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. Antigen-binding 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 beta-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.
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 onthe 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‐bindingB (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 totrigger 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 thelack 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.
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 beta-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.
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