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.     

Manipulation of the glycan-specific natural antibody repertoire for immunotherapy

Natural immunoglobulin derived from innate-like B lymphocytes plays important roles in the suppression of inflammatory responses and represents a promising therapeutic target in a growing number of allergic and autoimmune diseases. These antibodies are commonly autoreactive and incorporate evolutionarily conserved specificities, including certain glycan-specific antibodies. Despite this conservation, exposure to bacterial polysaccharides during innate-like B lymphocyte development, through either natural exposure or immunization, induces significant changes in clonal representation within the glycan-reactive B cell pool. Glycan-reactive natural antibodies (NAbs) have been reported to play protective and pathogenic roles in autoimmune and inflammatory diseases. An understanding of the composition and functions of a healthy glycan-reactive NAb repertoire is therefore paramount. A more thorough understanding of NAb repertoire development holds promise for the design of both biological diagnostics and therapies. In this article, we review the development and functions of NAbs and examine three glycan specificities, represented in the innate-like B cell pool, to illustrate the complex roles environmental antigens play in NAb repertoire development. We also discuss the implications of increased clonal plasticity of the innate-like B cell repertoire during neonatal and perinatal periods, and the prospect of targeting B cell development with interventional therapies and correct defects in this important arm of the adaptive immune system.     

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.     

IgD+IgM- B cells mount immune responses that exhibit altered antibody repertoire

IgM and IgD expression during B cell development and differentiation is strictly and developmentally controlled. Although studies have suggested subtle differences in B cell activation, tolerance, and affinity maturation when antigens ligate cell membrane IgM or IgD, the mechanisms that may explain these differences remain unknown and no drastic differences in immune responses have been reported in mice whose B cells selectively lack IgM or IgD. We now show that the antibody repertoire in IgM(-/-) mice is dramatically altered during the primary response to phosphorylcholine.In IgM(-/-) mice, B cells that are activated and differentiate into antibody-forming cells and germinal center B cells express VH genes other than the T15 genes that dominate in wild-type mice. The kinetics of the antigen-specific IgD primary antibody response in IgM(-/-) mice appears similar to that of IgG, but not to that of IgM in wild-type mice. Thus, our studies demonstrate that differences in the roles played by IgM and IgD in regulating the responsiveness and differentiation of B lymphocytes can have major biological consequences during adaptive immune responses.     

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.     

Disease etiology and diagnosis by TCR repertoire analysis goes viral

The importance of T‐cell receptor (TCR) repertoire diversity is highlighted in murine models of immunodeficiency and in many human pathologies. However, the true extent of TCR diversity and how this diversity varies in health and disease is poorly understood. In a previous issue of the European Journal of Immunology, Lossius et al. [Eur. J. Immunol. 2014. 44: 3439–3452] dissected the composition of the TCR repertoire in the context of multiple sclerosis (MS) using high‐throughput sequencing of TCR‐β chains in cerebrospinal fluid samples and blood. The authors demonstrated that the TCR repertoire of the CSF was largely distinct from the blood and enriched in EBV‐reactive CD8⁺ T cells in MS patients. Studies of this kind have long been hindered by technical limitations and remain scarce in the literature. However, TCR sequencing methodologies are progressing apace and will undoubtedly shed light on the genetic basis of T‐cell responses and the ontogeny of T‐cell‐mediated diseases, such as MS.     

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.     

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.     

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.     

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.     

Organ specific autoantigens and the autoreactiveT cell repertoire: the case of myelin oligodendrocyte glycoprotein

There is strong evidence that immunological self tolerance critically relies on the elimination of potentially autoaggressive T lymphocyte clones from the emerging immune repertoire during intrathymic T cell differentiation. These 'forbidden' T cells are deleted as a result of a confrontation with their specific self antigen as presented on medullary stroma cells. But this purging mechanism is remarkably leaky, allowing numerous autoreactive T cells to join the healthy immune repertoire. A paper in this issue of the European Journal of Immunology studies the effect of organ-specific autoantigen expression on the cognate T cell repertoire. Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in human multiple sclerosis, is used as a model self antigen. T cell receptor profiles in wild-type mice were compared with those in MOG-knock-out mice. Surprisingly, significant differences were not found suggesting that, in this particular case, autoantigen expression does not affect the autoreactive T cell repertoire.     

A population biological approach to understanding the maintenance and loss of the T‐cell repertoire during aging

The adaptive immune system requires a diverse T‐cell repertoire to be able to respond to a wide variety of pathogens. Worryingly, the repertoire diversity declines dramatically in old age. As thymic output generates novel T cells, the conventional view holds that a decrease in this output with age is responsible for the loss in the repertoire. However, many additional factors affect the repertoire such as homeostatic turnover and antigen‐dependent expansion in response to infection. Mathematical models taking a population biology perspective are important tools for understanding how the interplay between these factors affects the immune repertoire. These models suggest that thymic decline is not a major factor but rather that some combination of virus‐induced proliferation and T‐cell‐intrinsic genetic or epigenetic changes gives rise to the oligoclonal expansions that cause the decline in T‐cell diversity. We also discuss consequences for strategies to rejuvenate the immune repertoire in old age.     

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.