Selective in vitro inhibition of an antibody response to purified acetylcholine receptor by using antigen-ricin A chain immunotoxin

Purified acetylcholine receptor (AChR) covalently coupled to the catalytically toxic A chain of ricin has been used to selectively eliminate rat lymph node cells involved in in vitro anti-AChR antibody responses. The resulting inhibition was specific in view of the lack of such inhibition of anti-Keyhole limpet hemocyanin antibody responses. Furthermore, when fractionated B cell or T cell populations were treated with AChR-A chain, both populations were found to be sensitive to the specific cytotoxicity. However, T cell cytotoxicity required higher concentrations of the immunotoxin. Furthermore, when AChR-immune lymphocytes were treated with AChR-A chain in vitro, they became unable to mediate secondary adoptive transfer responses in vivo. The abrogation of the anti-AChR adoptive response correlated with the lack of muscle weakness characteristic of experimental autoimmune myasthenia gravis. Thus, it is possible, in principle, to eliminate clones of antigen-reactive lymphocytes with antigen-ricin A chain immunotoxins. This lets open the possibility of using such agents in immunotherapeutic approaches to autoimmune disease.     

Studies on the mechanism of suppression by T cells in an adoptive secondary response.

Suppression of antibody synthesis by lymphocytes was studied using an adoptive secondary response model in which human serum albumin (HSA)-primed lymphocytes (memory cells) from the thoracic ducts of inbred rats were inhibited in irradiated recipients by nonimmune lymphocytes after mixed cell transfer. This investigation extended earlier work and formally showed that the suppressor cells were peripheral thymus-derived lymphocytes, which could rapidly recirculate from the blood to lymph, were present in spleen but not in bone marrow, and that primed T cells lacked this property to inhibit. The suppressive effect was independent of antigen dose but was markedly influenced by the form of antigen used for challenge in that suppression was significantly abrogated with aggregated HSA or with soluble HSA in the presence of specific antibody. Suppressor cells were found to exert their effect maximally at the time of antigen injection, but became ineffective by 40 hr following challenge. The results are considered within a larger framework of cellular regulation of antibody synthesis.     

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.     

Selective suppression of allotype expression induced in vitro: maintenance of suppression following adoptive transfer

The question of the ultimate fate of lymphocytes subjected to treatment with anti-allotype antibody (Ab) has been investigated by means of an adoptive transfer system that uses noninbred rabbits matched for major histocompatibility antigens and mismatched for allotype. Suppression of b4 immunoglobulin (Ig) production was induced by incubating lymphocytes from b4b5 rabbits with anti-b4 in culture. Transfer of b4-suppressed cells to newborn recipients of allotype b6b6 resulted in stable chimerism of mixed donor-recipient allotypes, in which b4 Ig production remained suppressed. In recipients of non-Ab-treated cells, b4 Ig production predominated over b5, as had been the case in the intact donor. No evidence for stimulation of b4 Ig synthesis was seen, even when lymphocytes and serum from 1-week-old recipients were examined. When lymphoid cells of antigen-primed b4b5 donors were treated with anti-b4 in vitro and transferred, Ab production of the b4 type was specifically suppressed, with compensatory over-production by Ab-forming cells of the b5 type. The results reported here indicate that although anti-allotype Ab is not directly cytotoxic, a significant proportion of the b4-committed cells were irreversibly inactivated as a result of Ab pulse treatment.     

Study of the phenomenon of specific suppression of the immune response in an adoptive transfer system]

It was revealed that the administration of the spleen cells (SC) of syngeneic animals immunized with a high dose of sheep red blood cells (SRBC) to intact mice led to a marked specific suppression of the recipients' immune response. The donors' SC obtained on the 14th day after the intraperitoneal injection of SRBC had the greatest suppressive activity. The SC of intact animals and mice given rat erythrocytes preliminarily failed to influence the immune response of the intact recipients in their SRBC immunization. Treatment of immune SC with the anti-T-serum (ATS) or the anti-B-globulin (ABG) and the complement considerably decreased or completely eliminated the suppressive activity. Administration of a mixture of two immune SC suspensions one of which was ATS- and another ABC-treated did not produce any suppression of the immune response in the intact recipients. It is supposed that the suppressor cells in the given model were T-lymphocytes expressing the antigens, common of cross-reacting with the B-cells.     

Selective suppression by auto-anti-idiotypic antibody of B-cell idiotype repertoires generated after stimulation with the same hapten on T-dependent and T-independent carriers

In the present study, we investigated whether auto-anti-idiotypic antibody in the immune sera from old mice could recognize antitrinitrophenyl (TNP) plaque-forming cells (PFC) generated after stimulation with the T-dependent and T-independent forms of the hapten, TNP. Young and old C57BL/6J male mice were immunized with a variety of T-dependent (TNP-bovine gamma-globulin, TNP-BGG; TNP-keyhole Limpet hemocyanin, TNP-KLH; ovalbumin, OVA; bovine serum albumin, BSA; BGG) and T-independent (TNP-Brucella abortus, TNP-BA; TBP-Ficoll; TNP-polyacrylamide beads, TNP-PAA) antigens either in complete Freund's adjuvant (CFA) or in soluble form. Splenic anti-TNP or antiprotein PFC responses were assayed for anti-idiotype-blocked, hapten- or protein-augmentable IgM, IgG and IgA PFC, 1-2 weeks after immunization. It was found that 8-month-old mice produced significantly a higher percentage of hapten augmentable (26-42%) IgM PFC response to T-independent antigens as compared with the 2-month-old mice (3-6% augmentation). Similarly, old mice produced a significantly higher percentage of hapten or protein augmentable (25-129%) IgG PFC response to T-dependent antigens as compared with the 2-month-old group (2-6% augmentation). The data support the view that age-related regulation of auto-anti-idiotypic antibody is a general phenomenon for immune responses to T-dependent and T-independent antigens. Hapten-reversible inhibition of plaque formation was used to determine whether anti-idiotypic antibody containing antisera from old mice could inhibit B-cell idiotype repertoires generated after stimulation with the same hapten, TNP, on T-dependent and T-independent carriers. Pools of immune sera from 8-month-old mice primed with T-dependent TNP-BGG or TNP-KLH antigens but not with T-independent TNP-PAA or TNP-BA antigens, or with the proteins OVA, BSA, or BGG selectively inhibited IgM, IgG, and IgA anti-TNP PFC from 2-month-old mice that were previously primed with either TNP-BGG or TNP-KLH. In contrast, immune sera from old mice primed with TNP on either T-dependent or T-independent carriers inhibited anti-TNP PFC from mice primed with T-independent TNP-PAA or TNP-BA antigens. Immune sera from old mice primed with OVA or BSA only inhibited the respective antiprotein PFC. The immune sera from young mice did not show any appreciable inhibition of PFC generated after stimulation by any of the antigens studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of T15 idiotype dominance. II. Genes unlinked to the Igh locus regulate T15 dominance of the secondary adoptive transfer response to phosphocholine

We have studied the idiotype and fine specificity of the secondary immune response to phosphocholine (PC) in C57BL (B10, B10.D2, and B.C8) and BALB (BALB/c, BAB-14, and C.B20) congenic strains of mice. In vivo IgM responses of mice from these two genetic backgrounds differed in their T15 idiotypic representation. BALB strains expressed the T15 idiotype on greater than 90% of their IgM, PC-specific plaque-forming cells (PFC), whereas C57BL strains expressed the T15 idiotype on approximately 50% of their IgM PFC. All strains examined expressed greater than 75% PC-inhibitable, VHPC idiotype-positive, IgM PFC. The IgG3 and IgA memory responses were similar to the IgM memory response; BALB strains produced a higher proportion of T15+ PFC than C57BL strains; however, the majority of IgG3 and IgA PFC in all strains were VHPC+, and PC-inhibitable. In contrast, the IgG1 memory response was not dominated by T15+, VHPC+, PC-inhibitable PFC in any of the strains tested. The IgG1 PFC required nitrophenylphosphocholine (NPPC) for efficient inhibition. The IgG2 memory response generally mimicked the IgG1 response with respect to idiotype and specificity. These data demonstrate that the representation of the T15 idiotype in the anti-PC immune response is determined by genes outside both the MHC and Igh genetic loci. Control of T15 expression in secondary IgM, IgG3, and IgA anti-PC responses was examined by using a cell-mixing protocol with primed T and B cells from BALB/c and B10.D2 mice. T15 representation in these responses was determined by the genotype of the B cell, not by the genotype of the helper T cell. Similarly, the B cell genotype was responsible for the idiotypic profile of a primary, in vitro, T-dependent, anti-PC response.     

Selective suppression of Toll-like receptor 4 activation by chemokine receptor 4

The response of Toll-like receptor 4 (TLR4) to lipopolysaccharide (LPS) is thought vital for resisting infection. Since aberrant TLR4 signaling may initiate inflammatory conditions such as the sepsis syndrome, we sought a component of normal cells that might provide local control of TLR4 activation. We found that antibodies that block chemokine receptor 4 (CXCR4) function enhanced TLR4 signaling, while increased expression of CXCR4 or addition of the CXCR4 ligand SDF-1 suppressed TLR4 signaling induced by LPS. These findings suggest that CXCR4 could exert local control of TLR4 and suggest the possibility of new therapeutic approaches to suppression of TLR4 function.     

Mechanisms of idiotype suppression. V. Anti-idiotype antibody inhibits early B cell triggering induced by antigen

To investigate the relationship between antigen-mediated B cell commitment and induction of idiotype (id) suppression, anti-id antibody directed against the major id (TEPC-15 idiotype or T15id) of the anti-phosphorylcholine (PC) antibody was added at various time intervals to BALB/c spleen cell cultures stimulated with a T-independent PC antigen, R36a. The suppressive effect of anti-T15id antibody on the anti-PC response was rapidly decreased as addition of the antibody was delayed; when anti-T15id antibody was added 6 hr after the initiation of the cultures, only partial suppression was induced, whereas the addition of anti-id antibody after 24 hr did not result in significant suppression of the anti-PC response when compared with similar cultures treated with mock anti-id antibody. This acquisition of resistance to id suppression was completely inhibited by treatment with either sodium azide or colchicine, as well as at temperatures below 20 degrees C. The induction of resistance to id suppression during the preincubation period was dependent on the presence of an immunogenic level of specific antigen. This antigen-mediated B cell commitment did not appear to require macrophages because preincubation of macrophages with antigen did not affect the sensitivity of the B cells to anti-id antibody. These results support the possibility that anti-id antibody inhibits early B cell triggering, which involves an energy-dependent, epitope-mediated, lateral mobility of antigen receptors possibly followed by repolymerization of microtubules.     

Regulation of antibody response in different immunoglobulin classes. VI. Selective suppression of IgE response by administration of antigen-conjugated muramylpeptides.

The suppressive effect of antigen-conjugated muramylpeptides or 6-O-mycoloyl muramylpeptides selectively on the induction of IgE antibody response was demonstrated. Preadministration of DNP-mycoloyl muramylpeptides completely inhibited the induction of the anti-DNP IgE antibody response with DNP-ovalbumin (DNP-OA). The selective suppression of the IgE response was due to the induction of DNP-specific suppressor T cells by DNP-mycoloyl muramylpeptides, and the suppressor cells were shown to be radiosensitive. Preadministration of OA-conjugated muramylpeptides partially inhibited the primary and secondary induction of an anti-OA IgE antibody response. The suppressor effect was also due to the induction of OA-specific suppressor T cells. Application of allergen-conjugated muramylpeptides as therapeutic agents in human allergic diseases was suggested.     

Construction of an immunotoxin by linking a monoclonal antibody against the human epidermal growth factor receptor and a hemolytic toxin

Hybrid molecules obtained through conjugation of monoclonal antibodies and toxins constitute an approach under exploration to generate potential agents for the treatment of cancer and other diseases. A frequently employed toxic component in the construction of such immunotoxins is ricin, a plant toxin which inhibits protein synthesis at ribosomal level and so requires to be internalized by the cell. A hemolytic toxin isolated from the sea anemone Stichodactyla helianthus, which is active at the cell membrane level, was linked through a disulfide bond to the anti-epidermal growth factor receptor monoclonal antibody ior egf/r3. The resulting immunotoxin did not exhibit hemolytic activity except under reducing conditions. It was toxic for H125 cells that express the human epidermal growth factor receptor, but non-toxic for U1906 cells that do not express this receptor.     

Novel mechanisms of specific suppression of anti-hapten antibody response mediated by monoclonal anti-carrier antibody

The cellular mechanisms of the antibody-induced suppression of immune responses were analyzed in the keyhole limpet hemocyanin (KLH) system. Some of the monoclonal anti-KLH antibodies, like KLH-specific suppressor T cell factor (KLH-TsF), were demonstrated to suppress the anti-2,4-dinitrophenyl IgG but not IgM plaque-forming cell responses in a KLH-specific and H-2-restricted manner. The anti-KLH antibodies with suppressive activity reacted with, and in turn, stimulated the suppressor hybridoma (34S-281) with the anti-idiotypic receptor complementary to the idiotypic KLH-TsF of the inducer type. Moreover, because the suppressive activity of the anti-KLH antibody was completely abolished by the treatment of responding spleen cells with anti-Lyt-2 and complement, it was apparent that the suppressive antibody activated suppressor T cell pathways. The isotype or affinity of antibodies is not related to the suppressive activity, because suppressive and nonsuppressive antibodies possess a similar affinity belonging to the same Ig isotypes. It also has been demonstrated that the Fc portion is not the functional site, because the F(ab')2 fragment still has the activity. The antibody specificity is found to be important for determining whether the antibody is suppressive or not. In fact, anti-KLH 26, but not other antibodies without activity, recognizes the particular KLH epitope seen by KLH-TsF, and exclusively interacts with the anti-idiotypic suppressor T cells. Thus, the anti-idiotypic suppressor T cell receives signals both from the suppressive anti-KLH antibody and from KLH-TsF, and transmits the antibody-induced suppressor signals to the effector-suppressor pathway. The size of the repertoire of anti-idiotypic suppressor T cells involved in the suppression seems to be very limited, because only four out of 120 monoclonal anti-KLH antibodies were found to have suppressor activity. The possible mechanisms of the cell interaction mediated by the suppressive antibody are discussed.     

Selective in vitro inhibition of an antibody response to purified acetylcholine receptor by using anti-idiotypic antibodies coupled to the A chain of ricin

In vitro antibody responses by rat lymph node cells against purified acetylcholine receptor (AChR) were shown to be inhibitable by protein conjugates prepared with anti-idiotypic antibody and the toxic A chain of ricin. The idiotype specificity of the cytotoxicity was demonstrated by the inability of the same immunotoxin to inhibit an unrelated antibody response (anti-KLH) and by abrogation of specific toxicity in the presence of unconjugated anti-idiotype or antigen (AChR). Furthermore, immunotoxin prepared with an irrelevant antibody specificity failed to significantly inhibit either the anti-AChR or control antibody responses. Therefore, we suggest that idiotype-specific immunotoxins may be a useful addition or alternative to presently employed immunotherapies for autoimmune disease.     

Efficacy of an immunotoxin to folate receptor beta in the intra-articular treatment of antigen-induced arthritis

Introduction

We previously demonstrated that synovial sublining macrophages express folate receptor beta (FRβ). The aim of this study was to evaluate the efficacy of intra-articular administration of a recombinant immunotoxin to FRβ for treating rat antigen-induced arthritis.

Methods

A monoclonal antibody (mAb) to rat FRβ was produced by immunizing mice with B300-19 cells (murine pre-B cells) transfected with the rat FRβ gene. Recombinant immunotoxin was prepared by conjugating the Fv portion of the anti-rat FRβ mAb heavy chain with a truncated Pseudomonas exotoxin A and the Fv portion of the anti-rat FRβ mAb light chain. Antigen-induced arthritis was induced through intra-articular injection of methylated bovine serum albumin (mBSA) after two subcutaneous injections of mBSA and complete Freund''s adjuvant. Immunotoxin was intra-articularly injected into the arthritis joint every other day for seven days after arthritis onset. Joint swelling was measured and histological scores of inflammation, synovial thickness, cartilage, and bone destruction were determined. Immunohistochemistry was performed to detect osteoclast and osteoclast precursor FRβ-expressing macrophages and cathepsin K-positive cells on day 21.

Results

Intra-articular administration of the immunotoxin attenuated joint swelling (61% suppression; P < 0.01 compared to the control on day 21) and improved histological findings, particularly cartilage and bone destruction (scores of rats treated with control versus the immunotoxin: 2.2 versus 0.5; P < 0.01), by reducing the number of FRβ-expressing macrophages and cathepsin K-positive cells.

Conclusions

Intra-articular administration of an immunotoxin to FRβ is effective for improving rat antigen-induced arthritis.     

Inhibition of hematopoietic progenitor colony growth by a monoclonal antibody against the transferrin receptor: comparison of unconjugated antibody with an immunotoxin containing recombinant ricin A chain

We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb, a monoclonal antibody which recognizes an epitope on the human transferrin receptor, and compared the ability of 454A12 MoAb-rRA immunotoxin to inhibit the growth of erythroid burst-forming units (BFU-e) and myeloid colony-forming units (CFU-c) with unconjugated 454A12 MoAb. A significant reduction in BFU-e colony growth was observed at 0.001 microgram/ml of 454A12 MoAb-rRA versus 0.1 microgram/ml of unconjugated 454A12 MoAb (p = 0.005). Comparison of the effects of 454A12 MoAb-rRA and 454A12 MoAb on myeloid colony development gave markedly different results. Unconjugated antibody had no effect on CFU-c colony growth; in contrast, 0.01 microgram/ml of 454A12 MoAb-rRA reduced the number of colonies from 139 per 1 X 10(5) to 75 per 1 X 10(5) cells plated (p = 0.0005). No myeloid progenitor colonies developed at 0.1 microgram/ml of immunotoxin. These observations suggest that 454A12 MoAb-rRA inhibits growth by a potent, ricin A chain-mediated toxic effect on any proliferating cells expressing transferrin receptors, whereas the 454A12 MoAb exerts a selective inhibitory effect primarily on erythroid progenitors by perturbing the transferrin cycle. While growth factor receptors expressed on hematopoietic cells represent promising targets for immunotoxin therapy, our data indicate that an immunotoxin could inhibit cellular proliferation by a different mechanism than the corresponding unconjugated MoAb. Depending on the antibody used, these differences may be important in trials using immunotoxins for in vivo treatment or in vitro purging of malignant hematopoietic cells.     

Selective cytotoxic effect of an immunotoxin on human erythroid tumor cells]

The possibility of efficient directed elimination of human erythroblastoid cells by the conjugate of IgM-monoclonal antibody HAE9 directed against the erythroblast antigen and the A-chain of a plant toxin ricin has been demonstrated. The conjugate contained 2 molecules of A-chain per one antibody molecule. The efficiencies of the cytotoxic effect of native ricin and the conjugate were compared according to the number of binding sites on the surface of K562 cells as well as to the internalization rate of these molecules. As was shown, that the number of binding sites for the antibody approaches 2.7.10(4) molecules/cell, K a being equal to 1.7.10(8) M-1 while for ricin these indices constitute 2.4.10(5) and 4.6.10(8) M-1. Almost 100% of antibodies and 36% of ricin are internalized within 10 min at 37 degrees C. At a concentration 10(-11) of native ricin and 10(-10) of immunotoxin the 50% inhibition of growth of K562 cells carrying the erythroblast antigen on their surface is observed.     

Inhibition of adoptive secondary responses by lymphoid cell populations

The ability of normal and tolerant lymphoid cells to inhibit the adoptive secondary response was investigated in order to delineate the influence which the host can exert on a memory cell population as the host recovers from the effects of irradiation. LBN rats were irradiated with 500R whole body X-irradiation, reconstituted with either normal or tolerant lymphoid cells, then they were given immune spleen cells and challenged with soluble antigen (DNP-BGG). Cells capable of suppressing the adoptive secondary responses were shown to possess the following characteristics: 1) in nonimmune donors they were found in the greatest concentration in lymph nodes, followed by spleen and bone marrow and were practically absent in the thymus; 2) their numbers were not increased in donors rendered tolerant by long term treatment with deaggregated DNP-BGG plus cyclophosphamide nor in donors given large doses of DNP-BGG 48 to 72 hr before sacrifice; 3) in animals rendered tolerant by long term antigen treatment alone some enhancement of suppressor function was seen; 4) the suppressor cells could be shown among both glass wool adherent and nonadherent cells and 5) the nonantigen specific suppressor cells did not affect the kinetics of antibody formation nor the affinity of the antibody which was produced. These results are discussed in terms of the nature and source of the suppressor cell populations and their relevance to the control of secondary responses in intact animals.