Tinker, tailor, soldier, cell: the role of C-type lectins in the defense and promotion of disease.

C-type lectins (CTLs) represent a large family of soluble and membrane-bound proteins which bind calcium dependently via carbohydrate recognition domains (CRDs) to glycan residues presented on the surface of a variety of pathogens. The deconvolution of a cell's glycan code by CTLs underpins several important physiological processes in mammals such as pathogen neutralization and opsonization, leukocyte trafficking, and the inflammatory response. However, as our knowledge of CTLs has developed it has become apparent that the role of this innate immune family of proteins can be double-edged, where some pathogens have developed approaches to subvert and exploit CTL interactions to promote infection and sustain the pathological state. Equally, CTL interactions with host glycoproteins can contribute to inflammatory diseases such as arthritis and cancer whereby, in certain contexts, they exacerbate inflammation and drive malignant progression. This review discusses the 'dual agent' roles of some of the major mammalian CTLs in both resolving and promoting infection, inflammation and inflammatory disease and highlights opportunities and emerging approaches for their therapeutic modulation.

Therapeutic Targeting of Exportin-1 in Childhood Cancer.

Overexpression of Exportin-1 (XPO1), a key regulator of nuclear-to-cytoplasmic transport, is associated with inferior patient outcomes across a range of adult malignancies. Targeting XPO1 with selinexor has demonstrated promising results in clinical trials, leading to FDA approval of its use for multiple relapsed/refractory cancers. However, XPO1 biology and selinexor sensitivity in childhood cancer is only recently being explored. In this review, we will focus on the differential biology of childhood and adult cancers as it relates to XPO1 and key cargo proteins. We will further explore the current state of pre-clinical and clinical development of XPO1 inhibitors in childhood cancers. Finally, we will outline potentially promising future therapeutic strategies for, as well as potential challenges to, integrating XPO1 inhibition to improve outcomes for children with cancer.

Immunoglobulin isotype compositions of ABO specific antibodies are dependent on the individual patient blood group and blood group specificity: Results from a healthy donor cohort.

Antibodies specific for the blood group ABO system antigens are of clinical significance and immunological interest. Routine clinical methods typically employ direct or indirect haemagglutination methods to measure IgM and IgG, respectively. We have developed a simple, single tube method to quantify IgM, IgG, and IgA specific for A and B antigens in order to improve accuracy and reproducibility, and to investigate the relationships between ABO group antibody type, and antibody level. Plasma samples from 300 healthy blood donors were studied. Levels of IgM and IgG binding to reagent group A and B red cells were measure by agglutination (HA) and multi-colour flow cytometry (MC-FC). IgA was also measured by MC-FC. Our FC method was found to be significantly more reproducible than HA for the measurement of blood group A and B specific antibodies. We found statistically significant correlations between antibodies measured by GC-HA and MC-FC, but sufficient differences to indicate that these methods are not equivalent. By MC-FC, IgM, IgG and IgA levels and isotope profiles were found to be dependent on both the donor ABO type and the specificity of the antibody. This study demonstrated heterogeneity in the immunoglobulin class profiles of ABO-blood group specific antibodies within the healthy population. Differences in isotype profiles of ABO-blood group specific antibodies may indicate fundamental differences in the immune mechanisms that generate these antibodies. This is likely to be relevant to the clinical situations where management or diagnosis depend on ABO-specific antibody detection and measurement.

C-type lectin receptors MR and DC-SIGN are involved in recognition of hemocyanins, shaping their immunostimulatory effects on human dendritic cells.

Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1-biased cell-mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose-rich structures. It remains unclear whether these structures influence hemocyanin-induced immunostimulatory processes in human APCs. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C-type lectin receptors (CLRs). Here, we studied the interactions of these hemocyanins with two major mannose-binding CLRs on monocyte-derived human DCs: MR (mannose receptor) and DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose-sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins colocalized with MR and DC-SIGN, and were partly internalized through clathrin-mediated endocytosis. The hemocyanin-mediated proinflammatory cytokine response was impaired when using deglycosylated FLH and KLH compared to CCH. We further showed that hemocyanins bind to human MR and DC-SIGN in a carbohydrate-dependent manner with affinity constants in the physiological concentration range. Overall, we showed that these three clinically valuable hemocyanins interact with human mannose-sensitive CLRs, initiating an immune response and promoting a Th1 cell-driving potential.

C3d-positive donor-specific antibodies have a role in pretransplant risk stratification of cross-match-positive HLA-incompatible renal transplantation: United Kingdom multicentre study.

Anti-HLA-antibody characteristics aid to risk-stratify patients and improve long-term renal graft outcomes. Complement activation by donor-specific antibody (DSA) is an important characteristic that may determine renal allograft outcome. There is heterogeneity in graft outcomes within the moderate to high immunological risk cases (cross-match-positive). We explored the role of C3d-positive DSAs in sub-stratification of cross-match-positive cases and relate to the graft outcomes. We investigated 139 cross-match-positive living-donor renal transplant recipients from four transplant centres in the United Kingdom. C3d assay was performed on serum samples obtained at pretreatment (predesensitization) and Day 14 post-transplant. C3d-positive DSAs were found in 52 (37%) patients at pretreatment and in 37 (27%) patients at Day 14 post-transplant. Median follow-up of patients was 48 months (IQR 20.47-77.57). In the multivariable analysis, pretreatment C3d-positive DSA was independently associated with reduced overall graft survival, the hazard ratio of 3.29 (95% CI 1.37-7.86). The relative risk of death-censored five-year graft failure was 2.83 (95% CI 1.56-5.13). Patients with both pretreatment and Day 14 C3d-positive DSAs had the worst five-year graft survival at 45.5% compared with 87.2% in both pretreatment and Day 14 C3d-negative DSA patients with the relative risk of death-censored five-year graft failure was 4.26 (95% CI 1.79, 10.09). In this multicentre study, we have demonstrated for the first time the utility of C3d analysis as a distinctive biomarker to sub-stratify the risk of poor graft outcome in cross-match-positive living-donor renal transplantation.

Structure of the C1r-C1s interaction of the C1 complex of complement activation.

The multiprotein complex C1 initiates the classical pathway of complement activation on binding to antibody-antigen complexes, pathogen surfaces, apoptotic cells, and polyanionic structures. It is formed from the recognition subcomponent C1q and a tetramer of proteases C1r2C1s2 as a Ca2+-dependent complex. Here we have determined the structure of a complex between the CUB1-EGF-CUB2 fragments of C1r and C1s to reveal the C1r-C1s interaction that forms the core of C1. Both fragments are L-shaped and interlock to form a compact antiparallel heterodimer with a Ca2+ from each subcomponent at the interface. Contacts, involving all three domains of each protease, are more extensive than those of C1r or C1s homodimers, explaining why heterocomplexes form preferentially. The available structural and biophysical data support a model of C1r2C1s2 in which two C1r-C1s dimers are linked via the catalytic domains of C1r. They are incompatible with a recent model in which the N-terminal domains of C1r and C1s form a fixed tetramer. On binding to C1q, the proteases become more compact, with the C1r-C1s dimers at the center and the six collagenous stems of C1q arranged around the perimeter. Activation is likely driven by separation of the C1r-C1s dimer pairs when C1q binds to a surface. Considerable flexibility in C1s likely facilitates C1 complex formation, activation of C1s by C1r, and binding and activation of downstream substrates C4 and C4b-bound C2 to initiate the reaction cascade.

Direct quantitative measurement of the kinetics of HLA-specific antibody interactions with isolated HLA proteins.

HLA specific antibodies vary in their pathogenicity and this is likely to be the net effect of constant chain usage, quantity, specificity, and affinity. Here we have measured the affinity of human monoclonal antibodies for a range of HLA proteins. Purified antibodies and ligands allowed dynamic interactions to be measured directly by surface plasmon resonance. Physiochemical differences between pairs of ligands were quantified using electrostatic mismatch and hydrophobic mismatch scores. All antibodies were characterized by fast on-rates and slow off rates but with a wide range of association rates (kon, 3.63-24.25 × 105 per mol per second) and dissociation rates (koff, 0.99-10.93 × 10-3 per second). Dissociation constants (KD) ranged from 5.9 × 10-10 M to 3.0 × 10-8 M. SN320G6 has approximately a twenty-fold greater affinity for HLA A2 compared with SN607D8, but has a similar affinity for HLA-A2 and B57. In contrast, SN607D8 has greater than a twofold greater affinity for HLA-A2 compared with A68. Similarly, WK1D12 has about a threefold greater affinity for HLA-B27 compared with B7. The higher affinity interactions correlate with the specificity of stimulating antigen. This is the first study to directly measure the binding kinetics and affinity constants for human alloantibodies against HLA.

Manipulation of cytokine secretion in human dendritic cells using glycopolymers with picomolar affinity for DC-SIGN.

The human C-type lectin DC-SIGN (CD209) is a significant receptor on the surface of dendritic cells (DCs) - crucial components of host defense that bridge the innate and adaptive immune systems. A range of linear glycopolymers, constructed via controlled radical polymerization techniques have been shown to interact with DC-SIGN with affinities in the physiologically active range. However, these first generation glycopolymers possess limited structural definition and their effects on DCs were not known. Here we report the development of star-shaped mannose glycopolymers with the aim of targeting the clustered domain arrangement of DC-SIGN and these were shown to bind with picomolar affinity. Increased secretion of IL-10 with simultaneous decrease in secreted IL-12p70 occurred in activated DCs incubated with star-shaped glycopolymers - a cytokine secretion pattern characteristic of wound-healing tissue environments. Incorporating stellar architecture into glycopolymer design could be key to developing selective and very high-affinity therapeutic materials with distinct immunomodulatory and tissue repair potential.

Combinatorial peptide-based epitope mapping from Ebola virus DNA vaccines and infections reveals residue-level determinants of antibody binding.

Ebola virus (EBOV) infection is highly lethal and results in severe febrile bleeding disorders that affect humans and non-human primates. One of the therapeutic approaches for treating EBOV infection focus largely on cocktails of monoclonal antibodies (mAbs) that bind to specific regions of the EBOV glycoprotein (GP) and neutralize the virus. Recent structural studies using cryo-electron microscopy have identified key epitopes for several EBOV mAbs. While such information has yielded deep insights into antibody binding, limitations on resolution of these structures often preclude a residue-level analysis of EBOV epitopes. In this study, we performed combinatorial peptide-based epitope mapping of EBOV GP against a broad panel of mAbs and polyclonal sera derived from several animal species vaccinated with EBOV DNA and replicon vaccines and/or exposed to EBOV infection to identify residue-level determinants of antibody binding. The peptide-based epitope mapping obtained from a wide range of serum and mAb samples, combined with available cryo-EM structure reconstructions revealed fine details of antibody-virus interactions, allowing for a more precise and comprehensive mapping of antibody epitopes on EBOV GP. We show how these residue-level epitope definitions can be used to characterize antigenic variation across different filoviruses, and provide a theoretical basis for predicting immunity and cross-neutralization in potential future outbreaks.

Protein-Protein Interaction between Surfactant Protein D and DC-SIGN via C-Type Lectin Domain Can Suppress HIV-1 Transfer.

Surfactant protein D (SP-D) is a soluble C-type lectin, belonging to the collectin (collagen-containing calcium-dependent lectin) family, which acts as an innate immune pattern recognition molecule in the lungs at other mucosal surfaces. Immune regulation and surfactant homeostasis are salient functions of SP-D. SP-D can bind to a range of viral, bacterial, and fungal pathogens and trigger clearance mechanisms. SP-D binds to gp120, the envelope protein expressed on HIV-1, through its C-type lectin or carbohydrate recognition domain. This is of importance since SP-D is secreted by human mucosal epithelial cells and is present in the female reproductive tract, including vagina. Another C-type lectin, dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), present on the surface of the DCs, also binds to HIV-1 gp120 and facilitates viral transfer to the lymphoid tissues. DCs are also present at the site of HIV-1 entry, embedded in vaginal or rectal mucosa. In the present study, we report a direct protein-protein interaction between recombinant forms of SP-D (rfhSP-D) and DC-SIGN via their C-type lectin domains. Both SP-D and DC-SIGN competed for binding to immobilized HIV-1 gp120. Pre-incubation of human embryonic kidney cells expressing surface DC-SIGN with rfhSP-D significantly inhibited the HIV-1 transfer to activated peripheral blood mononuclear cells. In silico analysis revealed that SP-D and gp120 may occupy same sites on DC-SIGN, which may explain the reduced transfer of HIV-1. In summary, we demonstrate, for the first time, that DC-SIGN is a novel binding partner of SP-D, and this interaction can modulate HIV-1 capture and transfer to CD4+ T cells. In addition, the present study also reveals a novel and distinct mechanism of host defense by SP-D against HIV-1.

Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus the antibody response in mice.

We performed epitope mapping studies on the major surface glycoprotein (GP) of Ebola virus (EBOV) using Chemically Linked Peptides on Scaffolds (CLIPS), which form linear and potential conformational epitopes. This method identified monoclonal antibody epitopes and predicted additional epitopes recognized by antibodies in polyclonal sera from animals experimentally vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then constructed 2 DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to produce a membrane-bound oligopeptide was poorly immunogenic, a construct generating a secreted oligopeptide elicited strong antibody responses in mice. When this construct was administered as a boost to a DNA vaccine expressing the complete EBOV GP gene, the resultant antibody response was focused largely toward the less immunodominant epitopes in the oligopeptide. Taken together, the results of this work suggest a utility for this method for immune focusing of antibody responses elicited by vaccination.

Specific and Differential Binding of N-Acetylgalactosamine Glycopolymers to the Human Macrophage Galactose Lectin and Asialoglycoprotein Receptor.

A range of glycopolymers composed of N-acetylgalactosamine were prepared via sequential Cu(I)-mediated polymerization and alkyne-azide click (CuAAC). The resulting polymers were shown, via multichannel surface plasmon resonance, to interact specifically with human macrophage galactose lectin (MGL; CD301) with high affinity (KD = 1.11 µM), but they did not bind to the mannose/fucose-selective human lectin dendritic-cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN; CD209). The effect of sugar ligand valency on the binding (so-called "glycoside cluster effect") of poly(N-acetylgalactosamine) to MGL was investigated by varying first the polymer chain length (DP: 100, 64, 40, 23, 12) and then the architecture (4- and 8-arm star glycopolymers). The chain length did not have a significant effect on the binding to MGL (KD = 0.17-0.52 µM); however, when compared to a hepatic C-type lectin of a similar monosaccharide specificity, the asialoglycoprotein receptor (ASGPR), the binding affinity was more noticeably affected (KD = 0.37- 6.65 µM). These data suggest that known differences in the specific configuration/orientation of the carbohydrate recognition domains of MGL and ASGPR are responsible for the differences in binding observed between the different polymers of varied chain length and architecture. In the future, this model has the potential to be employed for the development of tissue-selective delivery systems.

A chemical genomics approach to drug reprofiling in oncology: Antipsychotic drug risperidone as a potential adenocarcinoma treatment.

Drug reprofiling is emerging as an effective paradigm for discovery of cancer treatments. Herein, an antipsychotic drug is immobilised using the Magic Tag® chemical genomics tool and screened against a T7 bacteriophage displayed library of polypeptides from Drosophila melanogaster, as a whole genome model, to uncover an interaction with a section of 17-ß-HSD10, a proposed prostate cancer target. A computational study and enzyme inhibition assay with full length human 17-ß-HSD10 identifies risperidone as a drug reprofiling candidate. When formulated with rumenic acid, risperidone slows proliferation of PC3 prostate cancer cells in vitro and retards PC3 prostate cancer tumour growth in vivo in xenografts in mice, presenting an opportunity to reprofile risperidone as a cancer treatment.

Enhanced non-vitreous cryopreservation of immortalized and primary cells by ice-growth inhibiting polymers.

Cell cryopreservation is an essential tool in modern biotechnology and medicine. The ability to freeze, store and distribute materials underpins basic cell biology and enables storage of donor cells needed for transplantation and regenerative medicine. However, many cell types do not survive freezing and the current state-of-the-art involves the addition of significant amounts of organic solvents as cryoprotectants, which themselves can be cytotoxic, or simply interfere with assays. A key cause of cell death in cryopreservation is ice recrystallization (growth), which primarily occurs during thawing. Here it is demonstrated that the addition of ice recrystalization inhibiting polymers to solutions containing low (non vitrifying) concentrations of DMSO enhance cell recovery rates by up to 75%. Cell functionality is also demonstrated using a placental cell line, and enhanced cryopreservation of primary rat hepatocytes is additionally shown. The crucial role of the polymers architecture (chain length) is shown, with shorter polymers being more effective than longer ones.

Generation and characterization of ß1,2-gluco-oligosaccharide probes from Brucella abortus cyclic ß-glucan and their recognition by C-type lectins of the immune system.

The ß1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic ß1,2-glucan (CßG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the ß1,2-gluco-oligosaccharides, with degrees of polymerization 2-13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the ß1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-binding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CßG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by ß1,2-glucans in mammalian systems.