previously been shown to be required for CSR, at least in part via expression of the activation-induced cytidine deaminase. Our studies show that impaired induction of E47, and subsequently activation-induced cytidine deaminase, contribute to poor CSR and production of secondary isotypes in senescence.

Interleukins IL-4 and IL-10 are considered to be central regulators for the limitation and eventual termination of inflammatory responses in vivo, based on their potent anti-inflammatory effects toward LPS-stimulated monocytes/macrophages and neutrophils. However, their role in T cell-dependent inflammatory responses has not been fully elucidated. In this study, we investigated the effects of both cytokines on the production of PGE(2), a key molecule of various inflammatory conditions, in CD40-stimulated human peripheral blood monocytes. CD40 ligation of monocytes induced the synthesis of a significant amount of PGE(2) via inducible expression of the cyclooxygenase (COX)-2 gene. Both IL-10 and IL-4 significantly inhibited PGE(2) production and COX-2 expression in CD40-stimulated monocytes. Using specific inhibitors for extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), we found that both kinase pathways are involved in CD40-induced COX-2 expression. CD40 ligation also resulted in the activation of NF-kappaB. Additional experiments exhibited that CD40 clearly induced the activation of the upstream kinases MAPK/ERK kinase 1/2, MAPK kinase 3/6, and I-kappaB in monocytes. IL-10 significantly inhibited CD40-induced activation of the ERK, p38 MAPK, and NF-kappaB pathways; however, inhibition by IL-4 was limited to the ERK pathway in monocytes. Neither IL-10 nor IL-4 affected the recruitment of TNFR-associated factors 2 and 3 to CD40 in monocytes. Collectively, IL-10 and IL-4 use novel regulatory mechanisms for CD40-induced prostanoid synthesis in monocytes, thus suggesting a potential role for these cytokines in regulating T cell-induced inflammatory responses, including autoimmune diseases.

During an immune response, B lymphocytes can switch expression of immunoglobulin (Ig) class (isotype) from IgM to IgG, IgE, or IgA. This Ig class switch is based on a deoxyribonucleic acid (DNA) recombination event that results in an exchange of the gene segments coding for the constant region of the Ig heavy chain, although the Ig heavy chain variable region is retained. This process changes the effector functions of the corresponding antibody (Ab). Much of our current understanding of the molecular mechanisms of class switch recombination is based on the analysis of B cells induced to switch class of Ig in vitro. In vitro, murine and human naive B cells can be activated with bacterial lipopolysaccharides, anti-CD40 or CD40L, to undergo class switch recombination. Cytokine signals can direct class switch recombination to distinct classes; for example, interleukin-4 will target murine IgG1 and IgE, and human IgG4 and IgE. Here we describe the technologies for the isolation of B lymphocytes, their activation to class switching, and the analysis of Ig class switching.

Defective expression of Fas leads to B cell autoimmunity, indicating the importance of this apoptotic pathway in eliminating autoreactive B cells. However, B cells with anti-self specificities occasionally escape such regulation in individuals with intact Fas, suggesting ways of precluding this apoptosis. Here, we examine whether coligation of the B cell Ag receptor (BCR) with the complement (C3)-binding CD21/CD19/CD81 costimulatory complex can enhance the escape of human B cells from Fas-induced death. This was warranted given that BCR-initiated signals induce resistance to Fas apoptosis, some (albeit not all) BCR-triggered events are amplified by coligation of BCR and the co-stimulatory complex, and several self Ags targeted in autoimmune diseases effectively activate complement. Using a set of affinity-diverse surrogate Ags (receptor-specific mAb:dextran conjugates) with varying capacity to engage CD21, it was established that BCR:CD21 coligation lowers the BCR engagement necessary for inducing protection from Fas apoptosis. Enhanced protection was associated with altered expression of several molecules known to regulate Fas apoptosis, suggesting a unique molecular model for how BCR:CD21 coligation augments protection. BCR:CD21 coligation impairs the generation of active fragments of caspase-8 via dampened expression of membrane Fas and augmented expression of FLIP(L). This, in turn, diminishes the generation of cells that would be directly triggered to apoptosis via caspase-8 cleavage of caspase 3 (type I cells). Any attempt to use the mitochondrial apoptotic protease-activating factor 1 (Apaf-1)-dependent pathway for apoptosis (as type II cells) is further blocked because BCR:CD21 coligation promotes up-regulation of the mitochondrial antiapoptotic molecule, Bcl-2.