Antigen-binding B cells and polyreactive antibodies

The present experiments were initiated to see if cells capable of binding antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self antigens were incubated with B cells from normal individuals. Antigenbinding cells were separated from non-antigen-binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound antigens than in the B cell subset that did not bind antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of antigens (e.g., insulin, IgGFc and β-galactosidase), whereas cell lines making monoreactive antibodies bound only a single antigen. The binding of antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%–57%) by both homogeneous and heterogeneous antigens. Both CD5⁺ and CD5^? antigen-binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5⁺ antigen-binding (85%) as compared to the CD5^? antigen-binding (50%) population. Comparison of CD5⁺ B cells that bound antigens with CD5⁺ B cells that did not bind antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of binding a variety of different antigens can make polyreactive antibodies and that antigen binding is a good marker for identifying polyreactive antibody-producing cells.     

Monoreactive and Polyreactive Rheumatoid Factors Produced by in Vitro Epstein-Barr Virus-Transformed Peripheral Blood and Synovial B Lymphocytes from Rheumatoid Arthritis Patients

The CD5 membrane molecule, initially identified as an exclusive T-cell marker, also defines a phenotypically and functionally distinct B-lymphocyte population. In normal individuals, CD5+ B cells are committed to secrete 'natural' polyreactive (auto)antibodies, that is antibodies, mainly IgM, endowed with multiple antigen-binding capabilities, including rheumatoid factor (RF) activity. At variance with this, in rheumatoid arthritis (RA) as well as in other autoimmune conditions, monoreactive autoantibodies binding with high affinity and specificity to a given self antigen have been isolated and the cells from which they originate differently related to the CD5+ B-lymphocyte subset. Here, we studied the proportions of CD5+ B cells and the characteristics, in terms of polyreactivity and monoreactivity, of RF produced by B lymphocytes in RA patients with classified disease activity. Our results suggest that patients with a more severe disease activity have higher proportions of CD5+ B cells and higher frequencies of B lymphocytes committed to secrete RF, with the characteristics of polyreactive antibodies. On the other hand, we did not find a significant difference between the proportions of peripheral B cells producing monoreactive RF in patients with high- versus patients with low-activity RA. However, in two highly active RA patients, we found that synovial fluid, compared with peripheral blood, was significantly enriched for (IgG and IgA) monoreactive RF-producing B cells.     

Molecular determinants of polyreactive antibody binding: HCDR3 and cyclic peptides

Human monoclonal antibody 63 (mAb63) is an IgM/lambda polyreactive antibody that binds to multiple self and non-self antigens. The molecular basis of polyreactivity is still unclear. The present study was initiated to prepare a recombinant Fab of mAb63 and use it to study the determinants involved in polyreactivity. The baculovirus system was employed to express large amounts of mAb63 Fab in Sf9 cells. Our experiments showed that infected Sf9 cells secreted a soluble 50-kD Fab heterodimer that bound to multiple self and non-self antigens. The antigen-binding activity of mAb63 Fab was inhibited by both homologous and heterologous antigens. To study in more detail the molecular determinants involved in polyreactivity, the heavy chain complementarity-determining region 3 (HCDR3), which is known to play a key role in the binding of monoreactive antibodies to antigens, was subjected to site-directed mutagenesis. A single substitution, alanine for arginine, at position 100A resulted in complete loss of antigen-binding activity. The 19 amino acids comprising the HCDR3 of mAb63 were then synthesized and a cyclic peptide prepared. The cyclic peptide showed the same antigen-binding pattern as the parental mAb63 and the recombinant mAb63 Fab. A five amino acid motif (RFLEW), present in the HCDR3 of mAb63, was found by searching the GenBank in three of 50 other human polyreactive antibodies, but in none of nearly 2500 human antibodies thought to be monoreactive. It is concluded that HCDR3 plays a major role in polyreactivity and that in some cases cyclic peptides comprising the HCDR3, by themselves, may be polyreactive.     

Characterization of murine polyreactive antigen-binding B cells: presentation of antigens to T cells

Monoclonal polyreactive antibodies (Ab) can bind, at low affinity, a variety of different self and non-self antigens (Ag). Recent studies in humans showed that polyreactive Ab are expressed on the surface of a subset of peripheral B lymphocytes and clonal analysis revealed that a variety of different Ag can bind to single cells expressing these Ab. To see if these polyreactive Ag-binding B (PAB) cells also are present in mice, fluorescein-conjugated Ag and FACS sorting were used to identify and separate PAB cells from non-polyreactive Ag-binding B cells. Depending on the Ag used for screening, up to one-third of mouse splenic B cells displayed polyreactive Ag-binding properties. Confirmation that the Ag actually bound to surface Ig came from treating PAB cells with anti-Ig which inhibited Ag binding by up to 80 %. Further studies showed that PAB cells could present Ag to Ag-specific T cells, but despite their Ag-presenting ability, PAB cells from normal mice failed to trigger Ag-specific T cells to proliferate. Analysis of the co-stimulatory molecules B7-1 and B7-2 showed that these molecules were not expressed on PAB cells from normal mice. These findings argue that the lack of co-stimulatory molecules on PAB cells is the most likely explanation for their failure to stimulate Ag-specific T cells. The ability of PAB cells from normal mice to bind and present Ag to Ag-specific T cells, without causing them to proliferate, suggests that PAB cells may contribute to the induction and / or maintenance of immunological tolerance.     

Half-life of polyreactive antibodies

Monoclonal polyreactive antibodies bind to a variety of self and foreign antigens. In contrast, monoclonal monoreactive antibodies bind to a single or restricted number of known antigens. The rate at which polyreactive antibodies are removed from the circulation compared to monoreactive antibodies has not been determined. In the present experiments, human monoclonal polyreactive and monoreactive antibodies of different isotypes were injected intravenously into mice and the clearance from the circulation was determined. The halflife of polyreactive IgM, IgA, and IgG antibodies was 8.0, 8.2, and 9.8 hr, respectively, compared to 35.4, 26.6, and 280 hr for monoreactive IgM, IgA, and IgG antibodies, respectively. Examination of tissue sections from animals given intravenous antibody showed substantial deposition of polyreactive, but not monoreactive, antibodies in several organs, the liver being the principal site of deposition. It is concluded that polyreactive antibodies are cleared from the circulation substantially faster than monoreactive antibodies.     

ANTIGEN BINDING TO LYMPHOID CELLS OF UNIMMUNIZED MICE

In order to determine the cell type responsible for the antigen-binding reaction in the bone marrow and spleen of mice, cells derived from pure in vitro derived colonies of neutrophils, eosinophils, macrophage-megakaryocytes and B lymphocytes were tested for their ability to bind fluorescent protein antigens. Only B lymphocytes bound antigen. An unexpectedly high percentage of bone marrow B lymphocytes (20%) bound a given antigen. This frequency was considerably higher than that found for spleen cells. As might be expected from such high binding frequencies, some cells bound two fluorchromated antigens when these are added together. As a direct test of the clonality of antigen binding to bone marrow B lymphocytes, whole colonies of B cells were tested for antigen binding of two non-cross-reacting protein antigens. The frequency of antigen-binding clones, including double antigen-binding clones, reflects exactly the frequencies observed for dispersed colony B cells and for in vivo derived Ig-bearing bone marrow B cells. The frequency of double antigen-binding colonies was equal to the product of the frequencies of the colonies binding each of the two antigens alone. No ‘mixed’ colonies containing single binding cells for each antigen were found. Thus, the ability to bind any two given antigens is a clonally distributed property of the bone marrow B lymphocyte population. Heterogenous receptors for multiple antigen binding on each cell are either randomly distributed among the B cell population, or homogenous antigen-binding receptors on each cell have a random chance of cross-reaction with the two antigens tested.     

Binding of polyreactive antibodies to self versus foreign antigens

Aside from their ability to bind to multiple antigens, the classic hallmark of polyreactive antibodies is their autoreactivity. Because of their ability to bind a number of common autoantigens, it has long been speculated that polyreactive antibodies are involved in the clearance of self-antigens. However, it has been demonstrated more recently that polyreactive antibodies are also capable of binding to some foreign and synthetic antigens. Although data regarding the relative reactivity of polyreactive antibodies with self versus foreign antigens is lacking, it is generally thought that both activities may play an important biological role. In this study, the relative reactivity of polyclonal human polyreactive IgM with human proteins and tissue extracts versus foreign (xenogeneic) proteins and tissue extracts was probed. The binding of affinity purified anti-ssDNA IgM from adult human serum and the binding of polyreactive IgM in human cord serum and in human adult serum were evaluated. Using competitive and direct binding assays, human polyreactive IgM were found to be generally more reactive with foreign (xenogeneic) proteins than with self or allogeneic proteins. These data shed light on the fundamental nature of polyreactive antibodies, and may provide additional insight into their putative biological roles.     

Somatic mutations can lead to a loss of superantigenic and polyreactive binding

Although antibodies have been assumed to bind a specific antigen, evidence exists showing that a single antibody can bind to multiple unrelated antigens. We previously studied a human monoclonal antibody expressing a mutated form of the V(H)3-73 gene and displaying anti-tubulin activity in a patient suffering from an immunocytic lymphoma. Despite its expression of a V(H)3 family member, this immunoglobulin failed to react with protein A (SpA), suggesting that somatic mutations could account for its change in specificity. To examine this possibility, we produced recombinant Ig expressing germ-line (IgM kappa-Germ) or the mutated form (IgM kappa-PER) of the V(H)3-73 fragment. Comparison of the respective affinities of the two Ig demonstrated that IgM kappa-Germ restores its SpA-binding capacity, and shows a moderate decrease in its affinity for tubulin. Interestingly, IgM kappa-Germ displayed polyreactive specificity for different autoantigens, which contrasted to the monospecific binding of IgM kappa-PER to tubulin. These results suggest that the monoreactive IgM kappa-PER antibody may be derived from a natural polyreactive antibody through somatic mutation. In addition, both temperature modification and mild denaturation succeeded in recovering the polyreactivity of IgM kappa-PER, which favors the view that conformational modifications of the tertiary structure of antibodies may play a key role in the genesis of polyreactivity.     

Polyreactive antigen-binding B (PAB-) cells are widely distributed and the PAB population consists of both B-1+ and B-1- phenotypes

B cells that make polyreactive antibodies (PAB+ cells) express polyreactive Ig receptors on their surface and can bind a variety of different antigens. The present study shows that PAB+ cells are widely distributed, are present in varying numbers in different lymphoid organs and that their phenotype varies depending on the organs from which they are isolated. Up to 10 times more cells in PAB+ enriched populations bind antigens as compared to PAB- populations. Comparison of PAB+ with B-1+ cells showed that a high percentage of PAB+ cells are B-1+, but that many PAB+ cells do not express B-1 cell surface markers and, in fact, are B-1-. It is concluded that the B cell population consists of PAB+/B-1+, PAB+/B-1-, PAB-/B-1+, and PAB-/B-1- cells. The presence of PAB+ cells in the thymus points to the possibility that PAB+ cells may carry endogenous host antigens from peripheral tissues to the thymus where they may contribute to immunological tolerance.     

Polyreactivity as an acquired artefact, rather than a physiologic property, of antibodies: evidence that monoreactive antibodies may gain the ability to bind to multiple antigens after exposure to low pH

Evidence is presented that monoreactive antibodies exposed to low pH may acquire the ability to bind to multiple antigens. M11, a murine, monoclonal, IgM(K) anti-goat IgG (GIgG) was purified from a hybridoma supernatant by elution at low pH from an anti-mu-Sepharose 4B affinity column. By measuring the specific antiGIgG activities and the affinity constants for the interactions of M11, pre- and post-affinity-purification, with GIgG, M11 was shown to be monoreactive before purification. Quite unexpectedly, however, the affinity-purified M11 reacted extensively with size-fractionated liver proteins when tested in an immunoblot, clearly indicating that it was polyreactive. It was concluded that the exposure to low pH had altered the M11 binding-site so as to allow it to bind to many different proteins. This phenomena provides an alternative basis for interpreting the polyreactivity observed following affinity-purification.     

The role of antigen in the selection of the human V3-23 immunoglobulin heavy chain variable region gene

The immune system mounts antibody responses using few of the available immunoglobulin variable region (IgV) genes with some, such as the V3-23 heavy chain gene, regularly over-represented in responses to many antigens. The reasons for the over-representation of some V genes have not been established; the process could be either stochastic or selective. We demonstrated previously that the V3-23 gene, which is over-represented in the primary B lymphocyte repertoire in humans, encodes antibodies with differing antigen-binding reactivities in transgenic mice that express the human V3-23 gene. The aim of the current study was to assess if V3-23 gene over-representation is stochastic or could be influenced by antigen exposure. Transgenic mice were immunized with human IgG-Fc (hIgG-Fc), bovine collagen type II (bCII) or tetanus toxoid (TT), and hybridomas secreting human mu chain-containing antibodies generated. These were tested for binding to the immunogens and a panel of self- and exogenous antigens. In hybridomas derived from hIgG-Fc-immunized mice, 53% secreted antibodies specific for hIgG-Fc. A similar proportion (54%) of hybridomas from bCII-immunized mice secreted antibody that bound to collagen. By contrast, only 21% of hybridomas from mice immunized with TT bound to tetanus toxoid. Intriguingly, chimaeric antibodies generated from mice immunized with bCII or TT were mainly polyreactive, similar to antibodies generated from naive transgenic mice. However, hybridomas generated from mice immunized with hIgG-Fc were mainly specific, reacting exclusively with hIgG-Fc. These results suggest that selection and eventual expansion of B lymphocytes expressing the V3-23 gene are likely to be determined by exposure to self- and/or environmental antigens.     

Polyreactivity of monoclonal antibodies produced against thyroid stimulating hormone (hTSH)

Monoclonal antibodies against human thyroid stimulating hormone (hTSH) are among the key reagents required in the development of an immunoradiometric assay (IRMA) for hTSH in serum. In this study we have produced and characterized twelve hTSH monoclonal antibodies. Hybridomas were generated by fusion of B-lymphocytes from mice immunized with hTSH and myeloma cells. Clones producing antibodies against hTSH were selected using ELISA (Enzyme Linked Immunosorbent Assay). Antibodies from the selected and cloned hybridoma cells were purified by affinity chromatography and their reactivities were tested by ELISA against a panel of antigens, i.e., hTSH, bovine serum albumin (BSA), and milk protein, and also by studying the binding of these monoclonal antibodies with the radioiodinated antigens (hTSH, BSA, and milk protein). It was observed that some of the hTSH monoclonal antibodies produced were polyreactive, reacting with hTSH as well as with unrelated antigen BSA, while others were monoreactive, reacting only to hTSH.     

CD5+ B lymphocytes, polyreactive antibodies and the human B-cell repertoire

The lock and key model of antigen-antibody reaction has traditionally been used to explain the specificity of antibodies and the need for antibody diversity. Recently it has become clear that certain antibodies are polyreactive and recognize a variety of self- and foreign antigens. It is now clear that these antibodies are made by a novel subset of B cells that bear the surface CD5 marker. Careful analysis has shown that about 20% of peripheral blood B lymphocytes in adults are CD5+ and, therefore, represent a major component of the normal human B-cell repertoire. The precise role of the antibodies produced by these cells is still not clear, but because of their polyreactivity they might function in clearing autoantigens from the circulation and/or as a rapid first line of defense against foreign invaders, such as bacteria and viruses. Sequence analysis showed that these antibodies use gene segments in germ-line configuration for their antigen-binding portion. In this article, Paolo Casali and Abner Notkins propose that polyreactive antibodies are what, for years, have been referred to as the 'natural antibodies' of serum and that under certain circumstances they may contribute to the pathogenesis of autoimmune diseases.     

Toll-like receptor driven B cell activation in the induction of systemic autoimmunity

Studies over the past decade have demonstrated a key role for pattern recognition receptors in the activation of autoreactive B cells. Self reactive B cells that manage to escape negative selection often express relatively low affinity receptors for self antigens (ignorant B cells), and can only be activated by integrating a relatively weak BCR signal with signals from additional receptors. Members of the toll-like receptor (TLR) gene family, and especially the nucleic acid binding receptors TLR 7, 8 and 9, appear to play a key role in this regard and promote the production of autoantibodies reactive with DNA- or RNA-associated autoantigens. These autoantibodies are able to form immune complexes with soluble or cell-bound ligands, and these immune complexes can in turn activate a second round of proinflammatory cells that further contribute to the autoimmune disease process. Recent data have emerged showing a pathogenic role for TLR7, with an opposing, protective role for TLR9. Targeting these disregulated pathways offers a therapeutic opportunity to treat autoimmune diseases without crippling the entire immune system. Further understanding of the role of specific receptors, cell subsets, and inhibitory signals that govern these TLR-associated pathways will enable future therapeutics to be tailored to specific categories of autoimmune disease.     

Affinity of T and B lymphocyte receptors for hapten determinants

The affinity of anti-hapten antibodies, produced by single cells, was studied by hapten inhibition of plaque-forming cells using the local hemolysis in gel assay. The shift in affinity of anti-hapten antibodies, that occurs with time after immunization, was parallelled by an analogous shift, at the cellular level, with regard to indirect plaque-forming cells (PFC). Thus, there was a gradual decrease of the hapten concentration which was needed to suppress indirect plaque formation with time after immunization, indicating a gradual increase in the number of high affinity cells. No such shift was observed with cells causing direct plaque formation. Analogous studies were performed with hapten-specific antigen-binding cells of T and B origin by using hapten-inhibition of rosette-forming cells. Hapten-specific antigen-binding cells were present in both cell populations. Lymphocyte binding of haptenated red cells could be specifically inhibited by free hapten. It was found that antigen-binding B lymphoid cells became gradually more susceptible to hapten inhibition with time after immunization, whereas no such change was observed in antigen-binding T lymphoid cells. Possible reasons for this discrepancy are discussed, as well as the implications for these findings regarding the specificity and structure of the T cell receptors.     

Structural aspects of human IgM antibodies expressed in chronic B lymphocytic leukemia.

BACKGROUND: Malignant B cells from patients with chronic B lymphocytic leukemia (B CLL) generally express both surface IgM and the pan T cell antigen CD5, a characteristic of the B1 population of B lymphocytes. The IgM on the surface of these B CLL cells is frequently polyreactive with respect to its capacity to recognize multiple structurally dissimilar antigens (Ag). OBJECTIVES: To understand the structural characteristics of the polyreactive binding sites of human IgM molecules expressed on B CLL cells by: (1) analyzing the nucleotide and protein sequences of the variable (V) domains of five IgM molecules expressed in cases of B CLL and; (2) utilizing these sequences to generate three-dimensional (3D) models of Fv (VL - VH) molecules. STUDY DESIGN: Peripheral blood leukocytes obtained from five cases of B CLL were tested for polyreactive binding properties by assessing their capacity to bind mouse IgG by indirect immunofluorescence. The V region genes of light and heavy chains were amplified using the polymerase chain reaction, subsequently cloned and their nucleotide sequences obtained. Translated amino acid sequences of the V domains were used to generate homology models of the Fv molecules. RESULTS: Low affinity binding of mouse IgG was demonstrated for all B CLL samples examined, confirming the polyreactive nature of the IgM expressed on these cells. There was an absence or minimal mutation within V region genes when compared to germline Ig genes. Junctional diversity was not observed for VL regions, although truncations and insertions were frequent in D minigenes of VH regions. The binding sites were predicted to form either relatively flat surfaces with occasional protrusions or cavities at the VL - VH domain interface. Aromatic side chains covered a large proportion of the potential binding surfaces in the models of B CLL Fv components. DISCUSSION: Primary DNA sequences can be categorized as germline, suggesting that the B cells involved in B CLL are germline or naive in origin. The medium to large HCDR3s provide the majority of probable contact residues for antigens. While prominent aromatic residues are likely to engage in binding patterns which are conserved (e.g. mouse Ig reactivity), the diverse binding sites predicted for B CLL-derived IgMs also have properties which are conducive to polyreactive antigen binding.     

Isolation of antigen-binding cells from unprimed mice. II. Evidence for monospecificity of antigen-binding cells.

Spleen cells from unimmunized mice were exposed to two contrastingly fluorescent antigens simultaneously. Antigen-binding cells of either specificity were isolated using a fluorescence-activated cell sorter (FACS). Purified cells binding one or the other of the antigens were then examined by fluorescence microscopy for the presence of bound antigen of the alternate specificity. No double binding cells were seen. If cells bear receptors of two or more specificities and these receptors are randomly distributed among antigen-binding cells, then of the 13 000 binding cells examined 82 were expected to bind both antigens. These results strongly suggest that antigen-binding cells bear receptors of only one specificity. In addition, by inference from the functional correlation between antigen-binding cells and precursor cells, the data support the contention that precursors of antibody-forming cells are monospecific.     

Specificity of Hapten-Reactive T and B Mouse Lymphocytes

Immunization of mice with hapten-protein conjugates leads to the appearance of increased numbers of hapten-specific antigen-binding cells of both T and B origin. as well as to the formation of anti hapten antibody-secreting cells. The affinity of cellular receptors of hapten-reactive T and B cells was studied after immunization with different antigen doses at different time intervals. It was found that the affinity of secreted antibodies of the IgG class as well as of B-cell receptors for hapten was dose- and time-dependent. The affinity of hapten-reactive T cells for the hapten itself was low, irrespective of dose and time. Binding strength of T-and B-cell receptors was also investigated with the aid of mono- and multivalent homologous and heterologous hapten-carner complexes. The affinity of T cells for the homologous monovalent ligand was considerably higher than for the hapten itself or a heterologous monovalent ligand. indicating carrier contribution to T-receptor affinity. The contribution of carrier to the affinity of 7S antibodies or B-cell receptors was relatively small The avidity of hapten-specific cells for multi valent ligands was high in both T and B cells, but the increment in avidity with such ligands compared to monovalent ligands was less pronounced in T cells     

Aberrant antibody affinity selection in SHIP‐deficient B cells

The strength of the Ag receptor signal influences development and negative selection of B cells, and it might also affect B‐cell survival and selection in the GC. Here, we have used mice with B‐cell‐specific deletion of the 5′‐inositol phosphatase SHIP as a model to study affinity selection in cells that are hyperresponsive to Ag and cytokine receptor stimulation. In the absence of SHIP, B cells have lower thresholds for Ag‐ and interferon (IFN)‐induced activation, resulting in augmented negative selection in the BM and enhanced B‐cell maturation in the periphery. Despite a tendency to spontaneously downregulate surface IgM expression, SHIP deficiency does not alter anergy induction in response to soluble hen‐egg lysozyme Ag in the MDA4 transgenic model. SHIP‐deficient B cells spontaneously produce isotype‐switched antibodies; however, they are poor responders in immunization and infection models. While SHIP‐deficient B cells form GCs and undergo mutation, they are not properly selected for high‐affinity antibodies. These results illustrate the importance of negative regulation of B‐cell responses, as lower thresholds for B‐cell activation promote survival of low affinity and deleterious receptors to the detriment of optimal Ab affinity maturation.