Lupus progression deteriorates oogenesis quality in MRL/lpr mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of the immune response against self antigens. Numerous reproductive complications, including reduced birth rate and complications for the mother and the fetus during pregnancy, have been observed in women with SLE. In the present study, we aimed to investigate the effect of SLE development on oocyte meiosis in lupus-prone mice. Lupus-prone MRL/lpr mice were used for the experiments: disease-free (4 weeks of age) and sick (20 weeks of age, virgin and postpartum). The immune response was monitored by flow cytometry, ELISpot, ELISA, and histology. Oocytes were analyzed by fluorescence microscopy based on chromatin, tubulin, and actin structures. The lupus-prone MRL/lpr mice developed age-dependent symptoms of SLE with increased levels of various autoantibodies, proteinuria, and renal infiltrates and a tendency for the immune response to worsen with changes in cell populations and the cytokine profile. The number and quality of oocytes were also affected, and the successful pregnancy rate of MRL/lpr mice was limited to only 60%. Isolated oocytes showed severe structural changes in all studied groups. Systemic alterations in immune homeostasis in SLE affect the quality of developing oocytes, which is evident from a young age. The data obtained is in line with the trend of reduced fertility in lupus-prone MRL/lpr mice. The phenomenon can be explained by changes in the microenvironment of the relevant organs and close connection between ovulation and inflammatory processes.

DNA and protein-generated chimeric molecules for delivery of influenza viral epitopes in mouse and humanized NSG transfer models.

Purified subunit viral antigens are weakly immunogenic and stimulate only the antibody but not the T cell-mediated immune response. An alternative approach to inducing protective immunity with small viral peptides may be the targeting of viral epitopes to immunocompetent cells by DNA and protein-engineered vaccines. This review will focus on DNA and protein-generated chimeric molecules carrying engineered fragments specific for activating cell surface co-receptors for inducing protective antiviral immunity. Adjuvanted protein-based vaccine or DNA constructs encoding simultaneously T- and B-cell peptide epitopes from influenza viral hemagglutinin, and scFvs specific for costimulatory immune cell receptors may induce a significant increase of anti-influenza antibody levels and strong CTL activity against virus-infected cells in a manner that mimics the natural infection. Here we summarize the development of several DNA and protein chimeric constructs carrying influenza virus HA317-41 fragment. The generated engineered molecules were used for immunization in intact murine and experimentally humanized NSG mouse models.

The Binding Landscape of Serum Antibodies: How Physical and Mathematical Concepts Can Advance Systems Immunology.

Antibodies constitute a major component of serum on protein mass basis. We also know that the structural diversity of these antibodies exceeds that of all other proteins in the body and they react with an immense number of molecular targets. What we still cannot quantitatively describe is how antibody abundance is related to affinity, specificity, and cross reactivity. This ignorance has important practical consequences: we also do not have proper biochemical units for characterizing polyclonal serum antibody binding. The solution requires both a theoretical foundation, a physical model of the system, and technology for the experimental confirmation of theory. Here we argue that the quantitative characterization of interactions between serum antibodies and their targets requires systems-level physical chemistry approach and generates results that should help create maps of antibody binding landscape.

Absolute Quantitation of Serum Antibody Reactivity Using the Richards Growth Model for Antigen Microspot Titration.

In spite of its pivotal role in the characterization of humoral immunity, there is no accepted method for the absolute quantitation of antigen-specific serum antibodies. We devised a novel method to quantify polyclonal antibody reactivity, which exploits protein microspot assays and employs a novel analytical approach. Microarrays with a density series of disease-specific antigens were treated with different serum dilutions and developed for IgG and IgA binding. By fitting the binding data of both dilution series to a product of two generalized logistic functions, we obtained estimates of antibody reactivity of two immunoglobulin classes simultaneously. These estimates are the antigen concentrations required for reaching the inflection point of thermodynamic activity coefficient of antibodies and the limiting activity coefficient of antigen. By providing universal chemical units, this approach may improve the standardization of serological testing, the quality control of antibodies and the quantitative mapping of the antibody-antigen interaction space.

Why current quantitative serology is not quantitative and how systems immunology could provide solutions.

Determination of the presence of antibodies against infectious agents, self-antigens, allogeneic antigens and environmental antigens is the goal of medical serology. Along with the standardization of these tests the community also started to use the expression "quantitative serology," referring to the fact that arbitrary units are used for the expression of results. In this review I will argue against the use of the term quantitative serology for current tests. Because each test and each antibody isotype determination uses its own references, the term semiquantitative better describes these methods. The introduction of really quantitative serology could both benefit from and drive forward systems immunological approach to immunity.

Anti-ANX A1 Antibody Therapy in MRL/lpr Murine Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease characterized by dysfunction of immune regulation, overproduction of inflammatory cytokines and attack on normal tissues by self-reactive cells and antibodies. The main role in the pathogenesis plays the autoreactive tandem of B-T cells, responsible for lupus progression and acceleration. Both activated B and T cells express a phospholipid binding protein Annexin A1 and abnormal levels of the protein were found in murine and human autoimmune syndromes, potentiating its role as a therapeutic target. Here, using anti-annexin A1 antibody we explore its property to modulate the autoimmune response in MRL/lpr mouse model of lupus. Anti-ANX A1 antibody was tested in vitro using spleen cells from MRL/lpr mice to determine the effect on lymphocyte activation, plasma cells differentiation, apoptosis and proliferation by flow cytometry and ELISpot assays. Subsequently, several groups of young (disease-free) and old (sick) MRL/lpr mice were treated with the antibody to determine the levels of panel auto-antibodies and cytokines, T cell arrest and migration. Treatment of splenocytes with anti-ANX A1 antibody inhibited T-cell activation and proliferation, suppressed anti-dsDNA antibody-producing plasma cells and affected B cell apoptosis. Administration of the antibody to MRL/lpr mice resulted to decreased autoantibody levels to various lupus antigens, suppressed T cell migration from lymph nodes and increased the levels of IL4 mRNA compared to the control group. Anti-ANX A1 antibody therapy suppresses B and T cell over-activation and down- modulates disease activity.

Multiple Levels of Immunological Memory and Their Association with Vaccination.

Immunological memory is divided into many levels to counteract the provocations of diverse and ever-changing infections. Fast functions of effector memory and the superposition of both quantitatively and qualitatively plastic anticipatory memory responses together form the walls of protection against pathogens. Here we provide an overview of the role of different B and T cell subsets and their interplay, the parallel and independent functions of the B1, marginal zone B cells, T-independent- and T-dependent B cell responses, as well as functions of central and effector memory T cells, tissue-resident and follicular helper T cells in the memory responses. Age-related limitations in the immunological memory of these cell types in neonates and the elderly are also discussed. We review how certain aspects of immunological memory and the interactions of components can affect the efficacy of vaccines, in order to link our knowledge of immunological memory with the practical application of vaccination.

Network Organization of Antibody Interactions in Sequence and Structure Space: the RADARS Model.

Adaptive immunity in vertebrates is a complex self-organizing network of molecular interactions. While deep sequencing of the immune-receptor repertoire may reveal clonal relationships, functional interpretation of such data is hampered by the inherent limitations of converting sequence to structure to function. In this paper, a novel model of antibody interaction space and network, termed radial adjustment of system resolution, RAdial ADjustment of System Resolution (RADARS), is proposed. The model is based on the radial growth of interaction affinity of antibodies towards an infinity of directions in structure space, each direction corresponding to particular shapes of antigen epitopes. Levels of interaction affinity appear as free energy shells of the system, where hierarchical B-cell development and differentiation takes place. Equilibrium in this immunological thermodynamic system can be described by a power law distribution of antibody-free energies with an ideal network degree exponent of phi square, representing a scale-free fractal network of antibody interactions. Plasma cells are network hubs, memory B cells are nodes with intermediate degrees, and B1 cells function as nodes with minimal degree. Overall, the RADARS model implies that a finite number of antibody structures can interact with an infinite number of antigens by immunologically controlled adjustment of interaction energy distribution. Understanding quantitative network properties of the system should help the organization of sequence-derived predicted structural data.

Targeting of Influenza Viral Epitopes to Antigen-Presenting Cells by Genetically Engineered Chimeric Molecules in a Humanized NOD SCID Gamma Transfer Model.

Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcγRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcγRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcγRI-expressing cells was confirmed by flow cytometry. The fusion protein induced a strong signal transduction on human monocytes via FcγRI. The expression vector pTriEx-3 Neo containing the described construct was used as a naked DNA vaccine and introduced directly to experimental humanized NOD SCID gamma mice with or without boosting with the expressed fusion protein. Immunization with the generated DNA chimeric molecules and prime-boost with the expressed recombinant proteins induced significant serum levels of anti-influenza immunoglobulin G antibodies and strong cytotoxic T lymphocyte activity against influenza virus-infected cells in humanized animals.

A generalized quantitative antibody homeostasis model: maintenance of global antibody equilibrium by effector functions.

The homeostasis of antibodies can be characterized as a balanced production, target-binding and receptor-mediated elimination regulated by an interaction network, which controls B-cell development and selection. Recently, we proposed a quantitative model to describe how the concentration and affinity of interacting partners generates a network. Here we argue that this physical, quantitative approach can be extended for the interpretation of effector functions of antibodies. We define global antibody equilibrium as the zone of molar equivalence of free antibody, free antigen and immune complex concentrations and of dissociation constant of apparent affinity: [Ab]=[Ag]=[AbAg]=KD. This zone corresponds to the biologically relevant KD range of reversible interactions. We show that thermodynamic and kinetic properties of antibody-antigen interactions correlate with immunological functions. The formation of stable, long-lived immune complexes correspond to a decrease of entropy and is a prerequisite for the generation of higher-order complexes. As the energy of formation of complexes increases, we observe a gradual shift from silent clearance to inflammatory reactions. These rules can also be applied to complement activation-related immune effector processes, linking the physicochemical principles of innate and adaptive humoral responses. Affinity of the receptors mediating effector functions shows a wide range of affinities, allowing the continuous sampling of antibody-bound antigen over the complete range of concentrations. The generation of multivalent, multicomponent complexes triggers effector functions by crosslinking these receptors on effector cells with increasing enzymatic degradation potential. Thus, antibody homeostasis is a thermodynamic system with complex network properties, nested into the host organism by proper immunoregulatory and effector pathways. Maintenance of global antibody equilibrium is achieved by innate qualitative signals modulating a quantitative adaptive immune system, which regulates molecular integrity of the host by tuning the degradation and recycling of molecules from silent removal to inflammatory elimination.

Corrigendum: A generalized quantitative antibody homeostasis model: antigen saturation, natural antibodies and a quantitative antibody network.

[This corrects the article DOI: 10.1038/cti.2016.90.].

A generalized quantitative antibody homeostasis model: antigen saturation, natural antibodies and a quantitative antibody network.

In a pair of articles, we present a generalized quantitative model for the homeostatic function of clonal humoral immune system. In this second paper, we describe how antibody production controls the saturation of antigens and the network of antibody interactions that emerges in the epitome space with the establishment of the immune system. Efficient control of antigens, be it self or foreign, requires the maintenance of antibody concentrations that saturate antigen to relevant levels. Simple calculations suggest that the observed diverse recognition of antigens by natural antibodies is only possible by cross-reactivity whereby particular clones of antibodies bind to diverse targets and shared recognition of particular antigens by multiple antibody clones contribute to the maintenance of antigen control. We also argue that natural antibodies are none else than the result of thymus-independent responses against immunological self. We interpret and explain antibody production and function in a virtual molecular interaction space and as a network of interactions. Indeed, the general quantitative (GQM) model we propose is in agreement with earlier models, confirms some assumptions and presumably provides the theoretical basis for the construction of a real antibody network using the sequence and interaction database data.

Detection of red blood cell surface antigens by probe-triggered cell collision and flow retardation in an autonomous microfluidic system.

Microfluidic devices exploit combined physical, chemical and biological phenomena that could be unique in the sub-millimeter dimensions. The current goal of development of Point-of-Care (POC) medical devices is to extract the biomedical information from the blood. We examined the characteristics of blood flow in autonomous microfluidic devices with the aim to realize sensitive detection of interactions between particulate elements of the blood and the appropriately modified surfaces of the system. As a model experiment we demonstrated the fast analysis of the AB0 blood group system. We observed that the accumulation of red blood cells immobilized on the capillary wall leads to increased lateral movement of the flowing cells, resulting in the overall selective deceleration of the red blood cell flow column compared to the plasma fraction. We showed that by monitoring the flow rate characteristics in capillaries coated with blood type reagents it is possible to identify red blood cell types. Analysis of hydrodynamic effects governing blood flow by Finite Element Method based modelling supported our observations. Our proof-of-concept results point to a novel direction in blood analysis in autonomous microfluidic systems and also provide the basis for the construction of a simple quantitative device for blood group determination.

A generalized quantitative antibody homeostasis model: regulation of B-cell development by BCR saturation and novel insights into bone marrow function.

In a pair of articles, we present a generalized quantitative model for the homeostatic function of clonal humoral immune system. In this first paper, we describe the cycles of B-cell expansion and differentiation driven by B-cell receptor engagement. The fate of a B cell is determined by the signals it receives via its antigen receptor at any point of its lifetime. We express BCR engagement as a function of apparent affinity and free antigen concentration, using the range of 10-14-10-3 M for both factors. We assume that for keeping their BCR responsive, B cells must maintain partial BCR saturation, which is a narrow region defined by [Ag]≈KD. To remain in this region, B cells respond to changes in [Ag] by proliferation or apoptosis and modulate KD by changing BCR structure. We apply this framework to various niches of B-cell development such as the bone marrow, blood, lymphoid follicles and germinal centers. We propose that clustered B cells in the bone marrow and in follicles present antigen to surrounding B cells by exposing antigen captured on complement and Fc receptors. The model suggests that antigen-dependent selection in the bone marrow results in (1) effector BI cells, which develop in blood as a consequence of the inexhaustible nature of soluble antigens, (2) memory cells that survive in antigen rich niches, identified as marginal zone B cells. Finally, the model implies that memory B cells could derive survival signals from abundant non-cognate antigens.

Annexin A1 as a target for managing murine pristane-induced systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a polygenic pathological disorder which involves multiple organs. Self-specific B cells play a main role in the lupus pathogenesis by generating autoantibodies as well as by serving as important autoantigen-presenting cells. Autoreactive T lymphocytes, on the other hand, are responsible for B cell activation and proliferation, and cytokine production. Therefore, both factors promote the idea that a down-modulation of activated self-reactive T and B cells involved in the pathogenic immune response is a reasonable approach for SLE therapy. Annexin A1 (ANX A1) is expressed by many cell types and binds to phospholipids in a Ca2+ dependent manner. Abnormal expression of ANX A1 was found on activated B and T cells in both murine and human autoimmunity, suggesting its potential role as a therapeutic target. While its role on T lymphocytes is through formyl peptide receptor-like molecules (FPRL), and the formed ANX A1/FPRL pathway modulates T cell receptor signalling, there is still no fool-proof data available for the role of ANX A1 in B cells. We employed a lupus model of Balb/c mice with pristane-induced SLE which very closely resembles human lupus. In the present study, we investigated the possibility to modulate the autoimmune response in a pristane-induced mouse model of SLE using an anti- ANX A1 antibody. Administration of this monoclonal antibody resulted in the inhibition of T-cell activation and proliferation, suppression of IgG anti-dsDNA antibody-secreting plasma cells and of proteinuria, decreased disease activity and prolonged survival compared to control group.

Characterization of NF-κB Reporter U937 Cells and Their Application for the Detection of Inflammatory Immune-Complexes.

Our study tested the hypothesis that immunoglobulins differ in their ability to activate the nuclear factor-κB pathway mediated cellular responses. These responses are modulated by several properties of the immune complex, including the ratio of antibody isotypes binding to antigen. Immunoassays allow the measurement of antigen specific antibodies belonging to distinct immunoglobulin classes and subclasses but not the net biological effect of the combination of these antibodies. We set out to develop a biosensor that is suitable for the detection and characterization of antigen specific serum antibodies. We genetically modified the monocytoid U937 cell line carrying Fc receptors with a plasmid encoding NF-κB promoter-driven GFP. This clone, U937-NF-κB, was characterized with respect to FcR expression and response to solid-phase immunoglobulins. Human IgG3, IgG4 and IgG1 induced GFP production in a time- and dose-dependent manner, in this order of efficacy, while IgG2 triggered no activation at the concentrations tested. IgA elicited no response alone but showed significant synergism with IgG3 and IgG4. We confirmed the importance of activation via FcγRI by direct stimulation with monoclonal antibody and by competition assays. We used citrullinated peptides and serum from rheumatoid arthritis patients to generate immune complexes and to study the activation of U937-NF-κB, observing again a synergistic effect between IgG and IgA. Our results show that immunoglobulins have distinct pro-inflammatory potential, and that U937-NF-κB is suitable for the estimation of biological effects of immune-complexes, offering insight into monocyte activation and pathogenesis of antibody mediated diseases.

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

Monovalent Fc receptor blockade by an anti-Fcγ receptor/albumin fusion protein ameliorates murine ITP with abrogated toxicity.

Patients with immune thrombocytopenia (ITP) commonly have antiplatelet antibodies that cause thrombocytopenia through Fcγ receptors (FcγRs). Antibodies specific for FcγRs, designed to inhibit antibody-FcγR interaction, had been shown to improve ITP in refractory human patients. However, the development of such FcγR-specific antibodies has stalled because of adverse events, a phenomenon recapitulated in mouse models. One hypothesis behind these adverse events involved the function of the Fc region of the antibody, which engages FcγRs, leading to inflammatory responses. Unfortunately, inhibition of Fc function by deglycosylation failed to prevent this inflammatory response. In this work, we hypothesize that the bivalent antigen-binding fragment regions of immunoglobulin G are sufficient to trigger adverse events and have reasoned that designing a monovalent targeting strategy could circumvent the inflammatory response. To this end, we generated a fusion protein comprising a monovalent human FcγRIIIA-specific antibody linked in tandem to human serum albumin, which retained FcγR-binding activity in vitro. To evaluate clinically relevant in vivo FcγR-blocking function and inflammatory effects, we generated a murine version targeting the murine FcγRIII linked to murine albumin in a passive murine ITP model. Monovalent blocking of FcγR function dramatically inhibited antibody-dependent murine ITP and successfully circumvented the inflammatory response as assessed by changes in body temperature, basophil activation, and basophil depletion. Consistent with our hypothesis, in vivo cross-linking of the fusion protein induced these inflammatory effects, recapitulating the adverse events of the parent antibody. Thus, monovalent blocking of FcγR function demonstrates a proof of concept to successfully treat FcγR-mediated autoimmune diseases.

Non-CpG Oligonucleotides Exert Adjuvant Effects by Enhancing Cognate B Cell-T Cell Interactions, Leading to B Cell Activation, Differentiation, and Isotype Switching.

Natural and synthetic nucleic acids are known to exert immunomodulatory properties. Notably, nucleic acids are known to modulate immune function via several different pathways and various cell types, necessitating a complex interpretation of their effects. In this study we set out to compare the effects of a CpG motif containing oligodeoxynucleotide (ODN) with those of a control and an inhibitory non-CpG ODN during cognate B cell-T cell interactions. We employed an antigen presentation system using splenocytes from TCR transgenic DO11.10 mice and the ovalbumin peptide recognized by the TCR as model antigen. We followed early activation events by measuring CD69 expression, late activation by MHC class II expression, cell division and antibody production of switched, and nonswitched isotypes. We found that both of the tested non-CpG ODN exerted significant immunomodulatory effects on early T cell and on late B cell activation events. Importantly, a synergism between non-CpG effects and T cell help acting on B cells was observed, resulting in enhanced IgG production following cognate T cell-B cell interactions. We propose that non-CpG ODN may perform as better adjuvants when a strong antigen-independent immune activation, elicited by CpG ODNs, is undesirable.

The endothelial deprotection hypothesis for lupus pathogenesis: the dual role of C1q as a mediator of clearance and regulator of endothelial permeability.

Systemic lupus erythematosus (SLE) is a heterogeneous multifactorial systemic autoimmune disease affecting several organs. SLE can start relatively early in life and results in impaired quality of life and shortened life expectancy because of a gradual disease progression leading to cardiovascular, renal and neoplastic disease. The basic mechanisms of the pathogenesis of the disease still remain to be clarified. It is clear that complement proteins play a key and complex role in the development of SLE. Complement component C1q has been known to be a fundamental component of lupus development, but most explanations focus on its role in apoptotic debris removal. Importantly, C1q was recently found to play a key role in the maintenance of vascular endothelial integrity. We suggest that apoptotic products, endothelial cells and extracellular matrix components, which display negatively charged moieties, compete for binding to molecules of the innate humoral immune response, like C1q. Genetic or acquired factors leading to an increased load of apoptotic cell debris and decrease or absence of C1q therefore interfere with the regulation of endothelial permeability and integrity. Furthermore, we suggest that lupus is the net result of an imbalance between the two functions of immune clearance and vascular endothelial integrity maintenance, an imbalance triggered and sustained by autoimmunity, which skews C1q consumption by IgG-mediated complement classical pathway activation on autoantigens. In this triangle of innate clearance, autoimmunity and endothelial integrity, C1q plays a central role. Hence, we interpret the pathogenesis of lupus by identifying three key components, namely innate immune clearance, autoimmunity and endothelial integrity and we establish a link between these components based on the protective role that innate clearance molecules play in endothelial renewal. By including the vasoprotective role of C1q in the interpretation of SLE development we attempt to provide novel explanations for the symptoms, organ damage, diagnostic and therapeutic difficulties of the disease.