BAFF and BAFF-Receptor in B Cell Selection and Survival

The BAFF-receptor (BAFFR) is encoded by the TNFRSF13C gene and is one of the main pro-survival receptors in B cells. Its function is impressively documented in humans by a homozygous deletion within exon 2, which leads to an almost complete block of B cell development at the stage of immature/transitional B cells. The resulting immunodeficiency is characterized by B-lymphopenia, agammaglobulinemia, and impaired humoral immune responses. However, different from mutations affecting pathway components coupled to B cell antigen receptor (BCR) signaling, BAFFR-deficient B cells can still develop into IgA-secreting plasma cells. Therefore, BAFFR deficiency in humans is characterized by very few circulating B cells, very low IgM and IgG serum concentrations but normal or high IgA levels

Ribosomes from Trypanosomatids: Unique Structural and Functional Properties

Trypanosomatids are a monophyletic group of protozoa that diverged early from the eukaryotic lineage, constituting valuable model organisms for studying variability in different highly conserved processes including protein synthesis. Moreover, several species of trypanosomatids are causing agents of endemic diseases in the third world. There are many evidences suggesting that translation in these organisms shows important differences with that of model organisms such as yeast and mammals. These unique features, which have a great potential relevance for both basic and applied research, will be discussed in this chapter.Fil: Juri Ayub, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Lapadula, Walter Jesús. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Hoebeke, Johan. Centre National de la Recherche Scientifique; FranciaFil: Smulski, Cristian Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Centre National de la Recherche Scientifique; Franci

Crystal Structure of the Complex mAb 17.2 and the C-Terminal Region of Trypanosoma cruzi P2β Protein: Implications in Cross-Reactivity

Patients with Chronic Chagas' Heart Disease possess high levels of antibodies against the carboxyl-terminal end of the ribosomal P2ß protein of Trypanosoma cruzi (TcP2ß). These antibodies, as well as the murine monoclonal antibody (mAb) 17.2, recognize the last 13 amino acids of TcP2ß (called the R13 epitope: EEEDDDMGFGLFD) and are able to cross-react with, and stimulate, the ß1 adrenergic receptor (ß1-AR). Indeed, the mAb 17.2 was able to specifically detect human β1-AR, stably transfected into HEK cells, by flow cytometry and to induce repolarisation abnormalities and first degree atrioventricular conduction block after passive transfer to naïve mice. To study the structural basis of this cross-reactivity, we determined the crystal structure of the Fab region of the mAb 17.2 alone at 2.31 Å resolution and in complex with the R13 peptide at 1.89 Å resolution. We identified as key contact residues on R13 peptide Glu3, Asp6 and Phe9 as was previously shown by alanine scanning. Additionally, we generated a model of human β1-AR to elucidate the interaction with anti-R13 antibodies. These data provide an understanding of the molecular basis of cross-reactive antibodies induced by chronic infection with Trypanosoma cruzi

Selective Blockade of Trypanosomatid Protein Synthesis by a Recombinant Antibody Anti-Trypanosoma cruzi P2β Protein

The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope

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

Multimerization of an Apoptogenic TRAIL-Mimicking Peptide by Using Adamantane-Based Dendrons

We have developed a straightforward strategy to multimerize an apoptogenic peptide that mimics the natural tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) by using adamantane‐based dendrons as multivalent scaffolds. The selective binding affinity of the ligands to TRAIL receptor 2 (TR2) was studied by surface plasmon resonance, thus demonstrating that the trimeric and hexameric forms of the peptide exert an increased affinity of about 1500‐ and 20 000‐fold, respectively, relative to the monomer. Moreover, only the trimeric and hexameric ligands were able to induce cell death in TR2 expressing cells (BJAB), thus confirming that a multivalent form of the peptide is necessary to trigger a substantial TR2‐dependent apoptotic response in vitro. These results provide interesting insight into the multivalency effect on biological ligand/receptor interactions for future therapeutic applications

Gel filtration analysis of BAFFR-Fc, TACI-Fc, Fn14-Fc and atacicept.

<p>Panel A. Superdex-200 elution profiles of the indicated receptors-Fc monitored at 280 nm. The elution position of molecular mass standards is indicated at the top of the profiles. Panel B. BMDMs were treated with TACI-Fc obtained before (total preparation) or after size exclusion chromatography (fraction 8 with high molecular mass aggregates and fraction 14 with dimeric TACI-Fc). Cells were also treated with atacicept (before size fractionation), a form of TACI-Fc unable to bind to Fc receptors. Cell activation was monitored by western blotting with an anti-phospho ERK1/2 antibody. Panel C. BAFF-sensitive BAFFR:Fas expressing cells were stimulated with the indicated concentration of BAFF 60-mer, in the presence or absence of atacicept at a fixed concentration of 300 ng/ml. Cell viability was monitored with a colorimetric assay.</p

Signalling by BAFFR-Fc and TACI-Fc also takes place in APRIL^−/− and BAFF^−/− BMDMs, and is not increased in BMDMs with uncleavable BAFF.

<p>Bone marrow-derived macrophages from various mice in the C57BL/6 background were stimulated for the indicated times with BAFFR-Fc, TACI-Fc or Fn14-Fc at 5 µg/ml, or with LPS at 50 ng/ml. Cells were lysed and analyzed by Western blotting with the indicated antibodies. Panel A: wt BMDMs. Panel B: APRIL^−/− BMDMs. Panel C: BAFF^−/− BMDMs. Panel D: BMDMs from BAFF^−/− mice overexpressing non-cleavable BAFF from a BAC transgene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061350#pone.0061350-Bossen2" target="_blank">[23]</a>. *: as judged by the anti-tubulin blot, less sample was accidentally loaded.</p

Impaired TACI-Fc signalling in FcRγ^−/− BMDMs.

<p>Bone marrow-derived macrophages from WT and FcRγ^−/− C57BL/6 mice were stimulated for the indicated times with TACI-Fc at 10 µg/ml, atacicept at 10 µg/ml, or with LPS at 50 ng/ml. Cell extracts were analyzed by western blotting with the indicated antibodies.</p