A functional mammalian display screen identifies rare antibodies that stimulate NK cell-mediated cytotoxicity.

Therapies that boost the antitumor immune response have shown a great deal of success. Although most of these therapies have focused on enhancing T cell functions, there is a growing interest in developing therapies that can target other immune cell subsets. Like T cells, natural killer (NK) cells are cytotoxic effector cells that play a key role in the antitumor response. To advance the development of NK-based therapies, we developed a functional screen to rapidly identify antibodies that can activate NK cells. We displayed antibodies on a mammalian target cell line and probed their ability to stimulate NK cell-mediated cytotoxicity. From this screen, we identified five antibodies that bound with high affinity to NK cells and stimulated NK cell-mediated cytotoxicity and interferon-γ (IFN-γ) secretion. We demonstrate that these antibodies can be further developed into bispecific antibodies to redirect NK cell-mediated cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. While antibodies to two of the receptors, CD16 and NCR1, have previously been targeted as bispecific antibodies to redirect NK cell-mediated cytotoxicity, we demonstrate that bispecific antibodies targeting NCR3 can also potently activate NK cells. These results show that this screen can be used to directly identify antibodies that can enhance antitumor immune responses.

Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins.

Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and ß2-adrenergic receptor (ß2AR) optobodies suppressed endogenous gelsolin activity and ß2AR signaling, respectively.

PD-L1 (Programmed Death Ligand 1) Regulates T-Cell Differentiation to Control Adaptive Venous Remodeling.

OBJECTIVE: Patients with end-stage renal disease depend on hemodialysis for survival. Although arteriovenous fistulae (AVF) are the preferred vascular access for hemodialysis, the primary success rate of AVF is only 30% to 50% within 6 months, showing an urgent need for improvement. PD-L1 (programmed death ligand 1) is a ligand that regulates T-cell activity. Since T cells have an important role during AVF maturation, we hypothesized that PD-L1 regulates T cells to control venous remodeling that occurs during AVF maturation. Approach and results: In the mouse aortocaval fistula model, anti-PD-L1 antibody (200 mg, 3×/wk intraperitoneal) was given to inhibit PD-L1 activity during AVF maturation. Inhibition of PD-L1 increased T-helper type 1 cells and T-helper type 2 cells but reduced regulatory T cells to increase M1-type macrophages and reduce M2-type macrophages; these changes were associated with reduced vascular wall thickening and reduced AVF patency. Inhibition of PD-L1 also inhibited smooth muscle cell proliferation and increased endothelial dysfunction. The effects of anti-PD-L1 antibody on adaptive venous remodeling were diminished in nude mice; however, they were restored after T-cell transfer into nude mice, indicating the effects of anti-PD-L1 antibody on venous remodeling were dependent on T cells. CONCLUSIONS: Regulation of PD-L1 activity may be a potential therapeutic target for clinical translation to improve AVF maturation.

Heterogeneity and longevity of antibody memory to viruses and vaccines.

Determining the duration of protective immunity requires quantifying the magnitude and rate of loss of antibodies to different virus and vaccine antigens. A key complication is heterogeneity in both the magnitude and decay rate of responses of different individuals to a given vaccine, as well as of a given individual to different vaccines. We analyzed longitudinal data on antibody titers in 45 individuals to characterize the extent of this heterogeneity and used models to determine how it affected the longevity of protective immunity to measles, rubella, vaccinia, tetanus, and diphtheria. Our analysis showed that the magnitude of responses in different individuals varied between 12- and 200-fold (95% coverage) depending on the antigen. Heterogeneity in the magnitude and decay rate contribute comparably to variation in the longevity of protective immunity between different individuals. We found that some individuals have, on average, slightly longer-lasting memory than others-on average, they have higher antibody levels with slower decay rates. We identified different patterns for the loss of protective levels of antibodies to different vaccine and virus antigens. Specifically, we found that for the first 25 to 50 years, virtually all individuals have protective antibody titers against diphtheria and tetanus, respectively, but about 10% of the population subsequently lose protective immunity per decade. In contrast, at the outset, not all individuals had protective titers against measles, rubella, and vaccinia. However, these antibody titers wane much more slowly, with a loss of protective immunity in only 1% to 3% of the population per decade. Our results highlight the importance of long-term longitudinal studies for estimating the duration of protective immunity and suggest both how vaccines might be improved and how boosting schedules might be reevaluated.

Migration-based selections of antibodies that convert bone marrow into trafficking microglia-like cells that reduce brain amyloid ß.

One goal of regenerative medicine is to repair damaged tissue. This requires not only generating new cells of the proper phenotype, but also selecting for those that properly integrate into sites of injury. In our laboratory we are using a cell-migration-based in vivo selection system to generate antibodies that induce cells to both differentiate and selectively localize to different tissues. Here we describe an antibody that induces bone marrow stem cells to differentiate into microglia-like cells that traffic to the brain where they organize into typical networks. Interestingly, in the APP/PS1 Alzheimer's disease mouse model, these induced microglia-like cells are found at sites of plaque formation and significantly reduce their number. These results raise the intriguing question as to whether one can use such antibody-induced differentiation of stem cells to essentially recapitulate embryogenesis in adults to discover cells that can regenerate damaged organ systems.

Structure-function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8.

Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, and VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138- and VACV-304-binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.

Antibody-based nanotechnology.

Antibodies are considered the hallmark of the adaptive immune system in that they mediate various key biological functions, such as direct neutralization and recruitment of effector immune cells to eliminate invading pathogens. Antibodies exhibit several unique properties, including high diversity (enabling binding to a wide range of targets), high specificity and structural integrity. These properties and the understanding that antibodies can be utilized in a wide range of applications have motivated the scientific community to develop new approaches for antibody repertoire analysis and rapid monoclonal antibody discovery. Today, antibodies are key modules in the pharmaceutical and diagnostic industries. By virtue of their high affinity and specificity to their targets and the availability of technologies to engineer different antibodies to a wide range of targets, antibodies have become the most promising natural biological molecules in a range of biotechnological applications, such as: highly specific and sensitive nanobiosensors for the diagnostics of different biomarkers; nanoparticle-based targeted drug delivery systems to certain cells or tissues; and nanomachines, which are nanoscale mechanical devices that enable energy conversion into precise mechanical motions in response to specific molecular inputs. In this review, we start by describing the unique properties of antibodies, how antibody diversity is generated, and the available technologies for antibody repertoire analysis and antibody discovery. Thereafter, we provide an overview of some antibody-based nanotechnologies and discuss novel and promising approaches for the application of antibodies in the nanotechnology field. Overall, we aim to bridge the knowledge gap between the nanotechnology and antibody engineering disciplines by demonstrating how technological advances in the antibody field can be leveraged to develop and/or enhance new technological approaches in the nanotechnology field.

Thalamic Reticular Nucleus in Caiman crocodilus: Immunohistochemical Staining.

The thalamic reticular nucleus in reptiles, Caiman crocodilus, shares a number of morphological similarities with its counterpart in mammals. In view of the immunohistochemical properties of this nucleus in mammals and the more recently identified complexity of this neuronal aggregate in Caiman, this nucleus was investigated using a number of antibodies. These results were compared with findings described for other amniotes. The following antibodies gave consistent and reproducible results: polyclonal sheep anti-parvalbumin (PV), monoclonal mouse anti-PV, and polyclonal sheep anti-glutamic acid decarboxylase (GAD). In the transverse plane, this nucleus is divided into two. In each part, a compact group of cells sits on top of the fibers of the forebrain bundle with scattered cells among these fibers. In the lateral forebrain bundle, this neuronal aggregate is represented by the dorsal peduncular nucleus and the perireticular nucleus while, in the medial forebrain bundle, these parts are the interstitial nucleus and the scattered cells in this fiber tract. The results of this study are the following. First, the thalamic reticular nucleus of Caiman contains GAD(+) and PV(+) neurons, which is similar to what has been described in other amniotes. Second, the morphology and distribution of many GAD(+) and PV(+) neurons in the dorsal peduncular and perireticular nuclei are similar and suggest that these neurons colocalize these markers. Third, neurons in the interstitial nucleus and in the medial forebrain bundle are GAD(+) and PV(+). At the caudal pole of the thalamic reticular nucleus, PV immunoreactive cells predominated and avoided the central portion of this nucleus where GAD(+) cells were preferentially located. However, GAD(+) cells were sparse when compared with PV(+) cells. This immunohistochemically different area in the caudal pole is considered to be an area separate from the thalamic reticular nucleus.

Rituximab, plasma exchange and immunoglobulins: an ineffective treatment for chronic active antibody-mediated rejection.

BACKGROUND: Chronic active antibody-mediated rejection (c-aABMR) is an important cause of allograft failure and graft loss in long-term kidney transplants. METHODS: To determine the efficacy and safety of combined therapy with rituximab, plasma exchange (PE) and intravenous immunoglobulins (IVIG), a cohort of patients with transplant glomerulopathy (TG) that met criteria of active cABMR, according to BANFF'17 classification, was identified. RESULTS: We identified 62 patients with active c-aABMR and TG (cg ≥ 1). Twenty-three patients were treated with the combination therapy and, 39 patients did not receive treatment and were considered the control group. There were no significant differences in the graft survival between the two groups. The number of graft losses at 12 and 24 months and the decline of eGFR were not different and independent of the treatment. A decrease of eGFR≥13 ml/min between 6 months before and c-aABMR diagnosis, was an independent risk factor for graft loss at 24 months (OR = 5; P = 0.01). Infections that required hospitalization during the first year after c-aABMR diagnosis were significantly more frequent in treated patients (OR = 4.22; P = 0.013), with a ratio infection/patient-year of 0.65 and 0.20 respectively. CONCLUSIONS: Treatment with rituximab, PE, and IVIG in kidney transplants with c-aABMR did not improve graft survival and was associated with a significant increase in severe infectious complications. TRIAL REGISTRATION: Agencia Española de Medicametos y Productos Sanitarios (AEMPS): 14566/RG 24161. Study code: UTR-INM-2017-01.

A Heterologous Model of Thrombospondin Type 1 Domain-Containing 7A-Associated Membranous Nephropathy.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target for autoimmunity in patients with membranous nephropathy (MN). Circulating autoantibodies from patients with THSD7A-associated MN have been demonstrated to cause MN in mice. However, THSD7A-associated MN is a rare disease, preventing the use of patient antibodies for larger experimental procedures. Therefore, we generated antibodies against the human and mouse orthologs of THSD7A in rabbits by coimmunization with the respective cDNAs. Injection of these anti-THSD7A antibodies into mice induced a severe nephrotic syndrome with proteinuria, weight gain, and hyperlipidemia. Immunofluorescence analyses revealed granular antigen-antibody complexes in a subepithelial location along the glomerular filtration barrier 14 days after antibody injection, and immunohistochemistry for rabbit IgG and THSD7A as well as ultrastructural analyses showed the typical characteristics of human MN. Mice injected with purified IgG from rabbit serum that was taken before immunization failed to develop any of these changes. Notably, MN developed in the absence of detectable complement activation, and disease was strain dependent. In vitro, anti-THSD7A antibodies caused cytoskeletal rearrangement and activation of focal adhesion signaling. Knockdown of the THSD7A ortholog, thsd7aa, in zebrafish larvae resulted in altered podocyte differentiation and impaired glomerular filtration barrier function, with development of pericardial edema, suggesting an important role of THSD7A in glomerular filtration barrier integrity. In summary, our study introduces a heterologous mouse model that allows further investigation of the molecular events that underlie MN.

Lipoxin A4 augments host defense in sepsis and reduces Pseudomonas aeruginosa virulence through quorum sensing inhibition.

Bacterial infections can quickly turn into sepsis, with its attendant clinical sequelae of inflammation, tissue injury, and organ failure. Paradoxically, sustained inflammation in sepsis may lead to immune suppression, because of which the host is unable to clear the existing infection. Use of agents that suppress the inflammatory response may accelerate host immune suppression, whereas use of traditional antibiotics does not significantly affect inflammation. In this study, we investigated whether lipoxin A4 (LXA4), a specialized, proresolution lipid mediator, could increase neutrophil phagocytic activity as well as reduce bacterial virulence. Using the mouse cecal ligation and puncture (CLP) model of sepsis, the administration of LXA4 (7 µg/kg i.v.) 1 h after surgery increased neutrophil phagocytic ability and Fcγ receptor I (CD64) expression. Ex vivo studies have confirmed that the direct addition of LXA4 to CLP neutrophils increased phagocytic ability but not CD64 expression. LXA4 did not affect neutrophils taken from control mice in which CD64 expression was minimal. Taken together with in vivo data, these results suggest that LXA4 directly augments CD64-mediated neutrophil phagocytic ability but does not directly increase neutrophil CD64 expression. Bacterial communication and virulence is regulated by quorum sensing inducers. In Pseudomonas aeruginosa, virulence is induced with release of various virulence factors, by N-3-oxododecanolyl homoserine lactone binding to the quorum sensing receptor, LasR. We show that LXA4 is an inhibitor of LasR in P. aeruginosa and that it decreases the release of pyocyanin exotoxin. These results suggest that LXA4 has the novel dual properties of increasing host defense and decreasing pathogen virulence by inhibiting quorum sensing.-Wu, B., Capilato, J., Pham, M. P., Walker, J., Spur, B., Rodriguez, A., Perez, L. J., Yin, K. Lipoxin A4 augments host defense in sepsis and reduces Pseudomonas aeruginosa virulence through quorum sensing inhibition.

Antisperm antibodies in repeat-breeding cows: Frequency, detection and validation of threshold levels employing sperm immobilization, sperm agglutination and immunoperoxidase assay.

Antisperm antibodies have been found in repeat-breeding(RB) cows, and those causing agglutination and/or immobilization of sperm are considered to be closely related to unexplained infertility. However, a standard protocol for identifying antisperm antibodies (ASA) in cattle is not validated. Therefore, an investigation was undertaken to evaluate sperm immobilization (SIT), sperm agglutination (SAT) and immunoperoxidase (IPT)assays for detection of ASA in serum and their respective threshold levels for confirmation. Animals (heifers, normally breeding, repeat-breeding and pregnant animals) that were free from IBR, brucellosis and uterine infections (screened by clinical examination) were included in the study. Sperm agglutinating, sperm immobilizing and antisperm antibodies evaluated by respective assay were significantly higher (p < .05) in RB cows compared to other groups. The SIT assay was able to identify 61% of RB caused by ASA, more than those employing SAT and IPT. Furthermore, a dilution rate of 1:5 and 1:80 (confirms 59.0 and 57.0% RB+ve)were sufficient to diagnose ASA by SAT and IPT, respectively. Results indicate the presence of __12.6% clumped spermatozoa and __ 2.6%(cut-off value) peroxidase-positive spermatozoa at 1:5 and 1:80 dilutions diagnosed with SAT and IPT, respectively, may be considered as repeaters arising out of ASA. Furthermore, study also showed the presence of lower incidence of ASA positivity in other groups of animals (heifer

Avanti lipid tools: connecting lipids, technology, and cell biology.

Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein-lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.

Antibodies as natural adjuvants.

Antibodies in complex with specific antigen can dramatically change the antibody response to this antigen. Depending on antibody class and type of antigen, >99 % suppression or >100-fold enhancement of the response can take place. IgM and IgG3 are efficient enhancers and operate via the complement system. In contrast, IgG1, IgG2a, and IgG2b enhance antibody and CD4(+) T cell responses to protein antigens via activating Fcγ-receptors. IgE also enhances antibody and CD4(+) T cell responses to small proteins but uses the low-affinity receptor for IgE, CD23. Most likely, IgM and IgG3 work by increasing the effective concentration of antigen on follicular dendritic cells in splenic follicles. IgG1, IgG2a, IgG2b, and IgE probably enhance antibody responses by increasing antigen presentation by dendritic cells to T helper cells. IgG antibodies of all subclasses have a dual effect, and suppress antibody responses to particulate antigens such as erythrocytes. This capacity is used in the clinic to prevent immunization of Rhesus-negative women to Rhesus-positive fetal erythrocytes acquired via transplacental hemorrage. IgG-mediated suppression in mouse models can take place in the absence of Fcγ-receptors and complement and to date no knock-out mouse strain has been found where suppression is abrogated.

Agonistic anti-CD40 induces thyrocyte proliferation and promotes thyroid autoimmunity by increasing CD40 expression on thyroid epithelial cells.

CD40 is expressed on cells of the immune system and in some tissues that are targets for autoimmune-mediated damage. It is not known if CD40 expression in target tissues plays a role in the pathology of autoimmune diseases. This study shows that agonistic anti-CD40 induces strong and sustained proliferation of thyroid epithelial cells (TECs), or thyrocytes, in IFN-γ(-/-) autoimmune-prone NOD and NOD.H-2h4 mice. TEC proliferation is accompanied by greatly increased expression of CD40 on TECs, development of fibrosis and hypothyroidism, and increased expression of proinflammatory molecules in thyroids. Bone marrow chimera experiments indicate that TEC expression of CD40 is required for anti-CD40-induced TEC proliferation, but lymphoid cells do not have to express CD40. TEC proliferation is reduced in wild-type mice given anti-CD40, presumably because they produce IFN-γ, which inhibits TEC proliferation. CD40 also increases on TECs during development of an autoimmune thyroid disease characterized by TEC hyperproliferation that develops spontaneously in IFN-γ(-/-) NOD.H-2h4 mice. TEC hyperproliferation development is accelerated in mice given agonistic anti-CD40. These studies provide new information regarding the role of target tissue expression of CD40 in development of autoimmunity and suggest that use of agonistic anti-CD40 for tumor therapy could result in autoimmune disease.

Structure of an integrin with an alphaI domain, complement receptor type 4.

We report the structure of an integrin with an alphaI domain, alpha(X)beta(2), the complement receptor type 4. It was earlier expected that a fixed orientation between the alphaI domain and the beta-propeller domain in which it is inserted would be required for allosteric signal transmission. However, the alphaI domain is highly flexible, enabling two betaI domain conformational states to couple to three alphaI domain states, and greater accessibility for ligand recognition. Although alpha(X)beta(2) is bent similarly to integrins that lack alphaI domains, the terminal domains of the alpha- and beta-legs, calf-2 and beta-tail, are oriented differently than in alphaI-less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the beta(2)-tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.

Structural biology of moonlighting: lessons from antibodies.

Protein moonlighting is the property of a number of proteins to have more than one function. However, the definition of moonlighting is somewhat imprecise with different interpretations of the phenomenon. True moonlighting occurs when an individual evolutionary protein domain has one well-accepted role and a secondary unrelated function. The 'function' of a protein domain can be defined at different levels. For example, although the function of an antibody variable fragment (Fv) could be described as 'binding', a more detailed definition would also specify the molecule to which the Fv region binds. Using this detailed definition, antibodies as a family are consummate moonlighters. However, individual antibodies do not moonlight; the multiple functions they exhibit (first binding a molecule and second triggering the immune response) are encoded in different domains and, in any case, are related in the sense that they are a part of what an antibody needs to do. Nonetheless, antibodies provide interesting lessons on the ability of proteins to evolve binding functions. Remarkably similar antibody sequences can bind completely different antigens, suggesting that evolving the ability to bind a protein can result from very subtle sequence changes.

U1-small nuclear ribonucleoprotein activates the NLRP3 inflammasome in human monocytes.

The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is a caspase-1-containing cytosolic protein complex that is essential for processing and secretion of IL-1ß. The U1-small nuclear ribonucleoprotein (U1-snRNP) that includes U1-small nuclear RNA is a highly conserved intranuclear molecular complex involved in splicing pre-mRNA. Abs against this self nuclear molecule are characteristically found in autoimmune diseases like systemic lupus erythematosus, suggesting a potential role of U1-snRNP in autoimmunity. Although endogenous DNA and microbial nucleic acids are known to activate the inflammasomes, it is unknown whether endogenous RNA-containing U1-snRNP could activate this molecular complex. In this study, we show that U1-snRNP activates the NLRP3 inflammasome in CD14(+) human monocytes dependently of anti-U1-snRNP Abs, leading to IL-1ß production. Reactive oxygen species and K(+) efflux were responsible for this activation. Knocking down the NLRP3 or inhibiting caspase-1 or TLR7/8 pathway decreased IL-1ß production from monocytes treated with U1-snRNP in the presence of anti-U1-snRNP Abs. Our findings indicate that endogenous RNA-containing U1-snRNP could be a signal that activates the NLRP3 inflammasome in autoimmune diseases like systemic lupus erythematosus where anti-U1-snRNP Abs are present.