Functional diversity and clonal frequencies of reactivity in the available antibody repertoire

The present experiments address functional antibody diversity and clonal distribution in murine available repertoires. IgM-containing supernatants were prepared by unbiased, polyclonal stimulation of resting splenic B cells from C57BL/6 mice, to ensure similar numbers of responding clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively assayed by limiting dilution analysis (LDA) on immunoblots of sodium dodecylsulfate polyacrylamide gel electrophoresis of homologous liver extracts, allowing to determine specific clonal frequencies towards the many hundred blotted antigens. The clonal frequency of reactivity of B cells with the extract was shown to be a bi-modal distribution of specific frequencies between 1/220 and 1/100 000. Cross-correlation analysis of reactivity to different bands in individual supernatants revealed low levels of cross-reactivity, suggesting that the blotted extract provides a very diverse set of antigens. Investigation of the affinity/concentration thresholds for detection of antigen-antibody interactions of our assay supports the notion that global repertoire analyses on immunoblots were highly discriminative and non-degenerate. Furthermore, reactivity patterns obtained with complex antibody mixtures correlated with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphocytes, indicating a minimal repertoire size that is orders of magnitude higher than previous theoretical proposals suggested, and extensively heterogeneous in the size of clonal specificities.     

Complement activation by (auto-) antibodies

The complement system is a key part of the innate immune system and plays an important role in the clearance of pathogens and apoptotic cells upon its activation. It is well known that both IgG and IgM can activate complement via the classical pathway by binding of C1q to the Fc regions of these immunoglobulins. Recent advances have shown that also IgA is capable of activating the complement system. Besides, more insight is gained into an additional role for antibodies in the activation of both the alternative and the lectin pathways. Mouse models have shown that auto-antibodies can activate the alternative pathway and induce in cell lysis and tissue damage.Besides the role of antibodies in complement activation, complement may also be a target for recognition by antibodies directed against autologous complement components. These auto-antibodies play a role in several diseases, especially vascular diseases. Understanding how antibodies interact with the complement system will allow the manipulation of this interaction to diminish pathological consequences of auto-antibodies and optimize the effect of therapeutic antibodies.In the current review, we discuss complement activation by (auto-) antibodies by the different pathways.     

Anti-nuclear antibody reactivity in lupus may be partly hard-wired into the primary B-cell repertoire

When monoclonal ANAs and non-ANAs generated from a genetically simplified mouse model of lupus, B6.Sle1, were recently compared, the ANAs exhibited three sequence motifs in their immunoglobulin heavy chains, including increased cationicity in CDR3 (“motif A”), reduced anionicity in CDR2 (“motif B”) and increased aspartate at H50 (“motif C”). The present study was designed to elucidate the extent to which these ANA-associated sequence motifs might be hard-wired into the primary B-cell repertoire in lupus. The immunoglobulin heavy chain sequence of total splenic B-cells, follicular B-cells and marginal zone B-cells from B6.Sle1 congenic mice and C57BL/6 controls were amplified by single-cell PCR and compared. Analysis of the primary immunoglobulin heavy chain repertoire indicated that the first two sequence motifs “A” and “B” were already encoded in the naïve repertoire of B6.Sle1^z mice, whereas the third motif “C” was introduced in part by somatic mutation. Site-directed mutagenesis confirmed that non-anionic CDR2 and cationic CDR3 residues in the immunoglobulin heavy chain facilitated nuclear antigen binding in concert, whereas aspartate at H50 strongly vetoed DNA-binding, while preserving nucleosome reactivity. Hence, anti-nuclear antibodies appear to arise as a consequence of two distinct processes—genetically programmed selection of specific CDR charge motifs into the primary immunoglobulin repertoire, with secondary contribution from somatic mutation. Polymorphisms in the lupus susceptibility gene Ly108 that impair central B-cell tolerance may be mechanistically responsible for these early repertoire differences in lupus.     

Expression and control of the natural autoreactive IgG repertoire in normal human serum

We have investigated the autoreactive repertoire expressed by serum IgG of healthy individuals of various age groups using a large panel of self antigens. Natural IgG autoantibodies against all self antigens of the panel were found in the purified IgG fraction of the serum of all donors that were tested. The mean binding activity to self antigens of IgG of pregnant women was higher than that of IgG purified from the serum of infants, young adults and aged individuals. No increase in IgG autoreactivity was observed with aging neither in the purified IgG fraction of serum nor in whole serum. Whereas autoantibody activity was easily detectable in purified IgG, it was low in serum. No difference was observed, however, between the binding activity of purified IgG and of IgG in serum in the case of foreign antigens nor in the case of anti-thyroglobulin autoantibodies of patients with Hashimoto's thyroiditis. Purified IgM from normal serum bound to F(ab')₂ fragments of autologous IgG in a dose-dependent fashion and inhibited the binding of autologous IgG to self antigens. Our results thus indicate that autologous IgM contributes to regulate expression of the natural IgG autoreactive repertoire through V region-dependent interactions, resulting in low levels of IgG autoreactivity in serum under physiological conditions.     

Selective impairment of serum antibody repertoires toward muscle and thymus antigens in patients with seronegative and seropositive myasthenia gravis

We analyzed the antibody (Ab) repertoires of IgM and IgG of patients with seropositive and patients with seronegative myasthenia gravis (MG) toward self antigens by means of a quantitative immunoblotting technique using normal human tissue extracts as sources of self antigens. Repertoires of reactivities of IgG and IgM with liver, kidney and stomach antigens were conserved between myasthenic patients and controls. IgG and IgM Ab repertoires toward muscle antigens differed significantly between patients with seropositive MG and healthy donors, as assessed by multiparametric statistical analysis. Patterns of Ab reactivities to muscle antigens were similar in patients with seronegative MG and healthy controls. Antibody repertoires of IgG and IgM toward thymus antigens of both seropositive and sero negative MG patients, differed significantly from those of healthy individuals. Our results indicate that MG is characterized by a selective impairment of self-reactive Ab repertoires toward muscle and thymus antigens. The observation that self-reactive Ab repertoires toward thymus antigens are similar in patients with seropositive and seronegative MG suggests that both forms of MG share common immunopathological features.     

Complement and cutaneous autoimmune blistering diseases

Cutaneous autoimmune blistering diseases are associated with tissue injury and fluid accumulation within the skin. The initial trigger for the organ-specific damage is autoantibodies targeting skin autoantigens, which are involved in cell–cell or cell–matrix adhesion in the skin. Pemphigus autoantibodies bind to desmosomal antigens and cause intraepidermal blisters, while pemphigoid autoantibodies interact with hemidesmosomal or hemidesmosome-associated antigens and lead to dermal–epidermal junction separation. Local complement activation is a common feature for these skin blistering diseases and some complement components are readily detected in the lesional skin and blister fluids. This review summarizes the current knowledge on the role the complement system in skin blister formation. Characterization of the pathogenically relevant complement cascade and relative contribution of different pathways into complement activation provides new insights of disease pathology and may help develop better therapeutic strategies for these potentially fatal cutaneous blistering disorders.     

Complement deficiency states and meningococcal disease

Analysis of complement deficiency states has supported the role of complement in host defense and elucidated diseases associated with defective complement function. Although neisserial infection plays a prominent role in these deficiency states, examination of individuals with late complement component deficiency (LCCD) reveals a particular propensity for recurrent meningococcal disease and provides important clues to the role of complement in neisserial infections. In response to meningococcal disease, LCCD individuals produce significantly greater amounts of antilipooligosaccharide (LOS) antibody which can kill group B meningococcus in a complement-sufficient in vitro system. Further studies of antibody cross-reactivity to other meningococci has led to a clearer understanding of its epitopic specificity. Nevertheless, epidemiologic evidence is consistent with the relative absence of protective immunity in LCCD persons following an episode of infection and supported by quantitation of antibody to capsular polysaccharide. However, compared to anti-LOS antibodies, anticapsular antibodies can offer immune protection to LCCD individuals via complement-dependent opsonophagocytosis-the only form of complement-mediated killing available to these persons. Thus vaccination of LCCD persons with capsular antigens is considered an important means of protecting these high-risk individuals against meningococcal disease.     

Probing the human natural autoantibody repertoire using an immunoscreening approach

While an increasing number of studies report the presence of antibodies capable of recognizing self-antigens, the function of these natural autoantibodies remains elusive. A variety of concepts has been advanced ranging from evolutionarily tolerated but non-functional natural autoantibodies to autoantibodies effecting various biological functions. Known IgM, IgG, and IgA natural autoantibodies are directed against various antigens, including nuclear and cell surface proteins. To explore further autoantibodies and their autoantigens, we employed an immunological screening method called SEREX recently used to characterize tumour-expressed antigens eliciting an immune response in patients [1]. Sera from 12 individuals were used to screen a cDNA expression library prepared from a cytogenetically normal meningioma to identify antigens reactive with normal human sera from individuals without obvious disease. Nineteen reactive normal antigen clones were identified representing 15 different antigens, including nine genes with known functions, five genes with unknown functions, and one gene with a novel sequence not present in the databases. Of the 12 individual normal sera tested, 75% were reactive to one or more of the 15 different antigens with two highly reactive sera demonstrating reactivity with 33% of the antigens. When screening the same meningioma expression library with serum from the patient, eight antigens were identified that were totally different from those identified using sera from normal individuals. This SEREX immunological screening method presents a new option for probing the natural autoantibody repertoire and identifying normal antigens whose functions may provide additional insights into how natural autoantibodies effect various biological functions.     

Global analysis of antibody repertoires. II. Evidence for specificity,self-selection and the immunological “homunculus” of antibodies in normal serum

The serum IgM repertoires of C57BL/6, DBA/2 and BALB/c mouse strains were analyzed using a recently developed global and quantitative assay that measures antibody reactivities to a very large number of antigens. A characteristic repertoire could be assigned to each strain. The different repertoires could be successfully classified with multivariate statistics. Many common reactivities were also observed among the different strains, which allows the definition of a mouse-specific repertoire. Analysis of human sera support this notion. To investigate the impact of minor genetic differences on the serum IgM repertoire, the congenic strains B10.D2/oSn and B10.D2/nSn, which differ in the expression of the C5 component of complement, were analyzed. The two strains could be separated based on the reactivity profiles obtained. The analysis of the results reveals that many antigenic proteins are not recognized at all by natural antibodies, while others are disproportionately reactive, the resulting patterns giving rise to what could be the definition of an “immunological homunculus”. The relevance of this type of analysis for clinical applications is discussed.     

Expression and regulation of complement receptors by human natural killer cells

Integration of cellular and humoral arms of the innate immune response is fundamental to the development of powerful effector functions in host defence as well as aberrant immune responses. Here, we provide evidence in support of the relationship between complement activation and NK cell functional modulation. We demonstrate that human NK cells and both CD56^brightCD16⁻ and CD56^dimCD16⁺ populations express receptors known to detect the biologically active peptides C3a and C5a (i.e. C3aR, C5aR, C5L2) and the covalently-bound fragments C3b and metabolites iC3b and C3d which serve in immune adhesion (e.g. CR3, CR4). We also show that several pathogen- or tumour/inflammation-related stimuli differentially regulated those complement receptor expression. Furthermore, our results suggest that C3 fragments (C3a, iC3b) have a negative regulatory effect on IFN-γ production in NK cells. This work provides extensive information of human complement receptors relevant to the integrated actions of complement and NK cells which has been suggested by animal studies. The observations may act as a resource that allows further understanding and exploitation of role of complement in human health and immune mediated diseases.     

Complement and cytokine based therapeutic strategies in myasthenia gravis

Myasthenia gravis (MG) is a T cell–dependent and antibody-mediated disease in which the target antigen is the skeletal muscle acetylcholine receptor (AChR). In the last few decades, several immunological factors involved in MG pathogenesis have been discovered mostly by studies utilizing the experimental autoimmune myasthenia gravis (EAMG) model. Nevertheless, MG patients are still treated with non-specific global immunosuppression that is associated with severe chronic side effects. Due to the high heterogeneity of AChR epitopes and antibody responses involved in MG pathogenesis, the specific treatment of MG symptoms have to be achieved by inhibiting the complement factors and cytokines involved in anti-AChR immunity. EAMG studies have clearly shown that inhibition of the classical and common complement pathways effectively and specifically diminish the neuromuscular junction destruction induced by anti-AChR antibodies. The inborn or acquired deficiencies of IL-6, TNF-α and TNF receptor functions are associated with the lowest EAMG incidences. Th17-type immunity has recently emerged as an important contributor of EAMG pathogenesis. Overall, these results suggest that inhibition of the complement cascade and the cytokine networks alone or in combination might aid in development of future treatment models that would reduce MG symptoms with highest efficacy and lowest side effect profile.     

Knock-out of the histidine decarboxylase gene modifies the repertoire of natural autoantibodies

Natural antibodies (NA) are antibodies produced in the absence of known immunization with specific antigens. NA are found in the blood of healthy humans and mice. Histamine influences many aspects of the immune response, including antibody production. However, the role of histamine in the generation of NA has not yet been studied. In this work, we used an ELISA assay to characterize the self-antigen binding repertoires of NA in wild type (WT) mice and in histidine decarboxylase knock-out (HDC-KO) mice, unable to synthesize histamine. We now report that HDC-KO and WT mice differed in the patterns of autoreactivity of their IgM and IgG NA. The NA in HDC-KO sera manifested a larger repertoire of IgM autoantibodies than did the WT sera. The self-antigens bound by IgM from HDC-KO mice included structural proteins, enzymes associated with cellular metabolism, double-stranded and single-stranded DNA, and tissue-specific antigens like insulin. There were relatively fewer differences in the NA repertoire of IgG autoantibodies of the mice: notably, the HDC-KO sera reacted with glutamic acid decarboxylase (GAD), an antigen associated with autoimmune diabetes. These results demonstrate that endogenous histamine can influence the self-reactivity of the NA repertoire.     

Correlation of antigenic epitope and antibody gene usage in the human immune response to Streptococcus pneumoniae type 23F capsular polysaccharide

The human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23F is predominated by antibodies which express either VkappaL6 or VkappaA23 light chain genes. In this report, we demonstrate that the antigenic epitopes recognized by these two families can be differentiated based on their specificity for the hapten l-rhamnose, a constituent sugar of the bacterial capsule polymer. The VkappaL6 family utilizes light chains with an unusually long third complementarity-determining region. These VkappaL6-encoded Fabs are 100-fold more sensitive to inhibition with l-rhamnose than VkappaA23 Fabs. VkappaL6 Fabs preferentially recognize the l-isomer of the hapten and can distinguish l-rhamnose from the structurally similar sugar l-mannose. We also demonstrate that the VkappaL6 family component of the antibody response recognizes the polysaccharide antigens of Group B streptococci (GBS), thereby accounting for the previously reported cross-reaction between PPS 23F-specific antisera and Group B streptococci.     

Studies on the T cell dependence of natural IgM and IgG antibody repertoires in adult mice

The present experiments address the thymic dependence of IgM and IgG natural antibody repertoires in adult euthymic and athymic BALB/c mice, as well as in athymic animals reconstituted with a fixed number of syngeneic T cells. Within 3 weeks of the transfer of 10⁷ syngeneic splenic T lymphocytes to athymic mice, the T cell compartment is essentially reconstituted in the peritoneal cavity (up to 80% of the numbers in euthymic animals), but is only 10–20% of controls in the spleen and lymph nodes. Early after transfer, there is an increase in the numbers of activated B cells and of immunoglobulin-secreting cells in the spleen, and within 1–2 weeks, the serum concentrations of IgG₁ and IgG_2a are fully reconstituted to control levels (30–40-fold increased). Multiparametric analyses of serum IgM and IgG repertoires revealed that euthymic and athymic mice share essentially all natural antibody reactivities towards syngeneic extracts of liver and muscle. When tested at the same immunoglobulin concentrations, however, nude sera consistently show higher values of reactivity in all detectable bands. The transfer of 10⁷ splenic T cells into athymic mice results in a general decrease of serum IgM reactivities, some of which become undetectable, and in alterations of the serum IgG repertoire as early as 1 week, and for at least 4 weeks after transfer. T cell transfer, however, fails to restore the euthymic IgM and IgG repertoires within 4 weeks. The present observations demonstrate that, after limited T cell reconstitution of nude mice, there is a rapid and quantitatively important increase of serum IgG₁ and IgG_2a production; the serum IgM reactivity repertoire is qualitatively similar in euthymic and athymic animals, but is generally decreased by T cell activity; and the serum IgG repertoire, which is qualitatively similar in euthymic and athymic animals, is amplified by T cell activity and partially altered by T cell transfer into athymic animals. These results raise questions on the mechanisms of B cell activation and natural antibody repertoire selection in T cell-deficient adult individuals.     

Generation and selection of an IgG-driven autoimmune repertoire during B-lymphopoiesis in Igmicro-deficient/lpr mice

Class switch recombination (CSR) is a well-regulated process that occurs in peripheral lymphoid tissue, and is thought of as an important factor constructing the memory repertoire. We have recently shown that CSR normally occurs during bone marrow (BM) development, and these isotype-switched B cells are negatively selected by Fas signaling. This novel pathway of B cell development may generate a primary repertoire driven by gamma-heavy receptors, the nature of which is yet unknown. To study this gammaH-driven repertoire we used mice lacking IgM-transmembrane tail exon ( micro MT), where B cell development is limited by their ability to undergo CSR. We already showed that lack of Fas signaling rescues development of a significant population of isotype-switched B cells and production of high titers of non-IgM serum antibodies in micro MT mice deficient in Fas ( micro MT/lpr), thereby providing a mouse model allowing the assessment of gammaH-driven repertoire. Using a tissue array and phage display epitope library we report here that IgG repertoire in micro MT/lpr mice is oligo-monoclonal, bearing self-tissue reactivity. This is supported by analysis of the Vkappa utilization in peripheral B cells from micro MT/lpr mice, which revealed a strikingly restricted repertoire. In contrast, micro MT/lpr B cells that are grown in non-selective BM cultures utilize a wide repertoire. These results suggest that the Fas pathway is an important regulator in the generation and selection of an autoimmune gammaH-driven repertoire in vivo.     

Complement Activation by Apoptotic Cells Occurs Predominantly via IgM and is Limited to Late Apoptotic (Secondary Necrotic) Cells

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation.

Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM.

We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.     

Analysis of human self-reactive antibody repertoires by quantitative immunoblotting

We review the use of a quantitative immunoblotting technique to characterize human self-reactive antibody repertoires in health and disease. The interactions of plasma IgM and IgG with tissue extracts as sources of self-antigens were analyzed by quantitative immunoblotting. Data were compared by means of multiparametric statistical analysis. The data summarized here demonstrate that natural self-reactive antibody repertoires of healthy individuals are restricted to a limited subset of immunodominant autoantigens that is selected early in development, and remains conserved between individuals through ageing. The selection of human natural self-reactive IgG antibody repertoires requires normal T-/B-cell interactions. The immunoblotting assay has the potential to distinguish between autoimmune diseases with organ-related oligoclonal expansion of self-reactive clones and those characterized by broad alterations of immunoregulation. However, organ-specific autoimmune diseases may be characterized by altered patterns of antibody repertoires unrelated to the target organ. The assay also revealed an unexpected defect in the regulatory function of self-reactive IgM on the expression of self-reactive IgG repertoires in several systemic and organ-specific autoimmune diseases. The results are discussed in the light of our current understanding of the processes of selection of self-reactive B-cells and the pathophysiology of autoimmunity.     

Homogenous expression of killer cell immunoglobulin-like receptors (KIR) on polyclonal natural killer cells detected by a monoclonal antibody to KIR2D

The activity of human natural killer (NK) cells is in part regulated by the expression of killer cell immunoglobulin (Ig)-like receptors (KIR) that recognize major histocompatibility complex (MHC) class I and can inhibit NK cell cytotoxicity. A monoclonal anti-KIR antibody was established and designated Lig1. Lig1 was shown to be specific for KIR in cell-surface staining and to react with all KIR2D, except KIR2DL4 which lacks a D1 domain, but not with KIR3D molecules in an enzyme-linked immunoadsorbent assay (ELISA) and Western blotting. Unlike other anti-KIR antibodies, Lig1 did not inhibit binding of KIR-Ig-fusion proteins to MHC-class I expressing cells nor did it interfere with KIR-mediated inhibition of NK cell cytotoxicity in a functional assay. Lig1 reacted with all NK cells in polyclonal NK populations from different donors, demonstrating that all NK cells express at least one KIR2D receptor.     

Genetic basis of the neonatal antibody repertoire: germline V-gene expression and limited N-region diversity

We report here on the molecular characterization of heavy and light chain V-regions of antibodies isolated from the highly connected idiotypic network of the newborn BALB/c mouse. Nucleotide sequence analysis of eight hybridomas confirmed their germline origin. Furthermore, in contrast to most hybridomas and myelomas derived from adult mice, the majority of these clones were found to lack N-region sequences. These data show that somatic processes amplifying junctional diversity are relatively inactive early in ontogeny, and that germline gene expression alone ensures idiotypic complementarities in the developing immune system.