Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.Resistance (R) proteins play a central role in the recognition-based immune system of plants. Unlike vertebrates, plants lack an adaptive immune system with highly specialized immune cells. Instead, they rely on an innate immune system in which each cell is autonomous. Two types of immune receptors can be distinguished in plants, pathogen-associated molecular patterns recognition receptors that detect conserved molecular patterns in plant pathogens and intracellular R proteins that recognize specific effectors employed by pathogens as modifiers of host metabolism or defense mechanisms (Jones and Dangl, 2006). Effector-triggered activation of R proteins leads to an array of protective responses, often culminating in programmed cell death at the site of infection (Greenberg and Yao, 2004), thereby preventing further ingress of the pathogen. Pathogens have evolved mechanisms to evade recognition by R proteins and to regain their virulence (Dodds and Rathjen, 2010). This continuous coevolutionary process between host and pathogen has resulted in a reservoir of highly diverse R proteins in plants, enabling them to counteract a wide range of pathogens and pests.The most common class of R proteins consists of nucleotide-binding (NB)-leucine-rich repeat (LRR) proteins with a tripartite domain architecture, which roughly corresponds to an N-terminal response domain (a coiled coil [CC] or Toll/Interleukin-1 receptor [TIR] domain) involved in downstream signaling, a central molecula...

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and was further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+ B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1.

Background: Direct targets for plant NLR proteins in immune signaling are largely unknown.Results: The Rx1 NLR protein of potato binds and distorts DNA following pathogen perception, resulting in immune activation.Conclusion: DNA is a direct signaling target for a plant NLR immune receptor.Significance: Plant NLR receptors might regulate immune transcriptional responses by directly interacting with plant chromatin.

SummaryTransforming growth factor beta (TGF‐β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF‐β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF‐β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF‐β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF‐β1 in the absence of the latency‐associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP‐TGF‐β1, we were able to show that processing of the latent complex by a furin‐like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP‐TGF‐β1, and co‐expression of human furin enabled the proteolytic processing of latent TGF‐β1. Engineering the plant post‐translational machinery by co‐expressing human furin also enhanced the accumulation of biologically active TGF‐β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.

Heterologous expression platforms of biopharmaceutical proteins have been significantly improved over the last decade. Further improvement can be established by examining the intrinsic properties of proteins. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with a short half-life that plays an important role in re-establishing immune homeostasis. This homodimeric protein of 36 kDa has significant therapeutic potential to treat inflammatory and autoimmune diseases. In this study we show that the major production bottleneck of human IL-10 is not protein instability as previously suggested, but extensive multimerisation due to its intrinsic 3D domain swapping characteristic. Extensive multimerisation of human IL-10 could be visualised as granules in planta. On the other hand, mouse IL-10 hardly multimerised, which could be largely attributed to its glycosylation. By introducing a short glycine-serine-linker between the fourth and fifth alpha helix of human IL-10 a stable monomeric form of IL-10 (hIL-10mono) was created that no longer multimerised and increased yield up to 20-fold. However, hIL-10mono no longer had the ability to reduce pro-inflammatory cytokine secretion from lipopolysaccharide-stimulated macrophages. Forcing dimerisation restored biological activity. This was achieved by fusing human IL-10mono to the C-terminal end of constant domains 2 and 3 of human immunoglobulin A (Fcα), a natural dimer. Stable dimeric forms of IL-10, like Fcα-IL-10, may not only be a better format for improved production, but also a more suitable format for medical applications.

Summary The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro‐inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF‐α, and Ustekinumab, directed against the interleukin‐12p40 subunit. We evaluated antibody yield, quality and N‐glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell‐based assay. However, IgA1κ‐based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ‐ and IgG1κ‐based Infliximab were enriched in oligomannose‐type N‐glycan structures. For IgG1κ‐based Ustekinumab and Adalimumab, the major N‐glycan type was the typical plant complex N‐glycan, biantennary with terminal N‐acetylglucosamine, β1,2‐xylose and core α1,3‐fucose. In contrast, the major N‐glycan on the IgA‐based antibodies was xylosylated, but lacked core α1,3‐fucose and one terminal N‐acetylglucosamine. This type of N‐glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant‐produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N‐glycan an antibody receives.

Secretory IgA (sIgA) is a crucial antibody in host defense at mucosal surfaces. It is a promising antibody isotype in a variety of therapeutic settings such as passive vaccination and treatment of inflammatory disorders. However, heterologous production of this heteromultimeric protein complex is still suboptimal. The challenge is the coordinate expression of the four required polypeptides; the alpha heavy chain, the light chain, the joining chain, and part of the polymeric-Ig-receptor called the secretory component, in a 4:4:1:1 ratio. We evaluated the transient expression of three sIgAκ variants, harboring the heavy chain isotype α1, α2m1, or α2m2, of the clinical antibody Ustekinumab in planta. Ustekinumab is directed against the p40 subunit that is shared by the pro-inflammatory cytokines interleukin (IL)-12 and IL-23. A sIgA variant of this antibody may enable localized treatment of inflammatory bowel disease. Of the three different sIgA variants we obtained the highest yield with sIgA1κ reaching up to 373 μg sIgA/mg total soluble protein. The use of a multi-cassette vector containing all four expression cassettes was most efficient. However, not the expression strategy, but the incorporation of the joining chain turned out to be the limiting step for sIgA production. Our data demonstrate that transient expression in planta is suitable for the economic production of heteromultimeric protein complexes such as sIgA.