Krisztián Papp

Anti-factor B autoantibody in dense deposit disease.

Dense deposit disease (DDD), also known as membranoproliferative glomerulonephritis type II, is a rare kidney disorder that is associated with dysregulation of the alternative pathway of complement. Autoantibodies against the C3bBb convertase termed C3 nephritic factor are common in DDD patients. Here we report an autoantibody that binds to complement factor B in a DDD patient who was negative for C3 nephritic factor. This anti-factor B autoantibody recognized an epitope within the Bb fragment and was able to bind to the C3bBb convertase. Upon binding, the anti-factor B autoantibody stabilized the convertase against both intrinsic and factor H-mediated extrinsic decay and thus enhanced C3 consumption. Functional analyses demonstrated that, in contrast to C3 nephritic factor, the anti-factor B autoantibody inhibited complement-mediated lysis in vitro due to inhibition of the C5 convertase and the terminal complement pathway. Analysis of C5a plasma levels indicated that not all C5 convertases are inhibited by the autoantibodies in the patient in vivo. Antigen array experiments confirmed the presence of anti-factor B autoantibodies and also revealed complement activating anti-C1q antibodies in the patients plasma. In summary, the present report describes a new autoantibody in DDD that binds to factor B and to the alternative pathway C3 convertase and alters the kinetics of complement activation and regulation.

B lymphocytes and macrophages release cell membrane deposited C3-fragments on exosomes with T cell response-enhancing capacity ☆

Recently exosomes have been shown to play important roles in several immune phenomena. These small vesicles contain MHC proteins along with co-stimulatory and adhesion molecules, and mediate antigen presentation to T cells. In the present study we show that upon incubation with autologous serum, murine macrophages and B cells--but not T lymphocytes--fix C3-fragments covalently to the cell membrane and release them on exosomes in a time dependent fashion. While in the case of human B lymphocytes CR2 has been shown to serve as the main C3b-acceptor site, here we clearly demonstrate that cells derived from CR1/2 KO animals also have the capacity to fix C3b covalently. This finding points to a major difference between human and murine systems, and suggests the existence of additional acceptor sites on the cell membrane. Here we show that C3-fragment containing exosomes derived from OVA loaded antigen presenting cells induce a significantly elevated T cell response in the presence of suboptimal antigen stimulus. These data reveal a novel function of cell surface-deposited C3-fragments and provide further evidence for the role of exosomes secreted by antigen presenting cells. Since fixation of C3b to plasma membranes can be substantial in the presence of pathogens; moreover tumor cells are also known to activate the complement system resulting in complement-deposition, C3-carrying exosomes released by these cells may play an important immunomodulatory role in vivo, as well.

Exploiting fluorescence for multiplex immunoassays on protein microarrays

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

Antigen microarrays: descriptive chemistry or functional immunomics?

Advances in protein microarray technology allow the generation of high content, reliable information about complex, multilevel protein interaction networks. Yet antigen arrays are used mostly only as devices for parallel immune assays describing multitudes of individual binding events. We propose here that the huge amount of immunological information hidden in the plasma of an individual could be better revealed by combining the characterization of antibody binding to target epitopes with improved estimation of effector functions triggered by these binding events. Furthermore, we could generate functional immune profiles characterizing general immune responsiveness of the individual by designing arrays incorporating epitope collections from diverse subsets of antibody targets.

Life on a microarray: assessing live cell functions in a microarray format

Microarray technology outgrew the detection of simple intermolecular interactions, as incubation of slides with living cells opened new vistas. Cell-based array technology permits simultaneous detection of several different cell surface molecules, allowing the complex characterization of cells with an amount of information that is hardly assessed by any other technique. Furthermore, binding of cells to printed antibodies or ligands may induce their activation, and consequently the outcome of these interactions, such as phosphorylation, gene expression, secretion of various products; differentiation, proliferation and apoptosis of the cells are also measurable on arrays. Moreover, since cells can be transfected with printed vectors, over- or under-expression of selected genes is also achievable simultaneously, creating a nice tool for assessing the function of a given gene. The enormously high-throughput cell-based microarray technology enables testing the effect of external stimuli on a scale that was earlier unthinkable. This review summarizes the possible applications of cell-based arrays.

On-chip Complement Activation Adds an Extra Dimension to Antigen Microarrays

Antibody profiling on antigen microarrays helps us in understanding the complexity of responses of the adaptive immune system. The technique, however, neglects another, evolutionarily more ancient apparatus, the complement system, which is capable of both recognizing and eliminating antigen and serves to provide innate defense for the organism while cooperating with antibodies on multiple levels. Complement components interact with both foreign substances and self molecules, including antibodies, and initiate a cascade of proteolytic cleavages that lead to the covalent attachment of complement components to molecules in nanometer proximity. By refining the conditions of antibody profiling on antigen arrays we made use of this molecular tagging to identify antigens that activate the complement system. Antigen arrays were incubated with serum under conditions that favor complement activation, and the deposited complement C3 fragments were detected by fluorescently labeled antibodies. We used genetically C3-deficient mice or inhibition of the complement cascade to prove that the technique requires complement activation for the binding of C3 to features of the array. We demonstrate that antigens on the array can initiate complement activation both by antibody-dependent or -independent ways. Using two-color detection, antibody and complement binding to the relevant spots was measured simultaneously. The effect of adjuvants on the quality of the immune response and binding of autoantibodies to DNA with concomitant complement activation in the serum of mice suffering from systemic autoimmune disease was readily measurable by this new method. We propose that measurement of complement deposition on antigen microarrays supplements information from antibody binding measurements and provides an extra, immune function-related fingerprint of the tested serum.

Immune Complex Signatures of Patients with Active and Inactive SLE Revealed by Multiplex Protein Binding Analysis on Antigen Microarrays

Systemic lupus erythematosus is characterized by dysfunctional clearance of apoptotic debris and the development of pathogenic autoantibodies. While the complement system is also involved in the disease no attempt has been made to generate a comprehensive view of immune complex formation from various autoantigens. We increased the complexity of autoantibody profiles by measuring the binding of two complement proteins, C3 and C4, in addition to two antibody classes, IgG and IgM, to a collection of autoantigens. These complement components covalently bind to those microarray features where antibodies and other serum components induce complement activation. Using this technology, we compared functional serum antibody profiles of control subjects (n = 31) and patients with lupus erythematosus (n = 61) in the active (n = 22) and inactive (n = 39) phase of the disease. Multivariate analysis was applied to identify contributions of binding data on 25 antigens to the discrimination of the study groups. Receiver operating characteristic analysis was used to portray the discriminative property of each measured parameter for each antigen in pairwise group comparisons. Complement C3 and C4 deposition increased on autoantibody targets in spite of the decreased serum complement concentrations, and decreased on other autoantigens, demonstrating the imbalance of complement function in patients with lupus erythematosus. Our observations confirmed previously known markers of disease and showed that C3 and C4 deposition data were at least as powerful as Ig binding data in separating the study groups.

FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.

Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients

Anti‐citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline‐ and arginine‐containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline‐peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline‐peptides identify antibody‐secreting cells in in vitro cultures of RA B cells. Recognition of citrulline‐ and arginine‐containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide‐specific microarray. B cells were purified from blood by negative selection; antibody‐producing cells were enumerated by ELISPOT assay. The panel composed of citrulline‐peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline‐peptide panel including the new short epitope peptide of filaggrin, fil311‐315, also identified nearly one‐third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide‐specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline‐containing filaggrin peptides (fil311–315 and fil306–326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline‐peptides of filaggrin and vimentin detect ACPA‐producing cells, and so could also be applied to study the B cells of RA patients.

Detection of complement activation on antigen microarrays generates functional antibody profiles and helps characterization of disease-associated changes of the antibody repertoire.

Humoral immune responses are traditionally characterized by determining the presence and quality of Abs specific for certain Ags. Arraying of large numbers of Ags allows the parallel measurement of Abs, generating patterns called Ab profiles. Functional characterization of these Abs could help draw an even more informative map of an immune response. To generate functional Ab profiles we simultaneously tested not only IgM, IgG, and IgA binding to, but also complement activation by, a panel of endogenous and exogenous Ags printed as microarrays, using normal and autoimmune human sera. We show that complement activation by a particular Ag in a given individual cannot be predicted by the measurement of Ag-specific Abs, despite a general correlation between the amount of Ag-bound Ab and the deposited C3 fragments. This is due to both differences in the isotypes that dominate in the recognition of an Ag and individual variations for a given isotype, resulting in altered complement activation potential. Thus, Ag-specific C3 deposition can be used as an additional parameter in immune response monitoring. This is exemplified by comparing the coordinates of Ags, used for the diagnosis of systemic lupus erythematosus, of normal and autoimmune serum samples in a two-dimensional space derived from C3 deposition and Ab binding. Since cleavage fragments of C3 mediate important immunological processes, we propose that measurement of their deposition on Ag microarrays, in addition to Ab profiling, can provide useful functional signature about the tested serum.