BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

TACI, an enigmatic BAFF/APRIL receptor, with new unappreciated biochemical and biological properties.

BAFF is a B cell survival factor that binds to three receptors BAFF-R, TACI and BCMA. BAFF-R is the receptor triggering naïve B cell survival and maturation while BCMA supports the survival of plasma cells in the bone marrow. Excessive BAFF production leads to autoimmunity, presumably as the consequence of inappropriate survival of self-reactive B cells. The function of TACI has been more elusive with TACI(-/-) mice revealing two sides of this receptor, a positive one driving T cell-independent immune responses and a negative one down-regulating B cell activation and expansion. Recent work has revealed that the regulation of TACI expression is intimately linked to the activation of innate receptors on B cells and that TACI signalling in response to multimeric BAFF and APRIL provides positive signals to plasmablasts. How TACI negatively regulates B cells remains elusive but may involve an indirect control of BAFF levels. The discovery of TACI mutations associated with common variable immunodeficiency (CVID) in humans not only reinforces its important role for humoral responses but also suggests a more complex role than first anticipated from knockout animals. TACI is emerging as an unusual TNF receptor-like molecule with a sophisticated mode of action.

Unique and conserved functions of B cell-activating factor of the TNF family (BAFF) in the chicken.

The chicken represents the best-characterized animal model for B cell development in the so-called gut-associated lymphoid tissue (GALT) and the molecular processes leading to B cell receptor diversification in this species are well investigated. However, the mechanisms regulating B cell development and homeostasis in GALT species are largely unknown. Here we investigate the role played by the avian homologue of B cell-activating factor of the tumor necrosis factor family (BAFF). Flow cytometric analysis showed that the receptor for chicken B cell-activating factor of the tumor necrosis factor family (chBAFF) is expressed by mature and immature B cells. Unlike murine and human BAFF, chBAFF is primarily produced by B cells both in peripheral lymphoid organs and in the bursa of Fabricius, the chicken's unique primary lymphoid organ. In vitro and in vivo studies revealed that chBAFF is required for mature B cell survival. In addition, in vivo neutralization with a decoy receptor led to a reduction of the size and number of B cell follicles in the bursa, demonstrating that, in contrast to humans and mice, in chickens BAFF is also required for the development of immature B cells. Collectively, we show that chBAFF has phylogenetically conserved functions in mature B cell homeostasis but displays unique and thus far unknown properties in the regulation of B cell development in birds.

Preferential binding of mouse mammary tumor virus to B lymphocytes.

Mouse mammary tumor virus (MMTV) has been shown to preferentially infect B lymphocytes in vivo. We have used recombinant envelope-coated fluospheres and highly purified MMTV particles to study the distribution of the viral receptors on fresh mouse lymphocytes. A preferential dose-dependent binding to B lymphocytes was observed which could be competed with neutralizing antibodies. In contrast, T-lymphocyte binding remained at background levels. These results strongly suggest a higher density of viral receptor molecules on B lymphocytes than on T lymphocytes and correlate with the preferential initial infection of B lymphocytes observed in vivo.

BCR and TLR signalling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas.

The genetics and pathogenesis of splenic marginal zone lymphoma are poorly understood. The lymphoma lacks chromosome translocation, and ~30% of cases are featured by 7q deletion, but the gene targeted by the deletion is unknown. A recent study showed inactivation of A20, a 'global' NF-kB negative regulator, in 1 of 12 splenic marginal zone lymphoma. To investigate further whether deregulation of the NF-kB pathway plays a role in the pathogenesis of splenic marginal zone lymphoma, we screened several NF-kB regulators for genetic changes by PCR and sequencing. Somatic mutations were found in A20 (6/46=13%), MYD88 (6/46=13%), CARD11 (3/34=8.8%), but not in CD79A, CD79B and ABIN1. Interestingly, these genetic changes are largely mutually exclusive from each other and MYD88 mutation was also mutually exclusive from 7q deletion. These results strongly suggest that deregulation of the TLR (toll like receptor) and BCR (B-cell receptor) signalling pathway may play an important role in the pathogenesis of splenic marginal zone lymphoma.

Phase II study of imatinib in patients with recurrent gliomas of various histologies: a European Organisation for Research and Treatment of Cancer Brain Tumor Group Study.

PURPOSE: To evaluate the safety and the efficacy of imatinib in recurrent malignant gliomas. PATIENTS: AND METHODS: This was a single-arm, phase II study. Eligible patients had recurrent glioma after prior radiotherapy with an enhancing lesion on magnetic resonance imaging. Three different histologic groups were studied: glioblastomas (GBM), pure/mixed (anaplastic) oligodendrogliomas (OD), and low-grade or anaplastic astrocytomas (A). Imatinib was started at a dose of 600 mg/d with dose escalation to 800 mg in case of no toxicity; during the trial this dose was increased to 800 mg/d with escalation to 1,000 mg/d. Trial design was one-stage Fleming; both an objective response and 6 months of progression-free survival (PFS) were considered a successful outcome to treatment. RESULTS: A total of 112 patients (51 patients with GBM, 25 patients with A, and 36 patients with OD) were enrolled. Imatinib was in general well tolerated. The median number of cycles was 2.0 (range, 1 to 43 cycles). Five patients had an objective partial response, including three patients with GBM; all had 6 months of PFS. The 6-month PFS rate was 16% (95% CI, 8.0% to 34.0%) in GBM, 4.0% (95% CI, 0.3% to 15.0%) in OD, and 9% (95% CI, 2.0% to 25.0%) in A. The exposure to imatinib was significantly lower in patients using enzyme-inducing antiepileptic drugs. The presence of ABCG2 point mutations were not correlated with pharmacokinetic findings. No somatic activating mutations of KIT or platelet-derived growth factor receptor-A or -B were found. CONCLUSION: In the dose range of 600 to 1,000 mg/d, single-agent imatinib is well tolerated but has limited antitumor activity in patients with recurrent gliomas.

Premature replacement of mu with alpha immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation.

Sequentially along B cell differentiation, the different classes of membrane Ig heavy chains associate with the Ig alpha/Ig beta heterodimer within the B cell receptor (BCR). Whether each Ig class conveys specific signals adapted to the corresponding differentiation stage remains debated. We investigated the impact of the forced expression of an IgA-class receptor throughout murine B cell differentiation by knocking in the human C alpha Ig gene in place of the S mu region. Despite expression of a functional BCR, homozygous mutant mice showed a partial developmental blockade at the pro-B/pre-BI and large pre-BII cell stages, with decreased numbers of small pre-BII cells. Beyond this stage, peripheral B cell compartments of reduced size developed and allowed specific antibody responses, whereas mature cells showed constitutive activation and a strong commitment to plasma cell differentiation. Secreted IgA correctly assembled into polymers, associated with the murine J chain, and was transported into secretions. In heterozygous mutants, cells expressing the IgA allele competed poorly with those expressing IgM from the wild-type allele and were almost undetectable among peripheral B lymphocytes, notably in gut-associated lymphoid tissues. Our data indicate that the IgM BCR is more efficient in driving early B cell education and in mucosal site targeting, whereas the IgA BCR appears particularly suited to promoting activation and differentiation of effector plasma cells.

Molecular insights into T cell activation : study of the TRAF, Bcl10 and Carma 1 proteins

Abstract : Activation of naïve T lymphocytes is essential for the onset of an adaptive immune response against a pathogenic threat. T lymphocytes are activated through the engagement of their highly specific cell surface antigen-receptor (TCR), together with co-stimulatory receptors, by activated antigen-presenting cells that display antigenic peptide fragments from the pathogen that they have detected. Dissection of the mechanisms that modulate TCR- and co-stimulation- induced signals is therefore crucial for the understanding of the molelcular basis of adaptive immune responses. Following antigen-receptor triggering, the Carma1, Bcl10 and Malt1 (CBM) proteins assemble into an oligomeric complex, which is essential for activation of the NF-κB and JNK signaling pathways in lymphocytes. In this work, by using human epithelial and lymphocytic cell lines, we identified the TNF-receptor-associated factor (TRAF) proteins TRAF3 and TRAF7 as new binding partners of Bcl10 and Carma1, respectively. We could show that TRAF3 is required for the proper transcriptional upregulation of IL-2 in activated T cells, and that endogenous TRAF3 is recruited to Bcl10 following TCR engagement. Although the mechanisms used by TRAF3 to modulate the transcriptional activation of the IL-2 promoter are not elucidated, the stimulus-dependent association ofTRAF3 with its direct binding partner Bcl10 suggests that TRAF3 is regulating Bcl10 function in TCR-activated lymphocytes. We also demonstrated that TRAF7 acts as a negative regulator of Carma1-induced NFκB-and AP1-dependent transcription by overexpression in 293T cells. These data suggest that TRAF7 could contribute to the negative regulation of TCR-dependent Carma1 functions. Finally, we showed that Carma1 is processed upon antigen-receptor triggering in B and T cell lines, as well as in primary human CTLs, and that this processing is dependent on the proteolytic activity of Malt1. Collectively, this work contributes to describe new proteins and regulatory mechanisms that modulate CBM-dependent functions in activated lymphocytes. Furthermore, it uncovers new tracks that could lead to a better molecular understanding of the complex interplay between the activatory and inhibitory regulators associated with the CBM complex. Résumé : L'activation des lymphocytes T naifs est une étape essentielle à la mise en place d'une réponse immunitaire adaptative pour combattre une infection. Après la détection d'un pathogène, les cellules présentatrices d'antigènes exposent à leur surface des fragments peptidiques provenant du pathogène, qui activent le récepteur à antigène (TCR) spécifique des lymphocytes T, ainsi que des molécules co-stimulatrices qui contribuent à l'activation complète des lymphocytes T. La caractérisation des mécanismes qui modulent les cascades de signaux émanant du TCR et des récepteurs de co-stimulation est essentielle à la compréhension du fonctionnement moléculaire de la réponse immunitaire adaptative. La ligation du TCR induit la formation d'un complexe oligomérique comprenant les protéines Carma1, Bcl10 et Malt1, qui est essentiel à l'activation des voies de signalisation cellulaires NF-κB et JNK induisant l'activation complète des lymphorctes T. Dans cette étude, à l'aide de lignées de cellules humaines épithéliales et lymphocytaires, nous avons identifié que deux protéines de la famille des TRAF (Tumor Necrosis Factor Receptor-Associated Factor), TRAF3 et TRAF7, s'associent à Bc110 et à Carma1, respectivement. Les TRAFs sont d'importants régulateurs des voies de signalisation dans les cellules du système immunitaire inné et adaptatif. Nous avons démontré que TRAF3 était important pour permettre la transcription de l'interleukine-2 (IL-2) dans les lymphocytes T activés, et que TRAF3 s'associait à Bc110 à la suite de la stimulation du TCR Les mécanismes que TRAF3 utilise pour moduler l'activation du promoteur de l'IL-2 ne sont pas connus, mais l'association de TRAF3 à Bc110 suite à la stimulation du TCR suggère que TRAF3 régule la fonction de Bc110. Nous avons également identifié TRAF7 comme un nouveau régulateur négatif des voies NF-κB et JNK induites par surexpression de la protéine Carma1. Nos données suggèrent que TRAF7 pourrait également contribuer à la régulation négative de la fonction de Carma1 dans les lymphocytes activés. Enfin, nous avons découvert que Carma1 était clivé suite à la stimulation du TCR, et que ce clivage dépendait de l'activité protéolytique de Malt1. Cette étude contribue ainsi à la description de nouvelles protéines et de nouveaux mécanismes qui modulent l'activité du complexe CBM dans les lymphocytes activés, et ouvre la voie à la caractérisation moléculaire de ces nouveaux mécanismes importants pour la régulation de la réponse immunitaire adaptative.

Constitutive activity and inverse agonism at the α(₁a) and α(₁b) adrenergic receptor subtypes.

The α(1b)-adrenergic receptor (AR) was, after rhodopsin, the first G protein-coupled receptor (GPCR) in which point mutations were shown to trigger constitutive (agonist-independent) activity. Constitutively activating mutations have been found in other AR subtypes as well as in several GPCRs. This chapter briefly summarizes the main findings on constitutively active mutants of the α(1a)- and α(1b)-AR subtypes and the methods used to predict activating mutations, to measure constitutive activity of Gq-coupled receptors and to investigate inverse agonism. In addition, it highlights the implications of studies on constitutively active AR mutants on elucidating the molecular mechanisms of receptor activation and drug action.

Antigen Receptor Signaling to NF-kappa B via CARMA1, BCL10, and MALT1

The signaling pathway controlling antigen receptor-induced regulation of the transcription factor NF-kappa B plays a key role in lymphocyte activation and development and the generation of lymphomas. Work of the past decade has led to dramatic progress in the identification and characterization of new players in the pathway. Moreover, novel enzymatic activities relevant for this pathway have been discovered, which represent interesting drug targets for immuno-suppression or lymphoma treatment. Here, we summarize these findings and give an outlook on interesting open issues that need to be addressed in the future.

Astressin B, a nonselective corticotropin-releasing hormone receptor antagonist, prevents the inhibitory effect of ghrelin on luteinizing hormone pulse frequency in the ovariectomized rhesus monkey.

Administration of ghrelin, a key peptide in the regulation of energy homeostasis, has been shown to decrease LH pulse frequency while concomitantly elevating cortisol levels. Because increased endogenous CRH release in stress is associated with an inhibition of reproductive function, we have tested here whether the pulsatile LH decrease after ghrelin may reflect an activated hypothalamic-pituitary-adrenal axis and be prevented by a CRH antagonist. After a 3-h baseline LH pulse frequency monitoring, five adult ovariectomized rhesus monkeys received a 5-h saline (protocol 1) or ghrelin (100-microg bolus followed by 100 microg/h, protocol 2) infusion. In protocols 3 and 4, animals were given astressin B, a nonspecific CRH receptor antagonist (0.45 mg/kg im) 90 min before ghrelin or saline infusion. Blood samples were taken every 15 min for LH measurements, whereas cortisol and GH were measured every 45 min. Mean LH pulse frequency during the 5-h ghrelin infusion was significantly lower than in all other treatments (P < 0.05) and when compared with the baseline period (P < 0.05). Pretreatment with astressin B prevented the decrease. Ghrelin stimulated cortisol and GH secretion, whereas astressin B pretreatment prevented the cortisol, but not the GH, release. Our data indicate that CRH release mediates the inhibitory effect of ghrelin on LH pulse frequency and suggest that the inhibitory impact of an insufficient energy balance on reproductive function may in part be mediated by the hypothalamic-pituitary-adrenal axis.

Toll-like receptor agonists synergize with CD40L to induce either proliferation or plasma cell differentiation of mouse B cells.

In a classical dogma, pathogens are sensed (via recognition of Pathogen Associated Molecular Patterns (PAMPs)) by innate immune cells that in turn activate adaptive immune cells. However, recent data showed that TLRs (Toll Like Receptors), the most characterized class of Pattern Recognition Receptors, are also expressed by adaptive immune B cells. B cells play an important role in protective immunity essentially by differentiating into antibody-secreting cells (ASC). This differentiation requires at least two signals: the recognition of an antigen by the B cell specific receptor (BCR) and a T cell co-stimulatory signal provided mainly by CD154/CD40L acting on CD40. In order to better understand interactions of innate and adaptive B cell stimulatory signals, we evaluated the outcome of combinations of TLRs, BCR and/or CD40 stimulation. For this purpose, mouse spleen B cells were activated with synthetic TLR agonists, recombinant mouse CD40L and agonist anti-BCR antibodies. As expected, TLR agonists induced mouse B cell proliferation and activation or differentiation into ASC. Interestingly, addition of CD40 signal to TLR agonists stimulated either B cell proliferation and activation (TLR3, TLR4, and TLR9) or differentiation into ASC (TLR1/2, TLR2/6, TLR4 and TLR7). Addition of a BCR signal to CD40L and either TLR3 or TLR9 agonists did not induce differentiation into ASC, which could be interpreted as an entrance into the memory pathway. In conclusion, our results suggest that PAMPs synergize with signals from adaptive immunity to regulate B lymphocyte fate during humoral immune response.

Induction of the alternative NF-κB pathway by lymphotoxin αβ (LTαβ) relies on internalization of LTβ receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTβR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTβR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTβR cellular trafficking as a process required for specific biological functions of NF-κB.

Hepatitis B Virus e Antigen Physically Associates With Receptor-Interacting Serine/Threonine Protein Kinase 2 and Regulates IL-6 Gene Expression.

We previously reported that hepatitis B virus (HBV) e antigen (HBeAg) inhibits production of interleukin 6 by suppressing NF-κB activation. NF-κB is known to be activated through receptor-interacting serine/threonine protein kinase 2 (RIPK2), and we examined the mechanisms of interleukin 6 regulation by HBeAg. HBeAg inhibits RIPK2 expression and interacts with RIPK2, which may represent 2 mechanisms through which HBeAg blocks nucleotide-binding oligomerization domain-containing protein 1 ligand-induced NF-κB activation in HepG2 cells. Our findings identified novel molecular mechanisms whereby HBeAg modulates intracellular signaling pathways by targeting RIPK2, supporting the concept that HBeAg could impair both innate and adaptive immune responses to promote chronic HBV infection.

Renal endothelin receptor type B upregulation in rats with low or high renin hypertension.

OBJECTIVES: To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats. METHODS: Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out. RESULTS: MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h. CONCLUSIONS: Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.