Auto- and Polyreactivity of IgM from CD5+ and CD5− Cord Blood B Cells

The presence of the CD5 (67 kDa) molecule on the surface of B cells has been considered a marker for cells producing auto- and polyreactive antibodies. Cord blood B lymphocytes (rich in CD5+ B cells) have been sorted into CD5 positive and negative populations by flow cytometry using monoclonal antibodies to CD20 and CD5. Clones of these populations were obtained by immortalization with Epstein-Barr virus. Clones derived from both CD5+ and CD5- B cells produced IgM which was auto- and polyreactive with a higher frequency of these specificities in the CD5+ population. These data indicate that expression of surface CD5 on cord blood B cells is not a definitive marker of an auto/polyreactive population.     

CD5+ B lymphocytes are the main source of antibodies reactive with non-parasite antigens in Trypanosoma congolense-infected cattle.

Mice infected with African trypanosomes produce exceptionally large amounts of serum IgM, a major part of which binds to non-trypanosome antigens such as trinitrophenol and single-strand DNA. In this paper, we describe that in cattle infected with Trypanosoma congolense and T. vivax, similar antibodies are found, although they bind mainly to protein antigens, such as beta-galactosidase, ovalbumin and ferritin. The parasite non-specific IgM antibodies appear around the same time as the parasite-specific antibodies, but their origin and function are not clear. We tested the hypothesis that CD5+ B cells (or B-1 cells), which increase during trypanosome infections in cattle, are responsible for production of antibodies to non-trypanosome antigens. Splenic CD5+ and CD5- B cells from infected cattle were sorted and tested in a single cell blot assay. The numbers of immunoglobulin-secreting cells were similar in both B-cell populations. However, antibodies with reactivity for non-trypanosome antigens were significantly more prevalent in the CD5+ B-cell fraction and were exclusively IgM. The preference for production of these antibodies by CD5+ B cells and the expansion of this subpopulation during infections in cattle, strongly suggest that CD5+ B cells are the main source of trypanosome non-specific antibodies. We propose that these antibodies are natural, polyreactive antibodies that are predominantly secreted by CD5+ B cells. Since B-1 cells are up-regulated in many states of immune insufficiency, the immunosuppression associated with trypanosome infections may be responsible for the increase of this subset and the concomitant increase in trypanosome non-specific antibodies.     

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.     

Properties of polyreactive natural antibodies to self and foreign antigens

Conclusions Natural polyreactive antibodies with the capacity to bind to both self and foreign antigens are present in the sear of healthy individuals. The B lymphocytes that secrete these antibodies belong to the subset of CD5+ B cells and may utilize a restricted set of VH and VL genes, strongly conserved during evolution. The role of these antibodies in the immune system is still a matter of debate. Further studies on their behavior in normal and autoimmune responses are required to elucidate this important issue.     

Spontaneous IgM Autoantibody Production In Vitro by B Lymphocytes of Normal Human Neonates

Human neonate B lymphocytes display unique phenotypic and functional characteristics: in addition to CD1c antigens, CD5+ and CD5- subsets both express activation markers such as CD23 and Bac-1. They proliferate strongly in the presence of various lymphokines (rIL-2, rIL-4, low molecular weight BCGF), but differentiate poorly in the presence of the same lymphokines, pokeweed mitogen and Epstein-Barr virus. It has also been reported that human neonate B lymphocytes produce polyreactive autoantibodies after in vitro activation by Staphylococcus aureus Cowan I and transformation by Epstein-Barr virus. We now show that, in the absence of in vitro stimulation, human neonate B lymphocytes produce polyreactive antibodies of the IgM isotype against several autoantigens. The B lymphocytes involved expressed membrane IgD, IgM, CD23 and CD11b molecules; CD5 expression was variable. This phenotype was consistently found on a minority of B lymphocytes and is similar to that of polyreactive autoantibody-producing B cells in mice. We also found that autoantibody production in vitro could occur in the absence of any T helper effect. The function of these autoantibodies is not clearly established, but their occurrence in a large proportion of human neonates strongly suggests that they play an important role in the development of the immune system.     

Antibody specificity and immunoglobulin VH utilization of human monoclonal CD5+ B cell lines

Human B lymphocytes that bear the CD5 antigen are relatively abundant in early ontogeny and comprise a small fraction of the B cell population in adults. The CD5 B cell subset has attracted much attention because of its possible involvement in autoimmune disease and certain B cell malignancies. To begin to understand the role of CD5 B cells in disease processes, we have generated a panel of ten human monoclonal B cell lines selected for expression of the CD5 antigen. These cell lines were obtained by Epstein-Barr virus transformation of B lymphocytes isolated from the spleen, liver and bone marrow of a 19-week-old fetus, from cord blood and from peripheral blood of healthy volunteers. In addition, one cell line was isolated from the spleen of a patient with chronic lymphocytic leukemia. Here, we describe the antibody and immunoglobulin V_H gene repertoire of this panel of CD5 B cell lines. The results of these experiments show that (a) some but not all CD5 B cell lines secrete polyreactive antibodies that bind to a variety of self- and xenoantigens and (b) members of the small V_H4, V_H5 and V_H6 gene families are overrepresented in this panel of cell lines. Nucleotide sequence analysis revealed the expression of V_H gene elements that have been previously reported in the preimmune B cell repertoire, in CD5 B cell tumors and in polyreactive antibodies.     

Polyreactive antigen-binding B cells in the peripheral circulation are IgD+ and B7−

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or β-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7⁻ cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.     

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.     

Possible immunoregulatory role for CD5 + B cells.

CD5 + B cells represent a subpopulation of B cells which have the characteristic of employing unmutated immunoglobulin variable region genes. These cells are found to be increased early in ontogeny. The percentage of CD5 + B cells is highest in the fetus and decreases after birth. The antibodies produced by CD5 + B cells are polyreactive and are the natural autoantibodies. These autoantibodies may not be pathogenic. CD5 + B cells are elevated in certain autoimmune disease states and are the malignant cell type in B-CLL, with a strong genetic component involved in determining elevated CD5 + B cell states. Elevated CD5 + B cells are found in immunodeficient states (young, aged, and autoimmune). CD5 + B cells may normally act as a first-line defense against invading foreign pathogens but are not involved in the specific immune response. There is some evidence, at least in newborns, that CD5 + B cells may affect the emerging B cell repertoire of conventional B cells via idiotype cascade. However, the action of CD5 + B cells in the newborn may be quite different than their activity in the adult. Nonimmunoglobulin-producing CD5 + B cells may be immunosuppressors. In this report, a unique subpopulation of CD5 + B cells was investigated. These cells were found only in the spleens of aged NZB mice. The CD5 + B cells were clonal and possessed extra chromosomes and did not appear to be producing antibodies. These cells were capable of rapid proliferation in unirradiated recipients. By taking advantage of this proliferative capability, the effect of exogenous clonal CD5 + B cells on recipient immune system was evaluated. Clonal CD5 + B cells from NZB mice were immunosuppressive and decreased the numbers of conventional B cells as well as the level of "natural antibodies." In summary, CD5 + B cells may play different roles in the immune system depending upon environment, age, and their differentiation state (i.e., proliferation versus antibody secretion). The natural antibody produced by CD5 + B cells may be involved in maintenance functions such as removal of dead cells and first-line defense mechanisms. In addition, CD5 + B cells may themselves regulate the immune system and produce a factor which is immunosuppressive. An understanding of the various functions of CD5 + B cells may elucidate fundamental immunoregulatory circuits.     

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.     

Organ reactive autoantibodies from non-immunized adult BALB/c mice are polyreactive and express non-biased VH gene usage

To examine the naturally activated autoreactive B cell repertoire, we analyzed a panel of hybridomas from unmanipulated adult BALB/c spleen cells for reactivity patterns and VH gene usage. We found a pattern of VH usage that was diverse and appeared to reflect the germline repertoire. Although all but one natural antibody hybridoma (NAb) were initially selected for organ rather than antigen binding, the majority of organ reactive IgM NAbs were polyreactive, expressing a broad range of reactivity patterns for both self and foreign antigens, that were unique for each NAb and were not indiscriminate. Our results are consistent with the hypothesis that many naturally activated adult B cells are highly polyreactive and that autoreactivity is a consequence of polyreactivity. We suggest that the population of NAbs exhibiting organ reactivity overlaps the populations of other IgM autoantibodies that have been described previously, and that these all derive from a pool of polyreactive IgM antibodies which are polyclonally activated in the early immune response. These polyreactive natural antibodies may represent a first line of defense and offer protection for the host against a variety of foreign agents.     

The role of CD5-expressing B cells in health and disease (review)

The CD5(+) B cell population is prominent in early life and produce low avidity and, thereby, polyreactive antibodies. CD5(+) B cells are receptive to cytokines and interleukin-10 seems to be influential in the regulation of some of these CD5(+) B cells. The question of whether CD5 is a marker of activation or a molecule specific for a B cell lineage remains unresolved because evidence in support or against a separate lineage are still a matter for debate. However, we suggest the possibility of different kind of CD5(+) B cells. Indeed, activated CD5(+) B cells do proliferate, following CD5 engagement, while resting CD5(+) B cells do not. Moreover, three ligands for CD5 have, thus far, been identified but their functional effects are yet unknown. CD5(+) B cells probably play a role in setting up the idiotype network, antigen presentation and tolerance induction. B cells of most of the chronic lymphoid leukemias express CD5 molecules and, surprisingly, these cells may be expanded in non-organ-specific autoimmune diseases, such as rheumatoid arthritis or primary Sj?gren's syndrome. CD5(+) B cells seems to be involved in the autoantibody production (this does not necessarily imply that pathogenic autoantibodies are produced by CD5(+) B cells) in autoimmune disease and particularly susceptible to transformation in lymphoproliferative disorders. Thus, this B cell population appears to play a key role at the crossroad of the non-organ-specific autoimmune diseases and B lymphoproliferative disorders.     

The CD5+ B cell: a B cell lineage with a central role in autoimmune disease?

It is apparent that B cells are heterogeneous with respect to, for example, the antigens they express on their surface, and the stimuli to which they can respond. It is still unclear to what extent these differences relate to the stage of differentiation (eg. virgin B cells differing from activated B cells or memory cells), or whether distinct developmental lineages might exist. It has been proposed by some authors that, in the mouse, B cells expressing the ly-1 antigen constitute a separate lineage. In man also, a minor population of B cells expresses detectable levels of the CD5 antigen, but far less information is available about these cells. Interest in the CD5+ and ly-1+ B cell subpopulations has been further stimulated by the suggestion that these cells might play a special role in autoimmune disease. Although, in mouse, ly-1+ B cells differ in several respects from ly-1- B cells, the main evidence that they form a separate lineage derives from experiments in which ly-1+ B cells could not be reconstituted with adult bone marrow. It should be borne in mind that the situation is quite different in humans where, following bone marrow transplantation, CD5+ B cells are rapidly restored. Moreover, in the irradiated mice, at least in some of the experiments ly-1+ B cells were in fact reconstituted by adult bone marrow. Furthermore, at least in humans, expression of CD5 can sometimes be induced. There is, as yet, no good evidence that human CD5+ B cells form a distinct lineage, and it is possible that CD5 expression depends upon microenvironmental influences acting on the B cell during its differentiation. Several interesting properties have been attributed to ly-1+ B cells, including the ability to provide help to other B cells, and the secretion of autocrine factors. However there is also evidence that these features are not exclusive to B cells expressing ly-1. It has also been suggested that ly-1+ B cells might be long-lived. It is not yet known whether some of the properties of ly-1+ B cells might be a direct result of their expressing this antigen; this may become more clear when the function of CD5 is elucidated. The suggestion that the repertoire of ly-1+ B cells might be biased towards the expression of certain V genes is very interesting. Many of the hybridomas from neonatal mice produce antibodies which are multi-specific, and therefore well suited to form a first line of defence against potential pathogens.(ABSTRACT TRUNCATED AT 400 WORDS)     

CD5 mRNA Expression and Auto-Antibody Production in Early Human B Cells Immortalized by EBV

A number of studies have suggested that lymphocytes producing polyreactive antibodies belong to the CD5+ B-cell subset. In this study we have examined CD5 at the cell surface and mRNA levels in EBV-driven cord blood and fetal liver clones previously characterized in terms of their antibody specificities. We show that EBV-immortalized cells can express surface CD5, and that some of the clones not expressing surface CD5 express it at the mRNA level. The complete absence of CD5 mRNA in some polyreactive clones is consistent with the proposition that the production of auto-antibodies and multispecific antibodies is not restricted to the CD5+ B-cell subset.     

The paradox of CD5-expressing B cells in systemic lupus erythematosus

The pathophysiological relevance of B cells for systemic lupus erythematosus (SLE), particularly those expressing the T-cell marker CD5, raises the question as to how they operate upon autoimmune processes. Based on their production of low-affinity multispecific antibodies (Abs), CD5(+) B lymphocytes, also referred to as B1 cells, have originally been endowed with the autoAb making. It has since been established that high-affinity Abs to double-stranded DNA are not generated by these cells, but rather by B2 cells. It does not appear that they have the exclusive rights to the production of pathogenic autoAbs. In the light of recent findings, CD5 plays a paradoxical role in preventing autoimmunity. Hence, misguided signaling through CD5 could lead to autoimmunity. This provocative view differs from the na?ve interpretation that the increased levels of B1 cells in SLE represent a direct source of autoAbs responsible for damaging organs.     

Repertoire of CD5+ and CD5- cord blood B cells: specificity and expression of VH I and VH III associated idiotopes.

Epstein-Barr (EBV)-immortalized B cell clones were established from CD5+ and CD5- cord blood B cells separated by flow cytometry. We have previously shown that IgM from many of the clones was polyreactive, exhibiting reactivity with a number of autoantigens. In this study, IgM produced by the clones was analysed by MoAb for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor paraproteins and from defined VH and V kappa subgroups of immunoglobulin heavy and light chains. IgM produced by clones established from CD5+ and CD5- B cells expressed the VH I associated idiotope G8. Furthermore, IgM produced by both sets of clones exhibited a similar frequency of VH III heavy chain subgroup expression, as determined by reactivity with staphylococcal protein A (SpA) and VH III-associated CRI expression (B6 and/or D12). In contrast, expression of the V kappa III-associated 17.109 CRI was significantly higher in IgM antibodies produced by clones established from CD5+ compared with the CD5- clones (32 versus 5%: P less than 0.05). Analysis of the VH and VL subgroup expression by IgM produced by the CD5+ and CD5- cord blood clones, and their autoantigen reactivity profile did not reveal restriction or selection within CD5+ and CD5- populations. However, our data suggest that differences may exist in the expression of certain germ-line genes between CD5+ and CD5- cord blood B cells and might indicate an expansion of CD5+ B cells within the fetal environment.     

Production of Heterohybridomas Secreting Autoreactive and Polyreactive Human Monoclonal Antibodies

Peripheral blood lymphocytes from two polytransfused renal dialysis patients were transformed by Epstein-Barr virus, fused to a heteromyeloma and cloned. Eight human monoclonal antibodies from the resulting clones were tested for their binding to a variety of antigens by ELISA, indirect immunofluorescence and immunoblotting. Antigens tested included B-cell lines, T and B lymphocytes, red blood cells, chronic lymphocytic leukaemic B cells, IgG, ssDNA, dsDNA, histones, nucleoprotamine, sperm nuclei, thymus and spleen extracts, MOLT4 cell lysates, affinity purified autoantigens, tetanus toxoid, bacterial lipopolysaccharide, insulin, and a tissue section screen. These human monoclonal antibodies reacted with more than one antigen to varying degrees and were autoreactive and polyreactive. One of these heterohybridoma cell lines exhibited cytoplasmic staining with an anti-CD5 monoclonal. Our findings support the concept that in adult individuals a subset of B cells produce heterogeneous IgM antibodies which can bind to a variety of different autoantigens and also to foreign antigens. These monoclonals were different from the autoantibodies usually seen in renal dialysis patients in the sense that they were not lymphocytotoxic.     

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.     

Properties and function of polyreactive antibodies and polyreactive antigen-binding B cells

The advent of hybridoma technology has made it possible to study in-depth individual antibody molecules. These studies have revealed a number of surprises that have and are continuing to change our view of the immune system. None of these was more surprising than the demonstration that many antibody molecules are polyreactive - that is they can bind to a variety of different and structurally unrelated self- and non-self-foreign antigens. These findings make it clear that self-reactivity is a common and not necessarily forbidden or pathogenic feature of the immune system and that the well-known broad antibacterial activity of natural antibodies is largely due to polyreactive antibodies. In this brief review we will discuss these insights and their impact on basic and clinical immunology.     

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.