Laure Willen

Human Cytomegalovirus Glycoprotein UL141 Targets the TRAIL Death Receptors to Thwart Host Innate Antiviral Defenses

Summary Death receptors (DRs) of the TNFR superfamily contribute to antiviral immunity by promoting apoptosis and regulating immune homeostasis during infection, and viral inhibition of DR signaling can alter immune defenses. Here we identify the human cytomegalovirus (HCMV) UL141 glycoprotein as necessary and sufficient to restrict TRAIL DR function. Despite showing no primary sequence homology to TNF family cytokines, UL141 binds the ectodomains of both human TRAIL DRs with affinities comparable to the natural ligand TRAIL. UL141 binding promotes intracellular retention of the DRs, thus protecting virus infected cells from TRAIL and TRAIL-dependent NK cell-mediated killing. The identification of UL141 as a herpesvirus modulator of the TRAIL DRs strongly implicates this pathway as a regulator of host defense to HCMV and highlights UL141 as a pleiotropic inhibitor of NK cell effector function.

Functional analysis of Ectodysplasin-A mutations causing selective tooth agenesis

Mutations of the Ectodysplasin-A (EDA) gene are generally associated with the syndrome hypohidrotic ectodermal dysplasia (MIM 305100), but they can also manifest as selective, non-syndromic tooth agenesis (MIM300606). We have performed an in vitro functional analysis of six selective tooth agenesis-causing EDA mutations (one novel and five known) that are located in the C-terminal tumor necrosis factor homology domain of the protein. Our study reveals that expression, receptor binding or signaling capability of the mutant EDA1 proteins is only impaired in contrast to syndrome-causing mutations, which we have previously shown to abolish EDA1 expression, receptor binding or signaling. Our results support a model in which the development of the human dentition, especially of anterior teeth, requires the highest level of EDA-receptor signaling, whereas other ectodermal appendages, including posterior teeth, have less stringent requirements and form normally in response to EDA mutations with reduced activity.

Mutation of the BAFF furin cleavage site impairs B‐cell homeostasis and antibody responses

B‐cell‐activating factor of the TNF family (BAFF)/BLyS contributes to B‐cell homeostasis and function in the periphery. BAFF is expressed as a membrane‐bound protein or released by proteolytic cleavage, but the functional importance of this processing event is poorly understood. Mice expressing BAFF with a mutated furin consensus cleavage site, i.e. furin‐mutant BAFF (fmBAFF), were not different from BAFF‐deficient mice with regard to their B‐cell populations and responses to immunization. It is however noteworthy that an alternative processing event releases some soluble BAFF in fmBAFF mice. Mild overexpression (∼5‐fold) of fmBAFF alone generated intermediate levels of B cells without improving humoral responses to immunization. Processed BAFF was however important for B‐cell homeostasis, as peripheral B‐cell populations and antibody responses were readily restored by administration of soluble BAFF trimers in BAFF‐deficient mice. However, the rescue of CD23 expression in B cells of BAFF‐deficient mice required both soluble BAFF trimers and fmBAFF, or a polymeric form of soluble BAFF (BAFF 60‐mer). These results point to a predominant role of processed BAFF for B‐cell homeostasis and function, and indicate possible accessory roles for membrane‐bound BAFF.

Biological Activity of Ectodysplasin A Is Conditioned by Its Collagen and Heparan Sulfate Proteoglycan-binding Domains

Mutations in the TNF family ligand EDA1 cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by defective development of skin appendages. The EDA1 protein displays a proteolytic processing site responsible for its conversion to a soluble form, a collagen domain, and a trimeric TNF homology domain (THD) that binds the receptor EDAR. In-frame deletions in the collagen domain reduced the thermal stability of EDA1. Removal of the collagen domain decreased its activity about 100-fold, as measured with natural and engineered EDA1-responsive cell lines. The collagen domain could be functionally replaced by multimerization domains or by cross-linking antibodies, suggesting that it functions as an oligomerization unit. Surprisingly, mature soluble EDA1 containing the collagen domain was poorly active when administered in newborn, EDA-deficient (Tabby) mice. This was due to a short stretch of basic amino acids located at the N terminus of the collagen domain that confers EDA1 with proteoglycan binding ability. In contrast to wild-type EDA1, EDA1 with mutations in this basic sequence was a potent inducer of tail hair development in vivo. Thus, the collagen domain activates EDA1 by multimerization, whereas the proteoglycan-binding domain may restrict the distribution of endogeneous EDA1 in vivo.

Molecular and therapeutic characterization of anti- Ectodysplasin A receptor (EDAR) agonist monoclonal antibodies.

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC50 of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.

Structure-function analysis of Eiger, the Drosophila TNF homolog.

Tumor necrosis factor (TNF) family ligands in vertebrates are type II transmembrane proteins with functions in the regulation of immunity, bone homeostasis and more [1]. Drosophila expresses a single TNF homologue, Eiger [2, 3], which contains in addition to the homotrimeric C-terminal TNF homology domain (THD) an extended extracellular portion harboring potential protease cleavage sites (Figure 1A-i). One of these sites (Val 145) was identified experimentally [4]. A second potential cleavage site (Arg 211) resembling a canonical furin consensus sequence (R-K-S-R compared to R-X-R/K-R) was cleaved by a furin-like activity when expressed in mammalian 293T cells (Supplementary information, Figure S1). Eiger modulates host responses to Salmonella infections and resistance against extracellular pathogens in Drosophila [5, 6]. When expressed in the eye, it induces cell death via a well-defined pathway (Figure 1B) [2, 3, 7]. The structural requirements for Eiger’s activity are however unknown. As reported previously [3], full-length Eiger expressed in the Drosophila eye under the control of GMR-Gal4 induced a severe apoptotic eye phenotype (Figure 1A-i and 1C-i). Expression of the entire extracellular domain (ecto-Eiger-60) also resulted in complete eye loss (Figure 1A-ii and 1C-iv). As anticipated, the THD that contains the receptor-binding domain in all vertebrate TNF ligands [8] was required for the activity (Figure 1A-vii and 1A-viii). However, shorter forms of soluble Eiger containing the THD alone (Figure 1A-v and 1A-vi) or corresponding to predicted processed forms (Figure 1A-iii and 1A-iv) were inactive despite protein expression, and also failed to interfere with death induced by full-length Eiger or ecto-Eiger-60 (data not shown). Moreover, Eiger lacking the stalk also failed to induce apoptosis (Figure 1A-ix). All these data establish both the THD and the membrane-proximal portion of the stalk (sequence 60145) as essential determinants of Eiger’s activity. The JNK pathway mediates the apoptotic activity of Eiger [2, 3, 7] (Figure 1B). Accordingly, the apoptotic activity of ecto-Eiger-60 was totally inhibited when expressed in the eye of dTAK1 hemizygous mutant flies (Figure 1C-v), similar to full-length Eiger expression (Figure 1C-ii). Interestingly, a novel “hanging eye” phenotype was observed when ecto-Eiger-60 was coexpressed in the eye with Puckered, a cell-autonomous inhibitor of Basket [9] (Figure 1C-vi and 1C-vi′). This phenotype, which was not observed when full-length Eiger was co-expressed with Puckered [3] (Figure 1C-iii and 1C-iii′), suggests that soluble Eiger (ecto-Eiger-60) can kill the surrounding tissue that is not protected by Puckered. This was confirmed by clonal analysis in the Drosophila wing imaginal disc. In this experiment, GFPpositive clones co-expressing ecto-Eiger-60 and Puckered induced caspase-3 activation in neighbouring cells where Puckered was not overexpressed (Figure 1D-iv, 1D-v and 1D-vi). In contrast, full-length Eiger failed to induce obvious activation of caspase-3 within or outside the clone when co-expressed with Puckered (Figure 1Di, 1D-ii and 1D-iii). These results demonstrate that both full-length Eiger and ecto-Eiger-60 induce a JNK-mediated cell death, but that soluble Eiger is able to act over longer distances. We conclude from these experiments that soluble processed forms of Eiger, if produced at all in the fly, are unlikely mediators of cell death and that the membrane-proximal portion of Eiger may contain a novel functional domain not previously described within the TNF family.

Tools and Techniques to Study Ligand–Receptor Interactions and Receptor Activation by TNF Superfamily Members

Ligands and receptors of the TNF superfamily are therapeutically relevant targets in a wide range of human diseases. This chapter describes assays based on ELISA, immunoprecipitation, FACS, and reporter cell lines to monitor interactions of tagged receptors and ligands in both soluble and membrane-bound forms using unified detection techniques. A reporter cell assay that is sensitive to ligand oligomerization can identify ligands with high probability of being active on endogenous receptors. Several assays are also suitable to measure the activity of agonist or antagonist antibodies, or to detect interactions with proteoglycans. Finally, self-interaction of membrane-bound receptors can be evidenced using a FRET-based assay. This panel of methods provides a large degree of flexibility to address questions related to the specificity, activation, or inhibition of TNF-TNF receptor interactions in independent assay systems, but does not substitute for further tests in physiologically relevant conditions.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties

Background: The B cell survival factors B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) can heteromerize. Results: BAFF-APRIL2 and APRIL-BAFF2 heteromers have distinct receptor-binding specificities and activities. Conclusion: BAFF-APRIL2 resembles APRIL, and APRIL-BAFF2 resembles BAFF but poorly activates the BAFF receptor. Significance: Heteromers should be taken into account when evaluating the physiology or pharmacological inhibition of BAFF and APRIL. The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

Generation and Characterization of Function-blocking Anti-ectodysplasin A (EDA) Monoclonal Antibodies That Induce Ectodermal Dysplasia

Background: The TNF family ligand EDA1 is required for development of hair, teeth, and many glands. Results: Mouse fetuses exposed to anti-EDA antibodies develop a permanent ectodermal dysplasia. Conclusion: Monoclonal anti-EDA antibodies block EDA at stoichiometric ratio in vitro and in vivo. Significance: Down-modulation of EDA has useful research and potentially therapeutic applications. Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

No Evidence That Soluble TACI Induces Signalling via Membrane-Expressed BAFF and APRIL in Myeloid Cells

Myeloid cells express the TNF family ligands BAFF/BLyS and APRIL, which exert their effects on B cells at different stages of differentiation via the receptors BAFFR, TACI (Transmembrane Activator and CAML-Interactor) and/or BCMA (B Cell Maturation Antigen). BAFF and APRIL are proteins expressed at the cell membrane, with both extracellular and intracellular domains. Therefore, receptor/ligand engagement may also result in signals in ligand-expressing cells via so-called “reverse signalling”. In order to understand how TACI-Fc (atacicept) technically may mediate immune stimulation instead of suppression, we investigated its potential to activate reverse signalling through BAFF and APRIL. BAFFR-Fc and TACI-Fc, but not Fn14-Fc, reproducibly stimulated the ERK and other signalling pathways in bone marrow-derived mouse macrophages. However, these effects were independent of BAFF or APRIL since the same activation profile was observed with BAFF- or APRIL-deficient cells. Instead, cell activation correlated with the presence of high molecular mass forms of BAFFR-Fc and TACI-Fc and was strongly impaired in macrophages deficient for Fc receptor gamma chain. Moreover, a TACI-Fc defective for Fc receptor binding elicited no detectable signal. Although these results do not formally rule out the existence of BAFF or APRIL reverse signalling (via pathways not tested in this study), they provide no evidence in support of reverse signalling and point to the importance of using appropriate specificity controls when working with Fc receptor-expressing myeloid cells.