Selective idiotype suppression of an adoptive secondary antiacetylcholine receptor antibody response by immunotoxin treatment before transfer

Acetylcholine receptor (AChR)-immune lymph node cells were treated with Id-specific immunotoxin before their transfer and antigenic challenge in adoptive recipient rats. In contrast to untreated, AChR-immune lymph node cells that, when challenged, produced high titers of serum anti-AChR antibody, in vitro anti-Id-ricin A chain treatment significantly inhibited anti-AChR antibody responses upon adoptive transfer. The inhibition was specific in that control lymph node cells immune to keyhole limpet hemocyanin were unaffected and totally responsive after treatment by the same immunotoxin. Furthermore, evidence is presented by isoelectric focusing analysis that the anti-Id antibody preparation used in this study contains all of the specificities required to eliminate the entire spectrum of AChR-reactive B cell clones.     

Study of idiotopic suppression induced by anti-cross-reactive idiotype monoclonal antibody in the anti-p-azophenylarsonate antibody response

A/J mice immunized with p-azophenylarsonate coupled to keyhole limpet hemocyanin produce antibodies expressing a cross-reactive idiotype (CRIA). The pretreatment of A/J mice with anti-idiotypic polyclonal or monoclonal antibody directed against the major cross-reactive idiotype (CRIA) borne by p-azophenylarsonate-specific antibody can lead to idiotypic suppression. In this study, we investigate this idiotypic suppression by using four mAb2 (E4, H8, E3, 2D3) recognizing distinct idiotopes whose expression is related to the presence of particular gene segments of the heavy chain V region. 2D3 expression has been related to the presence of some amino acid in the CDR2 region of the VH gene segment derived from the germ line VH IdCR11. So far, the latter is the only germ-line gene coding for CRIA+ antibody that has been identified in the A/J genome. E4 and H8 expression has been related to the use of a particular D segment, whereas E3 expression has been attributed to certain combinations of D and JH segments. Therefore, we might expect independent regulation of the expression of those various idiotopes in relation to the mechanism of gene recombination. Indeed, we observed that 2D3-suppressed A/J mice still produce the three other idiotopes, suggesting the recombination of those particular D and J segments with a different VH gene. Such a gene has been identified in the genome of BALB/c mice. A/J mice pretreated with one of the other three mAb2 are generally cosuppressed for the expression of E4, H8, and E3, but they still produce 2D3+ antibody. In this case, the IdCR11 VH germ-line gene is most probably recombined with different D and J segments. Molecular evidence for the existence of such molecules has also been presented in the literature. So our serologic data on idiotopic suppression in the arsonate system can be compared with recent data provided by molecular genetics.     

Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Different effects of cortisone on the humoral immune response to T-dependent and T-independent antigens.

The effect of the administration of cortisone on the murine humoral immune response to either thymus-dependent (TD) or -independent (TI) antigens was studied in vivo. Whereas the thymus-dependent immune response was markedly suppressed, the thymus-independent immune response was preserved. The opposing effect of steroids on these two types of immune responses appears to be due to the relative independence of thymus-independent antigens of a radioresistant cortisone-sensitive accessory cell.     

T-dependent suppression of the primary antibody response to sheep erythrocytes in mice infected with Trichinella spiralis.

Mice infected for 20 days with the parasitic mematode Trichinella spiralis had significantly reduced numbers of splenic antibody-forming cells (AFC) and decreased serum hemagglutinin titers following intraperitoneal immunization with sheep erythrocytes (SE). Similarly, when immunized in vitro to SE, cultures of splenocytes from infected mice developed fewer AFC than cultures of normal cells. Splenocytes from infected mice actively suppressed the in vitro response of normal cells to SE, and this in vitro suppression was abolished by lysis with anti-thy 1 antiserum and enhanced by lysis with anti-immunoglobulin antiserum. The addition of supernatant fluids from cultures of splenocytes from infected mice to cultures of normal cells on Day 0 of culture reduced by 70% the number of AFC produced by these cultures. These results indicate the presence of T-suppressor cells and suggest that antigen-induced suppression (antigenic competition) is one mechanism of Trichinella-induced suppression.     

Measurement and comparison of the proliferative and antibody response of neonatal, immature and adult murine spleen cells to T-dependent and T-independent antigens.

Mouse spleen cell antigenic responses to the thymic-dependent antigen sheep red blood cells (SRBC), and the thymic-independent antigens, E. Coli lipopolysaccharide (LPS) and pneumococcal polysaccharides Type I and II (SI, SII) were studied as as a function of age, employing both in vitro spleen cell stimulation and plaque-forming cell (PFC) assay systems. Primary spleen cell proliferative and PFC responses to SRBC, were either absent or meager in comparison to adult (8–12 weeks) values for the first 3 weeks of life. Thereafter responses rose achieving adult values between 4 and 8 weeks of age. The inability of young mice to respond to SRBC was not because of a different immunizing dose requirement for SRBC, since immunization with SRBC over a 200-fold range did not enhance their capability to respond. Also, addition of adherent cells or macrophages from adult mice did not enhance the immune responses of young mice. Furthermore, immunization of 2–4 week old mice with SRBC inhibited the secondary response to SRBC. In contrast, young murine spleen cell proliferative and PFC responses to SI, SII, and LPS were approximately the same as the adult by 7–14 days of life. These data suggest that B-cell immunologic activity, as measured by immunologic assays utilized in this study, develops much earlier than does T-cell responsiveness.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

Serum-mediated suppression of nonspecific B-cell activation. III. Selective inhibition of the polyclonal B-cell response by normal mouse serum

Normal, nonimmune adult serum is known to inhibit in vitro immune responses when present in sufficient amounts. The significance of inhibition of the immune response by serum, however, is not known. Previous work suggested that normal mouse plasma or serum (NMS) was selectively more inhibitory to nonantigen-specific (e.g., polyclonal) as compared to antigen-specific responses. This led to the hypothesis that constituents of serum (or plasma) may serve naturally to minimize the polyclonal type of antibody response, preserving immune specificity. The present study further examined the effect of NMS on polyclonal versus antigen-specific antibody responses. Under the in vitro assay conditions used, 0.5% NMS supported bacterial endotoxin (ET)-induced mitogenic and polyclonal B lymphocyte responses, antigen (SRBC, TNP-KLH)-specific antibody (IgM, IgG) responses, and antigen-induced or -specific T-lymphocyte proliferative responses, while 5% NMS inhibited all of these responses. However, antigen-specific T-lymphocyte responses could be restored by a 10-fold increase in the antigen concentration and antigen-specific antibody responses could be restored by the addition of ET (10 micrograms/ml) as adjuvant. On the other hand, the mitogenic response to ET remained suppressed regardless of ET concentration. Thus, despite significant reduction of the mitogenic and polyclonal properties of ET in 5% NMS (greater than 70% suppression), sufficient antigenic stimuli permitted optimal specific T- and B-cell responses. Many naturally occurring antigens, e.g., bacterial, fungal, and viral, have inherent B-cell mitogenic and polyclonal activity in addition to adjuvanticity and the presence of the serum inhibitory factor may serve to minimize their indiscriminate polyclonal stimulation of antibody.     

Studies on Fc receptor function. I. IgG-mediated inhibition of B lymphocyte activation by T-dependent and T-independent antigens.

Mouse aggregated IgG, when continuously present in cultures of mouse spleen cells immunized with sheep erythrocytes, causes a dose dependent inhibition of the generation of plaque forming cells with a maximum of about 90% at 400 μg IgG/culture. Unaggregated IgG induces a similar inhibition, whereas treatment with mouse albumin or F (ab¹)₂, under the same conditions, does not affect the generation of plaque forming cells.It has been reported that unaggregated IgG binds poorly to Fc receptors of B lymphocytes and thus should not be expected to inhibit PFC generation if the effect is at the level of B lymphocyte Fc receptors. Competitive binding experiments were carried out and showed that aggregated and unaggregated IgG compete similarly with ¹²⁵I-labeled aggregated IgG for binding to Fc receptors of mouse spleen cells.The same inhibition of PFC can be induced by aggregated IgG in cultures of B lymphocytes immunized with the T-independent antigen DNP-Ficoll. When IgG is absorbed extensively with sheep erythrocytes and added to cultures immunized with sheep erythrocytes, PFC generation is inhibited to a level comparable to that of nonabsorbed IgG.These results suggest that IgG binding to Fc receptors leads to a severe inhibition of the induction of PFC by both T-dependent and T-independent antigens. This and other work from our laboratory indicate that this effect may be at the level of B lymphocyte Fc receptors.Taken together with reports from several laboratories, the data presented here suggest that Fc receptors may have a regulatory role on the activation of B lymphocytes by antigens or mitogens.     

Hapten-IgG-induced suppression of the in vitro antibody response: role of hapten density and of antigenic challenge.

We have studied the suppression of the in vitro antibody response of mouse spleen cells to the trinitrophenyl (TNP) hapten by conjugates of TNP-human IgG (TNP-HGG). Normal mouse spleen cells were incubated with conjugates of HGG and of HGG fragments, with different hapten densities. They were then challenged with either a T-dependent or a T-independent antigen. Highly substituted (12 mol of TNP/mol of HGG) conjugates induced a dose-dependent suppression, apparent after short-term incubation at 4 °C, of both T-dependent and T-independent responses. Conjugates of Fab′₂ and Fab′ were as suppressive as conjugates of IgG, whereas conjugates of albumin, aggregated IgG, and β2 microglobulin lacked suppressive activity. In contrast, the lightly substituted conjugates TNP₈-HGG and TNP₂-Fab′₂ induced a time-dependent suppression, affecting only the T-dependent response. This suggests that the suppressive effect of hapten-IgG conjugates is mediated by two different mechanisms according to the density of hapten on the IgG carrier. When these conjugates are used as tolerogens in the in vivo situation, both mechanisms would operate to a variable extent, and this could account for the remarkable tolerogenic properties of hapten-IgG conjugates.     

Studies on Fc receptor function: II. Depressive effect of aggregated IgG2b on B lymphocyte activation by T-dependent and T-independent antigens

Mouse aggregated IgG2b, continuously present in mouse spleen cell cultures, markedly depressed, in a dose-dependent fashion, the direct plaque-forming cell response to sheep red blood cells (SRBC, T-dependent) and to DNP-Ficoll (T-independent). Pretreatment of mouse spleen cells with IgG2b for 30 min at 4 °C prior to culturing also markedly depressed the response to both antigens. Delayed addition of IgG2b to SRBC-immunized cultures caused an early and a late depression separated by a period when no significant depression was seen. Using DNP-Ficoll as antigen the late-occurring depression was observed only in Mishell-Dutton cultures, while it was not seen in microcultures. These data support a regulatory role of Fc receptors on the activation of B lymphocytes by antigens and suggest that Fc receptors may be important in at least two events during the differentiation of B lymphocytes into plasma cells: an early, short lived one and a later, longer-lasting event.     

Perturbation of the autoimmune network. II. Immunization with isologous idiotype induces auto-anti-idiotypic antibodies and suppresses the autoantibody response elicited by antigen: a serologic and cellular analysis

We report on the humoral and cellular events following autologous immunization against an idiotype (Id62) borne on a murine monoclonal autoantibody to thyroglobulin, and their impact on the autoantibody response to thyroglobulin. BALB/c mice with a state of active auto-anti-idiotypic immunity and challenged with thyroglobulin in complete Freund's adjuvant 2 wk after the last immunization with idiotype were found to have a suppressed autoantibody response. This suppression could be adoptively transferred to syngeneic x-irradiated recipients by using whole spleen cells from idiotype-primed mice. Transfer of separate T and B lymphocyte populations proved instrumental in disecting humoral from cellular events and in establishing that whereas B cells were required for transferring an intact anti-idiotype antibody response, T cells from idiotype-primed mice were necessary to transfer suppression. These findings contribute to our understanding of the interrelationship between antigen, idiotype, and anti-idiotype in the immune response to self-antigens, and the role of certain idiotypes in regulating autoimmune responses.     

Modulation of immune response by bacterial lipopolysaccharide (LPS): multifocal effects of LPS-induced suppression of the primary antibody response to a T-dependent antigen.

Spleen cells from mice injected with 2 to 50 microgram bacterial lipopolysaccharide (LPS) have a reduced capacity to make an antibody response in vitro to trinitrophenylated sheep erythrocytes (TNP-SRBC) when tested 1 to 7 days later. Recovery is gradual, and these cells are full functional 2 weeks after in vivo LPS treatment. Unresponsiveness resides in the nonadherent splenic cell populations, and can be shown to have a suppressive cell component, which is irradiation sensitive and has somme characteristics of a thymus-derived lymphocyte (T cell). In addition, neither bone marrow-derived lymphocytes (B cells) nor T cells in the spleens of LPS-treated mice are functionally normal in their abilities to cooperate during an antibody response in vitro. LPS-B cells cooperated poorly with nylon wool-enriched T cells from normal mice but cooperated well with irradiated carrier-primed T cells or nylon wool-purified splenic T cells from carrier-primed mice. LPS-T cells have a reduced capacity to interact with normal B cells and appear to contain a suppressor cell component. These results indicate that the effects of exposure of immunocompetent cells to LPS are multifocal and can include suppression as well as stimulation of antibody formation.     

Response of human B cells to Staphylococcus aureus Cowan I: T-independent proliferation and T-dependent differentiation to immunoglobulin secretion involve subsets separable by rosetting with mouse erythrocytes

We separated T-depleted mononuclear cells into subsets by rosetting with mouse erythrocytes and studied proliferation and differentiation responses to Staphylococcus aureus Cowan I (SAC), pokeweed mitogen (PWM), and a combination of the two polyclonal activators. All of the T cell-independent proliferation of unfractionated B cells in response to SAC was attributable to mouse erythrocyte rosette-forming cells (BMR+). BMR- cells were not stimulated to proliferate by SAC in the presence or absence of T cells, but did proliferate to PWM plus irradiated T cells. Co-stimulation of BMR+ cells with SAC and PWM in the presence of autologous T cells did not lead to immunoglobulin secretion. The B cells stimulated to divide by SAC apparently do not become responsive to B cell differentiation factors and are distinct from those that undergo T cell-dependent differentiation.     

Regeneration of the immunoglobulin heavy-chain repertoire after transient B-cell depletion with an anti-CD20 antibody

B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis. Nevertheless, the role of B cells in the pathogenesis of the disease is not clear. In particular, it is not known how the regeneration of the B-cell repertoire takes place. Two patients with active rheumatoid arthritis were treated with rituximab, and the rearranged immunoglobulin heavy-chain genes (Ig-V_H) were analysed to follow the B-cell regeneration. Patient A was treated with two courses of rituximab, and B-cell regeneration was followed over 27 months by analysing more than 680 Ig-V_H sequences. Peripheral B-cell depletion lasted 7 months and 10 months, respectively, and each time was accompanied by a clinical improvement. Patient B received one treatment course. B-cell depletion lasted 5 months and was accompanied by a good clinical response. B cells regenerated well in both patients, and the repopulated B-cell repertoire was characterised by a polyclonal and diverse use of Ig-V_H genes, as expected in adult individuals. During the early phase of B-cell regeneration we observed the expansion and recirculation of a highly mutated B-cell population. These cells expressed very different Ig-V_H genes. They were class-switched and could be detected for a short period only. Patient A was followed long term, whereby some characteristic changes in the V_H2 family as well as in specific mini-genes like V_H3–23, V_H 4–34 or V_H 1–69 were observed. In addition, rituximab therapy resulted in the loss of clonal B cells for the whole period.     

Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing

The humoral immune system in higher vertebrates is unique in its ability to generate highly diverse antibody responses against most pathogens as well as against certain malignancies. Several technologies have been developed to exploit this vast source of potentially therapeutic antibodies, including hybridoma technology, phage display and yeast display. Here, we present a novel, high-throughput technology (the Symplex Technology) for rapid direct cloning and identification of human antigen-specific high-affinity antibodies from single antibody-producing cells of immune individuals. The utility of the technology was demonstrated by isolation of diverse sets of unique high-affinity antibodies against tetanus toxoid and influenza virus from immunized volunteers. Hence, the Symplex Technology is a new method for the rapid isolation of high-affinity antibodies directly from humans.     

Studies on B-cell memory. IV. Effects of lipopolysaccharide on primary and secondary antibody responses to T-independent type-2 (TI-2) antigen in mice

Effects of LPS on primary and secondary antibody responses to typical TI-2 antigens were investigated in mice. Simultaneous injection of LPS with a TI-2 antigen showed only little adjuvant effect on the following primary antibody response to the antigen. In contrast, either a single or multiple injections of LPS, prior to the immunization with a TI-2 antigen, significantly augmented the following primary antibody response to the antigen. LPS, however, inhibited the development of B-cell memory to a TI-2 antigen when administered together with the antigen. Moreover, an injection of LPS in mice, which had strong IgM and IgG B-cell memories to a TI-2 antigen, caused disappearance or profound reduction of the memories. The results suggest that LPS produced by gram-negative bacteria exerts inhibitory effects on the development and continuation of B-cell memory to bacterial infections.