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.     

Fluoro-immuno-cytoadherence (FICA): A new method for the identification and enumeration of antigen-binding cells.

Antigen-coated particles of cross-linked dextran may be used for affinity chromatography of antibodies and for the fractionation of lymphoid cells with appropriate surface receptors. Furthermore, such particles serve as convenient substrates for quantitative immunofluorescence tests. The fluoro-immuno-cyto-adherence (FICA) is a simple technique which combines affinity chromatography and immunofluorescence, provides durable antigen-coated substrates and allows the identification, enumeration and characterization of lymphoid cells capable of binding an antigen, covalently linked via a spacer onto the surface of dextran beads. In the present study chicken thyroglobulin (TG) or bovine serum albumin (BSA) were coupled onto fluorescein and rhodamin-labelled or unlabelled Sephadex G-25 beads by means of spacer molecules. The specificity and degree of antigen-coating were controlled by indirect immunoflourescence. For the study of antigen-binding cells the different antigen-coated beads were mixed with suspensions of peripheral blood lymphoid cells from Obese strain (OS) chickens with spontaneous hereditary autoimmune thyroiditis, or with cells from BSA-immunized or unimmunized normal White Leghron chickens. Specific adherence of OS lymphocytes to TG-coated beads and of lymphocytes from BSA-immunized chickens to BSA-beads was found. The test and control preparations are observed simultaneously under the fluorescence microscope where the distinction of beads coated with different antigens can be made on the basis of the color of their fluorescence. Results obtained with the FICA technique are in good agreement with those of conventional rosette tests.     

Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

Immunoglobulin (Ig) bearing, antigen-binding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibody-secreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibody-secreting cells. These receptors are either lost from the B cell surface as they differentiate into antibody-secreting cells or are blocked in vivo by immune complexes or C3b.     

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.     

The development of antigen-binding lymphocytes in foetal tissues

The time of appearance and counts of antigen-binding cells, using radioiodine-labelled flagellin and haemocyanin, was studied in the human and mouse foetus at different gestational ages: the capacity for binding radioiodine-labelled antigens was equated with acquisition of immunological funciton. Cells resembling lymphocytes from human and mouse liver and bone marrow showed antigen binding at early gestational ages, but this binding could not be prevented with species-specific antisera to immunoglobulins. Specific antigen binding to lymphocytes was detected first with thymic lymphocytes, at gestational ages of 12 weeks in humans and 14 days in mice, then with splenic lymphocytes, at 16 weeks in humans and 17 days in mice, and still later with gut lymphocytes. Relative counts of antigen-binding cells in human foetal thymus were maximal at 16–22 weeks and decreased thereafter. Lymphocyte immunocompetence, as judged by the capacity specifically to bind antigen, develops rapidly after the appearance in thymus of cells with the morphology of lymphocytes; this seemed to occur at the equivalent foetal stage in the species studied.     

Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues: quantitation and specificity of binding

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from Salmonella adelaide. Cytophilic antibody as the cause of antigen binding was excluded.

The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood.

Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.

     

The expression of immunoglobulin determinants on the surface of antigen-binding lymphoid cells in mice. I. An analysis of light and heavy chain restrictions on individual cells

The expression of immunoglobulin light and heavy chain determinants on the surface of activated mouse spleen cells has been investigated using anti-immunoglobulin inhibition of antigen-binding reactions (rosette formation). The results indicate that the majority of binding cells appearing 4 to 7 days after a single injection of sheep erythrocytes have only one light chain type on their surface but simultaneously express more than one class of heavy chain class. As the response proceeds the proportion of heavy chain class “restricted” cells increases. Between days 15 to 30 following antigen injection the majority of binding cells have only one detectable heavy chain class on their surface. Control studies suggest that the appearance of more than one class of immunoglobulin on the surface of these cells is the result of active synthesis by the same individual cells and is not due to a passive absorption phenomenon. Experiments with antisera to non-immunoglobulin surface antigens indicate that the majority of antigen-binding cells inhibited by class specific sera are non, or minimally secreting B type lymphoid cells.     

Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. IV. Isolation of two groups of naturally occurring, idiotypic molecules with specific antigen-binding activity in the serum and urine of normal rats.

In the rat the major histocompatibility locus antigens are determined by the Ag-B locus. In the present article evidence is presented that the sera and urine of normal adult rats contain naturally occurring antibody-like molcules with reactivity to allogeneic Ag-B antigens. Such molecules can be shown to contain both antigen-binding capacity for the relevant antigens and the idiotypic markers signifying such specific reactivity. The molecules could be shown to be composed of two groups of molecules, one around 7S IgG in size and the other around 35,000 in molecular weight. Only the smaller molecules were found in the urine. From other data we know that the 7S molecules are produced by B lymphocytes and the 35,000-molecular-weight molecules by T cells. Purified natural anti-Ag-B factors, when inoculated into rabbits or chickens, lead to the production of specific anti-idiotypic antibodies that will selectively inactivate rat T lymphocytes with the capacity to react against the relevant Ag-B antigens while leaving other reactivity intact. We thus conclude that the present system allows the purification of naturally occurring idiotypic B- and T-cell products with antigen-binding specificity for further biochemical and functional analysis.     

Transition towards antigen-binding promiscuity of a monospecific antibody

Polyspecificity is defined as the ability of a given antibody molecule to bind a large panel of structurally diverse antigens. A fraction of circulating IgG in all healthy individuals acquires promiscuous antigen-binding activity only after a transient exposure to certain protein destabilizing factors. The molecular mechanisms of this phenomenon are not well understood. Exposures to protein destabilizing agents are common steps in immunoglobulin isolation and purification processes. We performed kinetic and thermodynamic analyses using surface plasmon resonance-based technique in order to characterize the interactions of a single mouse monoclonal antibody to its cognate antigen before and after induction of promiscuous antigen-binding activity. The obtained results, suggest that enhanced antigen binding activity induced by exposure to mild denaturing condition resulted from an increase in the structural flexibility of the antigen-binding site. Further pH and ionic strength-dependence analyses of the antibody/antigen interactions demonstrated that the transition to promiscuous antigen-binding was accompanied by a change in the type of non-covalent forces involved in the complex formation. Moreover, from this study, it is evident that an antibody molecule could use two distinct thermodynamic pathways for binding to the same antigen while retaining the same value of the binding affinity. The obtained results may contribute to the understanding of the molecular mechanisms that lay behind natural antibody polyspecificity.     

Binding of antigen-enzyme complex by antigen-binding cells as an approach for immunogdiagnosis.

An immunoenzyme method capable of detecting specific antigen-binding cells is developed. The method is based on the chemical coupling of the antigen to beta-galactosidase and binding of the conjugate to the corresponding antigen-binding cells. Human hydatid fluid was used as an antigen model, and cells derived from rabbits immunized with human hydatid fluid showed specific binding to human hydatid fluid-beta-galactosidase conjugate. The method is specific and reproducible and could be used as an immunodiagnostic test for various diseases in which little or no antibody is produced.     

Isolation of antigen-binding virgin and memory B cells

One major obstacle in studying the activation of antigen-specific B cells is the small number of B cells reactive with a particular antigenic epitope. In this report, we describe a method by which large numbers of highly purified antigen-binding cells can be obtained. We have shown that by varying the haptenation level of the erythrocytes used for rosetting, we can purify antigen-binding B cells which have different affinities for the antigenic epitope. Thus, memory cells (which have receptors of higher affinity) can be prepared and these cells are essentially free of contaminating virgin cells. The effects of varying the haptenation levels on the red cells used for purifying the B cells can, in turn, be related to the precursor frequency of secreting cells following their activation with T cells and antigen.     

Helper and suppressor functions of human mononuclear cells depleted of antigen-binding T8+ cells.

We have utilized the antigen-binding function of a subset of T8+ cells to remove these cells in vitro from human peripheral blood mononuclear cells. This was carried out by treating the cells with streptococcal antigen (SA), monoclonal anti-SA antibody and complement. The concentration of SA binding to T8+ cells differs with the HLA-DR type of the cells: 1 ng SA binds to DRw6+ cells and elicits T helper activity, whereas 1000 ng SA elicits T suppressor activity, in an assay for antibody-forming cells. After depletion of the antigen-binding cells by the SA-specific complement-dependent killing technique, the helper function of the DRw6+ cells was lost but suppression was elicited not only by 1000 ng but also by 1 ng SA. Similarly, DRw6- cells which bind 1000 ng SA to elicit helper activity and 1 ng to elicit suppression, when depleted of the SA-binding cells, resulted in loss of helper activity but again, suppression could be elicited by both 1000 and 1 ng SA. We suggest that treatment of mononuclear cells with antigen, the specific antibody and complement removes the T8+ antigen-binding cells which present antigen to T helper cells and results in the loss of helper function. Suppressor function is however, not only retained with the original concentration of SA but also expressed with that required to elicit helper function in the untreated cells. These findings are consistent with our hypothesis that the antigen-binding and presenting T8+ cells also function as contrasuppressor cells. Thus, the T8+ subset of cells have a dual function, to present antigen and to activate T helper cell function, and to prevent suppressor cells from inhibiting the helper cells.     

Difference between antigen-binding receptor repertoires in effector cytotoxic T lymphocytes and their secondary precursors (memory cells) specific to H-2Kb.

The fine specificity of antigen-binding receptors was compared in pCTL-2 and secondary effector CTL (cytotoxic T lymphocytes) induced in vivo with the H-2Kb alloantigen in recombinant inbred mice. The lymphocyte preparations were enriched by elution from macrophage monolayers of various origins, including the donor (B6 strain), the H-2Kb mutant bm1, the H-2Kk allele B10.A(4R) and the recipient strain B10.D2(R101) as a control. Anti-Kb pCTL-2 eluted from third-party bm1 or B10.A(4R) monolayers gave rise to CTL progeny that lysed, equally well, both donor TC and those third-party TC from whose monolayer the pCTL-2 had been eluted, but which were unable to lyse irrelevant third-party TC. The lytic activities of secondary CTL whose precursors had been eluted from bm1 or B10.A(4R) monolayers were 6 and 12 times lower, respectively, than pCTL-2 eluted from the donor monolayer. Opposite results were shown for receptors of enriched secondary anti-Kb effector CTL. Irrespective of their elution source, whether donor, mutant or allele variant, the eluted effector CTL were able to lyse the donor TC to a similar degree and much more than the given third-party TC; moreover, they retained cross-reactivity in all cases. It is suggested that CTL receptors are homogeneous in specificity for a whole composite immunodominant epitope and differ from each other only in the affinity/lability of the combining site. In contrast, pCTL-2 can be separated into fractions: receptors of each fraction are strictly specific (with high affinity) to a particular portion of the same composite CTL epitope. It seems likely that the pCTL-2 receptor antigen-binding site is modified during pCTL-2 in vivo differentiation into effector CTL.     

Functional heterogeneity of macrophages: subclasses of peritoneal macrophages with different antigen-binding activities and immune complex receptors

Cell suspensions prepared from 24-hour cultures of adherent peritoneal exudate cells from rabbits were separated into density subclasses on discontinuous gradients of Ficoll. Of the five subclasses obtained, macrophages comprised over 95 per cent of the cells in the two least dense subclasses and over 90 per cent of the cells in the subclasses of intermediate density. The most dense subclass contained approximately 80 per cent macrophages and 20 per cent lymphocytes.The antigen-binding properties of the subclasses were studied with a selected number of ¹²⁵I-labelled antigens in the presence and absence of added antibodies. In the absence of antibody the subclasses differed from one another in antigen-binding activities; these differences were independent of both the test antigens and the state of antigen aggregation. In the presence of specific antibody, two macrophage subclasses bound substantially more antigen than the other subclasses. The enhanced responses of these subclasses were confirmed by studying the cells'' capacity to form rosettes when incubated with sensitized chicken erythrocytes. The results indicated that macrophages in these two subclasses differed quantitatively and/or qualitatively from other macrophage subclasses with regard to immunoglobulin receptor sites.These studies demonstrate the feasibility of using gradient separation procedures to obtain functionally distinct subclasses of cells rich in macrophages. The availability of well-defined macrophage populations should permit more precise studies of macrophage functions in immunity.     

In vitro antigen-binding properties of coelomocytes of Eisenia foetida (Annelida)

Annelids are capable of cellular and humoral defence reactions against foreign antigens. The main aim of this study was to characterise the antigen-binding properties of coelomocytes of Eisenia foetida by means of quantitative autoradiography and direct measurement of radioactivity. It was found that the antigen-binding capacity was significantly increased after antigen stimulation. Furthermore, the preincubation of coelomocytes with non-labelled proteins reduced the binding of radiolabelled antigen. The highest level of inhibition was found when the same protein was used for preincubation. These results indicate that antigen-binding properties are to some extent specific.     

Rapid recovery of antigen-binding receptors on chicken B cells following anti-Ig serum treatment.

Rosette-forming cells (RFC) from the peripheral blood of sheep red blood cell (SRBC)-immunized chickens were characterized as B cells which manifest antigen-binding receptors but no antibody secretion. Lymphocytes were pretreated at 0 degrees C with anti-Ig serum, washed, incubated further at various temperatures and harvested for the rosette-forming cells (RFC) assay. Anti-Ig treatment blocked all RFC formation and providing that the cells were maintained at less than 10 degrees C regeneration did not occur up to 6 h. Complete recovery of RFC formation occurred within 10 min at 37 degrees C following treatment with low but not with high concentrations of anti-Ig serum. However, recovery from inhibition by high-dose anti-Ig treatment was achieved by including chicken IgG in the incubation medium. The recovery of receptors was reduced following "sandwich" treatment of lymphocytes. When cells were treated with anti-Ig antibody, washed and exposed to various drug inhibitors of "capping" the regeneration of RFC was prevented; in contrast, inhibitors of protein synthesis were ineffective. The results were interpreted in terms of dissociation of anti-Ig antibodies from the Ig receptor at an early stage of the ligand-induced receptor redistribution process.     

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.     

An antigen-binding assay to determine the specificity of monoclonal antibodies against influenza virus and mapping of epitopes

A simple antigen-binding assay is described for determining the specificity of monoclonal antibodies against A/Victoria/3/75 influenza virus. Monoclonal antibodies were bound to polyvinylchloride microtitre wells via protein A and anti-immunoglobulin serum. Radiolabelled cell extracts were then added and allowed to adsorb to the antigen. The bound material was eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. The method can also be used to study competitive binding of antibodies to a given antigen. In this case, one antibody was adsorbed to the wells as above and the competitor antibody was added in high excess with the radioactive antigen. The results obtained with this method are comparable to those obtained by competitive RIA or ELISA.     

Studies on the control of antibody synthesis. XIII. Preferential depeletion of precursors of high affinity antibody-secreting cells by specific immunoadsorbents.

Passage of spleen or bone marrow cells from non-immunized donors through a DNP-Ova coated Degalan bead immunoadsorbent column resulted in a specific depletion of the ability of the cell population to restore the anti-DNP antibody response to DNP-BGG by lethally irradiated mice. There was a preferential depletion of precursors of high affinity antibody-secreting cells which is consistent with the view that these cells bear high affinity antigen receptors. The cell population passed through the immunoadsorbent column was also preferentially depleted of the capacity to produce high affinity direct anti-DNP PFC in comparison with thigh affinity indirect anti-DNP PFC. These findings suggest, but do not prove, that different subpopulations of antigen-binding cells are precursors for direct and indirect PFC.     

A simplified technique for the evaluation of antigen-binding cells

A gamma counter technique was developed in an attempt to simplify the antigen-binding cell (ABC) assay for the detection of lymphocytes capable of binding ¹²⁵I-human encephalitogenic basic protein (¹²⁵I-HEProt), and for the detection of serum factors in the peripheral blood of multiple sclerosis (MS) patients capable of affecting the binding of ¹²⁵I-HEProt to the ABC. Appropriate amounts of ¹²⁵I-HEProt were added to sheep peripheral lymphocytes (SPL) and incubated at 0–4°C. The cells were counted for radioactivity and also smeared for autoradiography. Radioactivity (cpm) showed a positive linear relationship with increasing antigen dose from 2–80 ng of ¹²⁵I-HEProt. Binding of sheep red blood cells (SRBC) was significantly lower $\text{(p < 0.01)}$ than binding to SPL. Labelling of SPL was inhibited 80% by preincubation in excess unlabelled HEProt, but enhanced by preincubation in excess lysozyme, a basic protein of similar molecular weight and charge to HEProt. Thus, specificity of binding between SPL and ¹²⁵I-HEProt was shown. Autoradiographic examination revealed that an increase in the dosage of labelled antigen did not result in an increase in the number of labelled cells but rather in an increase in the number or density of grains per cell. This increase bore a linear relationship to the increase in cpm noted by the gamma counter technique. There was no significant difference in cpm between cell preparations washed in 100% foetal calf serum (FCS), gradients of FCS and Eisen's balanced salt solution (EBSS) and those washed in EBSS alone. Further, five or more washes in either FCS, FCS gradients, or EBSS did not significantly reduce the cpm of the cell pellet below that obtained with three washes. In preliminary studies, serum from MS patients when preincubated with SPL was able to significantly $\text{(p < 0.01)}$ inhibit the labelling of SPL by ¹²⁵I-HEProt. Investigations are underway to determine whether the inhibitory factor is circulating HEProt or an antigen cross-reactive with HEProt, a blocking antibody, or an inhibitory protein in serum other than gamma globulin.