Receptor Cross-linking Research Articles

Differential regulation of polysaccharide-specific antibody responses to isolated polysaccharides, conjugate vaccines, and intact Gram-positive versus Gram-negative extracellular bacteria

Bacterial capsular polysaccharides are major virulence factors and are key targets in a number of licensed anti-bacterial vaccines. Their major characteristics are their large molecular weight and expression of repeating antigenic epitopes that mediate multivalent B cell receptor cross-linking. In addition, since the majority of these antigens cannot associate with MHC-II they fail to recruit CD4+ T cell help and are referred to as T cell-independent antigens. In this review I will discuss a series of studies from my laboratory that have underscored the importance of understanding polysaccharide-specific antibody responses within the context in which the PS is expressed (i.e. in isolation, as a component of conjugate vaccines, and expressed naturally by intact bacteria). We have shown that multivalent B cell receptor crosslinking, as mediated by polysaccharides, uniquely determines the qualitative response of the B cell to subsequent stimuli, but by itself is insufficient to induce antibody secretion or class switching. For these latter events to occur, second signals must act in concert with primary signals derived from the B cell receptor. The co-expression of polysaccharide and protein within intact bacteria promotes recruitment of CD4+ T cell help for the associated PS-specific IgG response, in contrast to isolated polysaccharides. Further, the particulate nature of extracellular bacteria confers properties to the polysaccharide-specific IgG response that makes it distinct immunologically from soluble conjugate vaccines. Finally, the underlying biochemical and/or structural differences that distinguish Gram-positive and Gram-negative bacteria appear to play critical roles in differentially regulating the associated polysaccharide-specific IgG responses to these groups of pathogens. These studies have a number of implications for the understanding and future design of polysaccharide-based vaccines.

Rainbow trout CK9, a CCL25-like ancient chemokine that attracts and regulates B cells and macrophages, the main antigen presenting cells in fish.

CK9 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to mammalian CCL25. Although CK9 is known to be transcriptionally regulated in response to inflammation particularly in mucosal tissues, its functionality has never been revealed. In the current work, we have demonstrated that CK9 is chemoattractant for antigen presenting cells (APCs) expressing major histocompatibility complex class II (MHC II) on the cell surface. Among these APCs, CK9 has a strong chemotactic capacity for both B cells (IgM+ and IgT+) and macrophages. Along with its chemotactic capacities, CK9 modulated the MHC II turnover of B lymphocytes and up-regulated the phagocytic capacity of both IgM+ cells and macrophages. Although CK9 had no lymphoproliferative effects, it increased the survival of IgT+ lymphocytes. Furthermore, we have established that the chemoattractant capacity of CK9 is strongly increased after pre-incubation of leukocytes with a T-independent antigen, whereas B cell receptor (BCR) cross-linking strongly abrogated their capacity to migrate to CK9, indicating that CK9 preferentially attracts B cells at the steady state or under BCR-independent stimulation. These results point to CK9 being a key regulator of B lymphocyte trafficking in rainbow trout, able to modulate innate functions of teleost B lymphocytes and macrophages.

CK9, an ancient rainbow trout chemokine that attracts and regulates B lymphocytes and macrophages

CK9 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to mammalian CCL25. Although CK9 is known to be transcriptionally regulated in response to inflammation particularly in mucosal tissues, its functionality has never been revealed. In the current work, we have demonstrated that CK9 has a strong chemotactic capacity for both B cells (IgM and IgT) and macrophages. Along with its chemotactic capacities, CK9 modulated the MHC II turnover of B lymphocytes and up-regulated the phagocytic capacity of both IgM cells and macrophages. Although CK9 had no lymphoproliferative effects, it increased the survival of IgT lymphocytes. Furthermore, we have established that the chemoattractant capacity of CK9 is strongly increased after pre-incubation of leukocytes with a T-independent antigen, whereas B cell receptor (BCR) cross-linking strongly abrogated their capacity to migrate to CK9, indicating that CK9 preferentially attracts B cells at the steady state or under BCR-independent stimulation. These results point to CK9 being a key regulator of B lymphocyte mucosal trafficking in teleost fish, able to modulate innate functions of teleost B lymphocytes and macrophages.

Differential effects of interleukin 6 and lipopolysaccharide on teleost B lymphocytes

In mammals, interleukin-6 (IL-6) is a pleiotropic cytokine that has the capacity to induce the differentiation of B cells into antibody-secreting cells (ASCs). On the other hand, lipopolysaccharide (LPS) acts on B cells as a T cell independent antigen producing a polyclonal cell activation that goes along with an increased proliferation and antibody secretion. LPS is also known to enhance the B cell antigen-presenting capacity of B cells. Very few studies have examined how teleost B cells respond to these two stimuli, thus the aim of our work was to evaluate the stimulatory capacity of recombinant rainbow trout (Oncorhynchus mykiss) IL-6 on spleen B cells, comparing its effects to those induced by LPS. We found that both IL-6 and LPS induced the proliferation of spleen IgM + B cells and activated NF-κB in these cells as determined by NF-κB translocation to the nucleus. Additionally, IL-6 and LPSstimulated B cells mobilized more intracellular calcium after B cell receptor cross-linking. Although both IL-6 and LPS provoked a significant differentiation of B cells to ASCs as determined by ELISPOT, the levels of secreted IgM mRNA only increased in response to IL-6, suggesting different pathways for increased antibody secretion. Concerning effects on their antigen-presenting capacities, IL-6 and LPS showed quite different effects. We observed a significant down-regulation of MHC-II surface expression on IL-6-stimulated B cells, but a significant up-regulation with LPS in comparison with control cells. Accordingly, we found a significant increase of the percentage of phagocytic IgM + B cells when splenocytes were stimulated with LPS but not with IL-6. This work provides important insights on the B-stimulating capacities of IL-6 and LPS not previously explored in teleost fish.

CD13/aminopeptidase N is a negative regulator of mast cell activation.

Antigen-induced mast cell (MC) activation via cross-linking of IgE-bound high-affinity receptors for IgE (FcεRI) underlies type I allergy and anaphylactic shock. Comprehensive knowledge of FcεRI regulation is thus required. We have identified a functional interaction between FcεRI and CD13 in murine MCs. Antigen-triggered activation of IgE-loaded FcεRI results in cocapping and cointernalization of CD13 and equivalent internalization rates of up to 40%. Cointernalization is not unspecific, because ligand-driven KIT internalization is not accompanied by CD13 internalization. Moreover, antibody-mediated cross-linking of CD13 causes IL-6 production in an FcεRI-dependent manner. These data are indicative of a functional interaction between FcεRI and CD13 on MCs. To determine the role of this interaction, CD13-deficient bone marrow-derived MCs (BMMCs) were analyzed. Intriguingly, antigen stimulation of CD13-deficient BMMCs results in significantly increased degranulation and proinflammatory cytokine production compared to wild-type cells. Furthermore, in a low-dose model of passive systemic anaphylaxis, antigen-dependent decrease in body temperature, reflecting the anaphylactic reaction, is substantially enhanced by the CD13 inhibitor bestatin (-5.9 ± 0.6°C) and by CD13 deficiency (-8.8 ± 0.6°C) in contrast to controls (-1.2 ± 1.97°C). Importantly, bestatin does not aggravate anaphylaxis in CD13-deficient mice. Thus, we have identified CD13 as a novel negative regulator of MC activation in vitro and in vivo-Zotz, J. S., Wölbing, F., Lassnig, C., Kauffmann, M., Schulte, U., Kolb, A., Whitelaw, B., Müller, M., Biedermann, T., Huber, M. CD13/aminopeptidase N is a negative regulator of mast cell activation.

Cultured Neurons on Fiduciary Markers and Small Quantum Dots with Monomeric Streptavidin for Super-Resolution Microscopy

Given the sub-diffraction limited size of neuronal synapses, it is desirable to image them with nanometer-scale super-resolution fluorescence microscopy. Here we image live cultured neurons with a highly stable small quantum dots, attached to a glutamatergic receptor via a new monofunctional streptavidin. On single synapses, it is able to achieve < 10 nm accuracy in 3 dimensions over the course of 15 minutes or more. Simultaneously, we have imaged a post-synaptic protein using a photoactivatable fluorescent protein, with a resolution of < 20 nm in x-y and <50 nm in z. By using a fiduciary pattern, passivated with poly-lysine and Laminin, the neuron was stable. Stage-drift could be corrected to < 1 nm in x and y, and < 15 nm in z-dimension. Chromatic aberration could be corrected to ∼ 3 nm. Small quantum dots, 85% likelihood, in contrast to commercially available quantum dots, which can only label synaptic glutamatergic receptors to <15%. The use of monofunctional streptavidin ensures that problems with cross-linking of receptors don’t occur. The use of super-resolution of small quantum dots argues that the role of inter-synaptic diffusion has been significantly over-stated.

The Binding Lifetime of Syk Kinase to the Immune Receptor FcεRI Regulates the Efficiency of Signal Propagation

The recruitment of intracellular signaling molecules to membrane receptors is a critical step in signal transduction. While many of the proteins involved in signaling cascades are known, how the spatiotemporal response of these protein-protein interactions regulates signal propagation is still not well defined. Using single molecule imaging in live cells, we visualized recruitment of the cytoplasmic tyrosine kinase Syk to the high affinity IgE receptor, FceRI. FceRI serves as the primary immunoreceptor on mast cells and basophils, and its activation results in the release of mediators associated with the allergic response. Using TIRF microscopy, we tracked individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FceRI activation. Analysis of these single molecule trajectories allowed us to characterize Syk mobility and binding lifetime over a range of receptor activating conditions. We find that receptor crosslinking causes a dose dependent increase in Syk residence time at the membrane, and that the mobility of Syk depends on the activating antigen dose. We also investigated the structure-function relationship of Syk interactions with FceRI. Binding of Syk to the receptor is thought to transition it from an autoinhibited to an active kinase. Phosphorylation of Syk within the receptor binding region (Y130) has been suggested to regulate its interactions by promoting a structural rearrangement. We show that mutation of this site to mimic constitutive phosphorylation (Y130E) decreases the Syk-FceRI interaction time. This subtle change in protein dynamics results in altered Syk phosphorylation kinetics that subsequently impair cellular responses, including deficiencies in calcium release and a complete loss of secretion. This data highlights the importance of finely-tuned protein-protein interactions in regulating cellular outcomes.

Non-T cell activation linker (NTAL) proteolytic cleavage as a terminator of activatory intracellular signals.

Non-T cell activation linker is an adaptor protein that is tyrosine phosphorylated upon cross-linking of immune receptors expressed on B lymphocytes, NK cells, macrophages, basophils, or mast cells, allowing the recruitment of cytosolic mediators for downstream signaling pathways. Fas receptor acts mainly as a death receptor, and when cross-linked with Fas ligand, many proteins are proteolytically cleaved, including several signaling molecules in T and B cells. Fas receptor triggering also interferes with TCR intracellular signals, probably by means of proteolytic cleavage of several adaptor proteins. We have previously found that the adaptor linker for activation of T cells, evolutionarily related to non-T cell activation linker, is cleaved upon proapoptotic stimuli in T lymphocytes and thymocytes, in a tyrosine phosphorylation-dependent fashion. Here, we describe non-T cell activation linker proteolytic cleavage triggered in human B cells and monocytes by Fas cross-linking and staurosporine treatment. Non-T cell activation linker is cleaved, producing an N-terminal fragment of ∼22 kDa, and such cleavage is abrogated in the presence of caspase 8/granzyme B and caspase 3 inhibitors. Moreover, we have identified an aspartic acid residue at which non-T cell activation linker is cleaved, which similar to linker for activation of T cells, this aspartic acid residue is located close to tyrosine and serine residues, suggesting an interdependence of phosphorylation and proteolytic cleavage. Consistently, induction of non-T cell activation linker phosphorylation by pervanadate inhibits its cleavage. Interestingly, the truncated isoform of non-T cell activation linker, generated after cleavage, has a decreased signaling ability when compared with the full-length molecule. Altogether, our results suggest that cleavage of transmembrane adaptors constitutes a general mechanism for signal termination of immune receptors.

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking.

Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE). In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR) as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease.

D-102 Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicity

Presenting vaccine antigens in particulate form can improve their immunogenicity by enhancing B cell receptor cross-linking and B cell activation. We describe ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664) to improve upon the promising neutralizing antibody responses obtained with BG505 SOSIP.664 alone. With the aim of increasing neutralization breadth we also generated SOSIP.664-ferritin nanoparticles from different viral strains from subtypes A, B and C. Trimer- bearing nanoparticles were significantly more immunogenic than trimers in both mice and rabbits. Furthermore, rabbits immunized with the trimer- bearing nanoparticles induced significantly higher neutralizing antibody responses than when the same trimers were delivered as soluble proteins. Thus, nanoparticle-displayed native-like trimers might be valuable in the quest for an HIV-1 vaccine aimed at inducing broadly neutralizing antibodies.

The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as “central” interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, “peripheral” interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.

Receptor Crosslinking in Drug Delivery: Detour to the Lysosome?

A universal method for delivery of bioactive molecules to lysosomes is highly desirable for a broad range of clinical applications. For instance, lysosomal targeting is important for delivery of ligand–drug conjugates and therapeutic strategies that involve degradation of growth-inducing oncogenic receptors. In this issue of Molecular Therapy, Moody and colleagues report a simple and generic method to achieve crosslinking of cell-surface receptors, which triggers internalization and subsequent redirected trafficking of the ligand–cargo complex to lysosomes.1 Crosslinking was achieved by employing biotinylated ligands that were caused to form complexes using (multivalent) streptavidin after receptor binding. The authors show that this method is applicable to otherwise noninternalized ligand–receptor complexes, such as trastuzumab, the antibody specific to Her2 (used for therapy of Her2-positive breast cancer). Similarly, the method was also able to target transferrin (Tf) to lysosomes. Although the method has only limited in vivo applications, the results should spur the development of alternative crosslinking methods and agents that could facilitate the development of more efficient strategies of macromolecular and/or targeted drug delivery, immunotherapy of cancer, and receptor ablation therapy.

Receptor Crosslinking: A General Method to Trigger Internalization and Lysosomal Targeting of Therapeutic Receptor:Ligand Complexes

A major unmet clinical need is a universal method for subcellular targeting of bioactive molecules to lysosomes. Delivery to this organelle enables either degradation of oncogenic receptors that are overexpressed in cancers, or release of prodrugs from antibody–drug conjugates. Here, we describe a general method that uses receptor crosslinking to trigger endocytosis and subsequently redirect trafficking of receptor:cargo complexes from their expected route, to lysosomes. By incubation of plasma membrane receptors with biotinylated cargo and subsequent addition of streptavidin to crosslink receptor:cargo–biotin complexes, we achieved rapid and selective lysosomal targeting of transferrin, an anti-MHC class I antibody, and the clinically approved anti-Her2 antibody trastuzumab. These three protein ligands each target a receptor with a distinct cellular function and intracellular trafficking profile. Importantly, we confirmed that crosslinking of trastuzumab increased lysosomal degradation of its cognate oncogenic receptor Her2 in breast cancer cell lines SKBR3 and BT474. These data suggest that crosslinking could be exploited for a wide range of target receptors, for navigating therapeutics through the endolysosomal pathway, for significant therapeutic benefit.

Nanoparticle-allergen interactions mediate human allergic responses: protein corona characterization and cellular responses.

BackgroundEngineered nanomaterials (ENMs) interact with different biomolecules as soon as they are in contact, resulting in the formation of a biomolecule ‘corona’. Hence, the ‘corona’ defines the biological identity of the ENMs and could affect the response of the immune system to ENM exposure. With up to 40 % of the world population suffering from type I allergy, a possible modulation of allergen effects by binding to ENMs is highly relevant with respect to work place and consumer safety. Therefore, the aim of this present study was to gain an insight into the interactions of gold nanoparticles with different seasonally and perennially occurring outdoor and indoor allergens.MethodsGold nanoparticles (AuNPs) were conjugated with the major allergens of birch pollen (Bet v 1), timothy grass pollen (Phl p 5) and house dust mite (Der p 1). The AuNP-allergen conjugates were characterized by means of TEM negative staining, dynamic light scattering (DLS), z-potential measurements and hyperspectral imaging. Furthermore, 3D models were constructed, based on the characterization data, to visualize the interaction between the allergens and the AuNPs surface. Differences in the activation of human basophil cells derived from birch/grass pollen- and house dust mite-allergic patients in response to free allergen and AuNP-allergen conjugates were determined using the basophil activation assay (BAT). Potential allergen corona replacement during BAT was controlled for using Western blotting. The protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was assessed, by an enzymatic activity assay and a cellular assay pertaining to lung type II alveolar epithelial cell tight junction integrity.ResultsThe formation of a stable corona was found for all three allergens used. Our data suggest, that depending on the allergen, different effects are observed after binding to ENMs, including enhanced allergic responses against Der p 1 and also, for some patients, against Bet v 1. Moreover elevated protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was found.ConclusionIn summary, this study presents that conjugation of allergens to ENMs can modulate the human allergic response, and that protease activity can be increased. Graphical Cross-linking of IgE receptors and degranulation of human basophils due to epitope alignment of nanoparticle-coated allergens. Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0113-0) contains supplementary material, which is available to authorized users.

Recent Advances in Celiac Disease from TTG to Gluten in Pee.

Recent Advances in Celiac Disease from TTG to Gluten in Pee.

Intravenous Immunoglobulin (IVIG) Attenuates TNF-Induced Pathologic Bone Resorption and Suppresses Osteoclastogenesis by Inducing A20 Expression.

Investigations on the therapeutic effects of intravenous immunoglobulin (IVIG) have focused on the suppression of autoantibody and immune complex-mediated inflammatory pathogenesis. Inflammatory diseases such as rheumatoid arthritis are often accompanied by excessive bone erosion but the effect of IVIG on osteoclasts, bone-resorbing cells, has not been studied. Here, we investigate whether IVIG directly regulates osteoclast differentiation and has therapeutic potential for suppressing osteoclast-mediated pathologic bone resorption. IVIG or cross-linking of Fcγ receptors with plate-bound IgG suppressed receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteoclastogenesis and expression of osteoclast-related genes such as integrin β3 and cathepsin K in a dose-dependent manner. Mechanistically, IVIG or plate-bound IgG suppressed osteoclastogenesis by downregulating RANKL-induced expression of NFATC1, the master regulator of osteoclastogenesis. IVIG suppressed NFATC1 expression by attenuating RANKL-induced NF-κB signaling, explained in part by induction of the inflammatory signaling inhibitor A20. IVIG administration attenuated in vivo osteoclastogenesis and suppressed bone resorption in the tumor necrosis factor (TNF)-induced calvarial osteolysis model. Our findings show that, in addition to suppressing inflammation, IVIG directly inhibits osteoclastogenesis through a mechanism involving suppression of RANK signaling. Direct suppression of osteoclast differentiation may provide beneficial effects on preserving bone mass when IVIG is used to treat rheumatic disorders.

T-cell activation or tolerization: the Yin and Yang of bacterial superantigens.

Bacterial superantigens (SAg) are exotoxins from pathogens which interact with innate and adaptive immune cells. The paradox that SAgs cause activation and inactivation/anergy of T-cells was soon recognized. The structural and molecular events following SAg binding to antigen presenting cells (APCs) followed by crosslinking of T-cell receptors were characterized in detail. Activation, cytokine burst and T-cell anergy have been described in vitro and in vivo. Later it became clear that SAg-induced T-cell anergy is in part caused by SAg-dependent activation of T-regulatory cells (Tregs). Although the main focus of analyses was laid on T-cells, it was also shown that SAg binding to MHC class II molecules on APCs induces a signal, which leads to activation and secretion of pro-inflammatory cytokines. Accordingly APCs are mandatory for T-cell activation. So far it is not known, whether APCs play a role during SAg-triggered activation of Tregs. We therefore tested whether in SAg (Streptococcal pyrogenic exotoxin A) -treated APCs an anti-inflammatory program is triggered in addition. We show here that not only the anti-inflammatory cytokine IL-10 and the co-inhibitory surface molecule PD-L1 (CD274) but also inhibitory effector systems like indoleamine 2,3-dioxygenase (IDO) or intracellular negative feedback loops (suppressor of cytokine signaling molecules, SOCS) are induced by SAgs. Moreover, cyclosporine A completely prevented induction of this program. We therefore propose that APCs triggered by SAgs play a key role in T-cell activation as well as inactivation and induction of Treg cells.

Novel insights from 3D models: the pivotal role of physical symmetry in epithelial organization.

3D tissue culture models are utilized to study breast cancer and other pathologies because they better capture the complexity of in vivo tissue architecture compared to 2D models. However, to mimic the in vivo environment, the mechanics and geometry of the ECM must also be considered. Here, we studied the mechanical environment created in two 3D models, the overlay protocol (OP) and embedded protocol (EP). Mammary epithelial acini features were compared using OP or EP under conditions known to alter acinus organization, i.e. collagen crosslinking and/or ErbB2 receptor activation. Finite element analysis and active microrheology demonstrated that OP creates a physically asymmetric environment with non-uniform mechanical stresses in radial and circumferential directions. Further contrasting with EP, acini in OP displayed cooperation between ErbB2 signalling and matrix crosslinking. These differences in acini phenotype observed between OP and EP highlight the functional impact of physical symmetry in 3D tissue culture models.

Design of smart HPMA copolymer-based nanomedicines

The state-of-the art in water-soluble macromolecular therapeutics has been reviewed. First the design principles for polymer-drug conjugates are discussed followed by two recent developments in the field: a) The design, synthesis and properties of backbone degradable N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates. The enhanced intravascular half-life of such conjugates creates a concentration gradient (blood vs. tumor) for an extended time interval resulting in increased solid tumor accumulation by enhanced permeability and retention (EPR) effect with concomitant increase in efficacy. b) Drug-free macromolecular therapeutics is a new paradigm in macromolecular therapeutics. Apoptosis in malignant cell is induced by crosslinking of cell surface non-internalizing receptors. Crosslinking of receptors is mediated by the biorecognition of two nanoconjugates containing high-fidelity complementary motifs (peptides or oligonucleotides). Results for the treatment of B cell lymphomas in animal models and patient cells demonstrate the high translational potential of this approach.