CVID-Associated B Cell Activating Factor Receptor Variants Change Receptor Oligomerization, Ligand Binding, and Signaling Responses.

PURPOSE: Binding of the B cell activating factor (BAFF) to its receptor (BAFFR) activates in mature B cells many essential pro-survival functions. Null mutations in the BAFFR gene result in complete BAFFR deficiency and cause a block in B cell development at the transition from immature to mature B cells leading therefore to B lymphopenia and hypogammaglobulinemia. In addition to complete BAFFR deficiency, single nucleotide variants encoding BAFFR missense mutations were found in patients suffering from common variable immunodeficiency (CVID), autoimmunity, or B cell lymphomas. As it remained unclear to which extent such variants disturb the activity of BAFFR, we performed genetic association studies and developed a cellular system that allows the unbiased analysis of BAFFR variants regarding oligomerization, signaling, and ectodomain shedding. METHODS: In addition to genetic association studies, the BAFFR variants P21R, A52T, G64V, DUP92-95, P146S, and H159Y were expressed by lentiviral gene transfer in DG-75 Burkitt's lymphoma cells and analyzed for their impacts on BAFFR function. RESULTS: Binding of BAFF to BAFFR was affected by P21R and A52T. Spontaneous oligomerization of BAFFR was disturbed by P21R, A52T, G64V, and P146S. BAFF-dependent activation of NF-κB2 was reduced by P21R and P146S, while interactions between BAFFR and the B cell antigen receptor component CD79B and AKT phosphorylation were impaired by P21R, A52T, G64V, and DUP92-95. P21R, G64V, and DUP92-95 interfered with phosphorylation of ERK1/2, while BAFF-induced shedding of the BAFFR ectodomain was only impaired by P21R. CONCLUSION: Although all variants change BAFFR function and have the potential to contribute as modifiers to the development of primary antibody deficiencies, autoimmunity, and lymphoma, P21R is the only variant that was found to correlate positively with CVID.

Antibodies That Block or Activate Mouse B Cell Activating Factor of the Tumor Necrosis Factor (TNF) Family (BAFF), Respectively, Induce B Cell Depletion or B Cell Hyperplasia.

B cell activating factor of the TNF family (BAFF), also known as B lymphocyte stimulator, is a ligand required for the generation and maintenance of B lymphocytes. In this study, the ability of different monoclonal antibodies to recognize, inhibit, or activate mouse BAFF was investigated. One of them, a mouse IgG1 named Sandy-2, prevented the binding of BAFF to all of its receptors, BAFF receptor, transmembrane activator and calcium modulating ligand interactor, and B cell maturation antigen, at a stoichiometric ratio; blocked the activity of mouse BAFF on a variety of cell-based reporter assays; and antagonized the prosurvival action of BAFF on primary mouse B cells in vitro A single administration of Sandy-2 in mice induced B cell depletion within 2 weeks, down to levels close to those observed in BAFF-deficient mice. This depletion could then be maintained with a chronic treatment. Sandy-2 and a previously described rat IgG1 antibody, 5A8, also formed a pair suitable for the sensitive detection of endogenous circulating BAFF by ELISA or using a homogenous assay. Interestingly, 5A8 and Sandy-5 displayed activities opposite to that of Sandy-2 by stimulating recombinant BAFF in vitro and endogenous BAFF in vivo These tools will prove useful for the detection and functional manipulation of endogenous mouse BAFF and provide an alternative to the widely used BAFF receptor-Fc decoy receptor for the specific depletion of BAFF in mice.

Thioether analogues of disulfide-bridged cyclic peptides targeting death receptor 5: conformational analysis, dimerisation and consequences for receptor activation.

Cyclic peptides containing redox-stable thioether bridges might provide a useful alternative to disulfide-bridged bioactive peptides. We report the effect of replacing the disulfide bridge with a lanthionine linkage in a 16-mer cyclic peptide that binds to death receptor 5 (DR5, TRAIL-R2). Upon covalent oligomerisation, the disulfide-bridged peptide has previously shown similar behaviour to that of TNF-related apoptosis inducing ligand (TRAIL), by selectively triggering the DR5 cell death pathway. The structural and biological properties of the DR5-binding peptide and its desulfurised analogue were compared. Surface plasmon resonance (SPR) data suggest that these peptides bind DR5 with comparable affinities. The same holds true for dimeric versions of these peptides: the thioether is able to induce DR5-mediated apoptosis of BJAB lymphoma and tumorigenic BJELR cells, albeit to a slightly lower extent compared to its disulfide homologue. NMR analysis revealed subtle variation in the conformations of the two peptides and suggests that the thioether peptide is slightly less folded than its disulfide homologue. These observations could account for the different capability of the two dimers to cluster DR5 receptors on the cell surface and to trigger apoptosis. Nevertheless, our results suggest that the thioether peptide is a potential candidate for evaluation in animal models.

Cysteine-rich domain 1 of CD40 mediates receptor self-assembly.

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a K(D) of 0.6 µM. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state.

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.

Protocol to study the oligomeric organization of single-span transmembrane peptides using molecular dynamics simulations.

Herein, you will find detailed information for the preparation of a coarse-grained array of peptides embedded in a lipid membrane. It contains all the steps to set up and run a molecular dynamic simulation using a coarse-grained approach. We provide analytical tools and scripts for generating a residue-level contact matrix between multiple peptides, as well as geometric analysis of arrangements between multiple peptides. This protocol was designed to study the organization of transmembrane peptides in an unbiased manner using computational approaches. For complete details on the use and execution of this protocol, please refer to Smulski et al. (2022).

BAFFR activates PI3K/AKT signaling in human naive but not in switched memory B cells through direct interactions with B cell antigen receptors.

Binding of BAFF to BAFFR activates in mature B cells PI3K/AKT signaling regulating protein synthesis, metabolic fitness, and survival. In humans, naive and memory B cells express the same levels of BAFFR, but only memory B cells seem to survive without BAFF. Here, we show that BAFF activates PI3K/AKT only in naive B cells and changes the expression of genes regulating migration, proliferation, growth, and survival. BAFF-induced PI3K/AKT activation requires direct interactions between BAFFR and the B cell antigen receptor (BCR) components CD79A and CD79B and is enhanced by the AKT coactivator TCL1A. Compared to memory B cells, naive B cells express more surface BCRs, which interact better with BAFFR than IgG or IgA, thus allowing stronger responses to BAFF. As ablation of BAFFR in naive and memory B cells causes cell death independent of BAFF-induced signaling, BAFFR seems to act also as an intrinsic factor for B cell survival.

Differential trafficking of ligands trogocytosed via CD28 versus CTLA4 promotes collective cellular control of co-stimulation.

Intercellular communication is crucial for collective regulation of cellular behaviors. While clustering T cells have been shown to mutually control the production of key communication signals, it is unclear whether they also jointly regulate their availability and degradation. Here we use newly developed reporter systems, bioinformatic analyses, protein structure modeling and genetic perturbations to assess this. We find that T cells utilize trogocytosis by competing antagonistic receptors to differentially control the abundance of immunoregulatory ligands. Specifically, ligands trogocytosed via CD28 are shuttled to the T cell surface, enabling them to co-stimulate neighboring T cells. In contrast, CTLA4-mediated trogocytosis targets ligands for degradation. Mechanistically, this fate separation is controlled by different acid-sensitivities of receptor-ligand interactions and by the receptor intracellular domains. The ability of CD28 and CTLA4 to confer different fates to trogocytosed ligands reveals an additional layer of collective regulation of cellular behaviors and promotes the robustness of population dynamics.

Ligand-independent oligomerization of TACI is controlled by the transmembrane domain and regulates proliferation of activated B cells.

In mature B cells, TACI controls class-switch recombination and differentiation into plasma cells during T cell-independent antibody responses. TACI binds the ligands BAFF and APRIL. Approximately 10% of patients with common variable immunodeficiency (CVID) carry TACI mutations, of which A181E and C172Y are in the transmembrane domain. Residues A181 and C172 are located on distinct sides of the transmembrane helix, which is predicted by molecular modeling to spontaneously assemble into trimers and dimers. In human B cells, these mutations impair ligand-dependent (C172Y) and -independent (A181E) TACI multimerization and signaling, as well as TACI-enhanced proliferation and/or IgA production. Genetic inactivation of TACI in primary human B cells impaired survival of CpG-activated cells in the absence of ligand. These results identify the transmembrane region of TACI as an active interface for TACI multimerization in signal transduction, in particular for ligand-independent signals. These functions are perturbed by CVID-associated mutations.

Antibody covalent immobilization on carbon nanotubes and assessment of antigen binding.

Controlling the covalent bonding of antibodies onto functionalized carbon nanotubes is a key step in the design and preparation of nanotube-based conjugates for targeting cancer cells. For this purpose, an anti-MUC1 antibody (Ab) is linked to both multi-walled (MWCNTs) and double-walled carbon nanotubes (DWCNTs) using different synthetic strategies. The presence of the Ab attached to the nanotubes is confirmed by gel electrophoresis and thermogravimetric analysis. Most importantly, molecular recognition of the antigen by surface plasmon resonance is able to determine similar Ab binding capacities for both Ab-DWCNTs and Ab-MWCNTs. These results are very relevant for the design of future receptor-targeting strategies using chemically functionalized carbon nanotubes.

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2.

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF-2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1ß, TcP2α, TcP2ß and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two-hybrid (Y2H) protein-protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1ß-TcP2α and TcP1ß-TcP2ß. Moreover P2 but not P1 proteins were able to homo-oligomerize. In addition, the region comprising amino acids 210-270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2ß were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF-2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF-2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0.

Coarse Grained Molecular Dynamic Simulations for the Study of TNF Receptor Family Members' Transmembrane Organization.

The Tumor Necrosis Factor (TNF) and the TNF receptor (TNFR) superfamilies are composed of 19 ligands and 30 receptors, respectively. The oligomeric properties of ligands, both membrane bound and soluble, has been studied most. However, less is known about the oligomeric properties of TNFRs. Earlier reports identified the extracellular, membrane-distal, cysteine-rich domain as a pre-ligand assembly domain which stabilizes receptor dimers and/or trimers in the absence of ligand. Nevertheless, recent reports based on structural nuclear magnetic resonance (NMR) highlight the intrinsic role of the transmembrane domains to form dimers (p75NTR), trimers (Fas), or dimers of trimers (DR5). Thus, understanding the structural basis of transmembrane oligomerization may shed light on the mechanism for signal transduction and the impact of disease-associated mutations in this region. To this end, here we used an in silico coarse grained molecular dynamics approach with Martini force field to study TNFR transmembrane homotypic interactions. We have first validated this approach studying the three TNFR described by NMR (p75NTR, Fas, and DR5). We have simulated membrane patches composed of 36 helices of the same receptor equidistantly distributed in order to get unbiassed information on spontaneous proteins assemblies. Good agreement was found in the specific residues involved in homotypic interactions and we were able to observe dimers, trimers, and higher-order oligomers corresponding to those reported in NMR experiments. We have, applied this approach to study the assembly of disease-related mutations being able to assess their impact on oligomerization stability. In conclusion, our results showed the usefulness of coarse grained simulations with Martini force field to study in an unbiased manner higher order transmembrane oligomerization.

BAFF/APRIL System Is Functional in B-Cell Acute Lymphoblastic Leukemia in a Disease Subtype Manner.

BAFF, APRIL and their receptors regulate the survival, maturation and homeostasis of mature B-cells. Despite the lack of a functional role of BAFF/APRIL system during normal early B-cell development, previous studies indicated a contribution of these molecules in the pathogenesis of B-lineage acute lymphoblastic leukemia (B-ALL). Here, we evaluated the expression of this system in B-ALL and its involvement in spontaneous and drug-induced apoptosis of B-lymphoblasts, taking into consideration the distinct disease subtypes. We found that BAFFR is the most predominant aberrantly expressed receptor in B-ALL and that its expression, along with BCMA and APRIL, positively correlates with the maturation stage of B-lymphoblasts. Moreover, the binding of the E2A-PBX1 chimeric protein to the BAFFR promoter suggests that the transcriptional activator promotes the increase in BAFFR expression observed in about 50% of pre-B-ALL patients carrying the t (1, 19) translocation. BAFF binding to BAFFR led to the processing of NF-κB2 p100 in pre-B ALL cells suggesting that BAFFR can activate the NF-κB2 pathway in pre-B ALL cells. Surprisingly, we found that BAFF treatment promotes the cell death of primary BCR-ABL+ BAFFR+ pre-B-lymphoblasts in adult B-ALL. It also enhances glucocorticoid-induced apoptosis in the E2A-PBX1+ pre-B-ALL cell line 697. These data suggest that BAFF/BAFFR signaling in B-ALL cells differs from normal B cells and that it may affect the pathogenesis of the disease.

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity.

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sdelta. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A(51) residue on 28Sdelta. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.

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.

A loop region of BAFF controls B cell survival and regulates recognition by different inhibitors.

The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors.

Hetero-oligomerization between the TNF receptor superfamily members CD40, Fas and TRAILR2 modulate CD40 signalling.

TNF receptor superfamily members (TNFRSF) such as CD40, Fas and TRAIL receptor 2 (TRAILR2) participate to the adaptive immune response by eliciting survival, proliferation, differentiation and/or cell death signals. The balance between these signals determines the fate of the immune response. It was previously reported that these receptors are able to self-assemble in the absence of ligand through their extracellular regions. However, the role of this oligomerization is not well understood, and none of the proposed hypotheses take into account potential hetero-association of receptors. Using CD40 as bait in a flow cytometry Förster resonance energy transfer assay, TNFRSF members with known functions in B cells were probed for interactions. Both Fas and TRAILR2 associated with CD40. Immunoprecipitation experiments confirmed the interaction of CD40 with Fas at the endogenous levels in a BJAB B-cell lymphoma cell line deficient for TRAILR2. TRAILR2-expressing BJAB cells displayed a robust CD40-TRAILR2 interaction at the expense of the CD40-Fas interaction. The same results were obtained by proximity ligation assay, using TRAILR2-positive and -negative BJAB cells and primary human B cells. Expression of the extracellular domains of Fas or TRAILR2 with a glycolipid membrane anchor specifically reduced the intrinsic signalling pathway of CD40 in 293T cells. Conversely, BJAB cells lacking endogenous Fas or TRAILR2 showed an increased NF-κB response to CD40L. Finally, upregulation of TRAILR2 in primary human B cells correlated with reduced NF-κB activation and reduced proliferation in response to CD40L. Altogether, these data reveal that selective interactions between different TNFRSF members may modulate ligand-induced responses upstream signalling events.

BAFF- and TACI-Dependent Processing of BAFFR by ADAM Proteases Regulates the Survival of B Cells.

B cell activating factor (BAFF) provides B cells with essential survival signals. It binds to three receptors: BAFFR, TACI, and BCMA that are differentially expressed by B cell subsets. BAFFR is early expressed in circulating B cells and provides key signals for further maturation. Here, we report that highly regulated BAFFR processing events modulate BAFF responses. BAFFR processing is triggered by BAFF binding in B cells co-expressing TACI and it is executed by the metalloproteases ADAM10 and ADAM17. The degree of BAFF oligomerization, the expression of ADAM proteins in different B cell subsets, and the activation status of the cell determine the proteases involved in BAFFR processing. Inhibition of ADAM10 augments BAFF-dependent survival of primary human B cells, whereas inhibition of ADAM17 increases BAFFR expression levels on germinal center B cells. Therefore, BAFF-induced processing of BAFFR regulates BAFF-mediated B cell responses in a TACI-dependent manner.