Analysis of the Repertoire of Anti-Idiotypic B-Cell Responses to Self-I-Ak- or -I-Ek-Reactive Monoclonal Antibodies in A.TL Mice

Twenty anti-idiotypic antisera (anti-Ids) were produced in A.TL mice to self-I-Ak or -I-Ek-reactive monoclonal antibodies (mAbs), constructed in the A.TH anti-A.TL combination. The reactivity of these anti-Ids was examined in a panel of 31 anti-Iak A.TH mAbs, using direct idiotype binding, cross-competitive inhibition of idiotype binding, and isoelectrofocusing (IEF) assays. Among 13 anti-Ids produced against anti-I-Ak mAbs, one only recognized individual idiotypic specificities (IdIs) on its corresponding mAb, while the 12 others identified homologous IdIs and recurrent idiotypic specificities also expressed on heterologous anti-I-Ak and/or I-Ek mAbs. Two sets of major cross-reactive idiotypes (IdXs) were characterized on two groups of mAbs recognizing public Ia.1, I-Ak,f,u and r) or private (Ia.2, I-Ak) determinants clustered in two spatially distinct epitope regions of the I-Ak molecule, respectively. By contrast, most (5/7) of the anti-Ids raised against mAbs recognizing polymorphic or monomorphic (Ia.7-like) I-Ek determinants displayed specificity apparently restricted to their corresponding mAb IdIs. This finding contrasted with the previous characterization, using xenogeneic anti-idiotypic reagents, of an interstrain IdX expressed on all mAbs defining Ia.7-like determinants in the IEk epitope group I. These data indicate that A.TL mice can readily develop anti-idiotypic responses towards self Ia-reactive mAb minor idiotypes (IdIs) and that recognition of anti-Iak mAb IdXs in such mice is preferentially observed when anti-I-Ak mAbs are used as immunogens.     

Anti-Idiotypic Antibodies of Reovirus as Biochemical and Immunological Mimics

Among the autoantibodies that are known to play a role in the pathogenesis or autoimmune diseases, antibodies to DNA (anti-DNA) have been the subject of much study. Several interesting observations have resulted. The ability to make antibodies that bind DNA is not abnormal. Normal mice and humans can produce antibodies that bind DNA. On the other hand, large quantities of antibodies to DNA are found in the sera of patients with systemic lupus erythematosus (SLE), and complement-lixing antibodies to double-stranded (ds) DNA cause some of the tissue lesions, especially glomerulonephritis (GN). Why, then, do some individuals make anti-DNA that deposits in glomeruli, skin, and other tissue, resulting in organ damage? It is likely that disease results from a combination of several factors— ability to make pathogenic antibody subsets, inability to downregulate those subsets, and “tissue susceptibility” to injury from those antibodies and their immune complexes. This chapter will focus on the characteristics of pathogenic antibody subsets and their regulation     

Detection of Cells Producing Anti-Idiotypic Antibody to Thyroid Stimulating Hormone-Reactive Antibodies

BALB/c mice were immunized either with bovine (b) or human (h) thyroid stimulating hormone (TSH) or with one of several mouse monoclonal anti-hTSH antibodies. The binding of biotinylated mouse monoclonal anti-hTSH to sections of spleen, kidney, liver and lung was assessed with fluorescein isothiocyanate labelled avidin. Clusters of cells in the spleens of mice immunized with bTSH bound the labelled monoclonal anti-TSH. We suggest that these cells synthesized auto-anti-idiotypic antibodies to anti-TSH.     

Detection of Cells Producing Anti-Idiotypic Antibody to Thyroid Stimulating Hormone-Reactive Antibodies

BALB/c mice were immunized either with bovine (b) or human (h) thyroid stimulating hormone (TSH) or with one of several mouse monoclonal anti-hTSH antibodies. The binding of biotinylated mouse monoclonal anti-hTSH to sections of spleen, kidney, liver and lung was assessed with fluorescein isothiocyanate labelled avidin. Clusters of cells in the spleens of mice immunized with bTSH bound the labelled monoclonal anti-TSH. We suggest that these cells synthesized auto-anti-idiotypic antibodies to anti-TSH.     

Anti-Idiotypic B-Cell Lines from a Patient with Monoclonal Gammopathy of Undetermined Significance

The B-cell repertoire in a patient with benign monoclonal gammopathy of unknown significance was studied using Epstein-Barr virus transformation of peripheral lymphocytes. The presence of anti-idiotypic B cells producing monoclonal antibodies that reacted with idiotypic determinants on the monoclonal immunoglobulin was verified. The two monoclonal anti-idiotypic antibodies studied were of the IgM-kappa type. Such anti-idiotypic antibodies may be part of an idiotypic network regulation of the monoclonal B-cell population.     

Genetic Control of B-Cell Responses

The responsiveness of spleen cells from C3H/HeJ and C3H/Tif mice to lipopolysaccharide (LPS) was compared. Around 1,000-fold higher concentration of LPS were required to minimally activate HeJ cells, as compared with Tif high-responder cells, to both proliferation and polyclonal antibody secretion. However, HeJ cells did respond to higher LPS concentrations (100 and 1000 mug/ml). This selective pattern of responsiveness to LPS was also observed in the polyclonal responses of strains to LPS in vivo. Furthermore, the unresponsiveness of HeJ spleen cells was found to depend on a pure B-cell defect in the capacity to interact and/or generate triggering signals on interaction with LPS. Thus, adherent cells, thymus-derived lymphocytes, serum factors, and other non-specific conditions inherent in spleen cell suspensions of low-responder mice were not responsible for suppressing a putative B-cell response to LPS. The present findings are compatible with the possibility that the defect in C3H/HeJ B cells reflects the absence of a structure on the cell surface membrane that is functionally responsible for mediating LPS triggering.     

Genetic Control of B-Cell Responses

Spleen cells from C3H/HeJ mice fail to develop both proliferative responses and increased polyclonal antibody secretion in the presence of concentrations of the B-cell mitogen lipopolysaccharide that are optimal for the induction of B-cell responses in conventional strains This unresponsiveness is selective for lipopolysaccharide, since C3H/HeJ spleen cells respond normally to two other polyclonal B-cell activators–dextran-sulphate and purified protein derivative of tuberculin. These findings are interpreted as indicating a selective defect in the B-cell sub-population that responds to lipopolysaccharides in conventional strains.     

Anti-Idiotypic Antibodies in Patients with Different Clinical Forms of Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Patients with PCM show a wide spectrum of clinical and pathological manifestations depending on both host and pathogen factors. Two clinical forms of the disease are recognized: the acute or juvenile form and the chronic or adult form. The major antigenic component of the parasite is a glycoprotein of 43 kDa (gp43). All patient sera present antibodies against gp43 (anti-gp43) and, as demonstrated before by our group, spontaneous anti-idiotypic (anti-Id) antibodies (Ab2) can be detected in patient sera with high titers of anti-gp43. Since it has been postulated that anti-Id antibodies may have a modulating function, we decided to purify and characterize anti-Id antibodies in this system. The possible correlation of Ab2 titers with different clinical forms of disease was also verified. Results showed that purified human anti-Id antibodies (human Ab2) recognized specifically the idiotype of some murine monoclonal anti-gp43 (17c and 3e) but not others (40.d7, 27a, and 8a). Spontaneous anti-Id antibodies were found in all clinical forms of disease. The majority of patients (88%, n = 8) with the acute form of PCM had high titers of Ab2. However, among patients with the multifocal chronic form of the disease, only 29% (n = 14) had high titers of Ab2; 70% (n = 10) of patients with the unifocal chronic form had low titers of Ab2. A correlation between Ab2 titers and anti-gp43 titers was observed before and during antimycotic treatment. Our results suggest that titers of anti-Id antibodies correlate with the severity of PCM in humans.     

Anergy and Suppression in B-Cell Responses

Two main ideas have been put forward to explain the unexpectedly low anti-hapten antibody titres which can result from pre-priming a mouse with carrier before hapten-carrier immunization. The first involves the interaction of a network of idiotype-specific suppressor T cells, the second instead arguing for the role of intrinsic B-cell anergy. This paper proposes that the data available can equally be interpreted as reflecting the suboptimal interaction between T and B cells at differing stages of maturity, provided that memory B cells can be divided into two subsets. Further, it is suggested that these considerations must be taken into account in the analysis of B-cell anergy in receptor transgenic mice.     

Specific Suppression of MLC and CML by Anti-Idiotypic Antibodies in the Mouse

Rabbit antisera directed against idiotypic determinants of alloreactive mouse CBA anti-C57BL/6 T blasts were raised in the following manner: first, a rabbit serum directed against nonspecific CBA blasts cells was prepared by injecting CBA concanavalin A blasts three times at monthly intervals into a rabbit. Second, specific CBA anti-C57BL/6 T lymphoblasts were induced in a mixed lymphocyte culture (MLC), were purified by gravity sedimentation through a fetal calf serum gradient, and, finally, were incubated with the anti-blast serum from the first step. During this incubation, presumably all epitopes of the blast cell population were blocked by anti-blast antibodies, except for the greatly amplified set of CBA anti-C57BL/6 alloreactive idiotypes. The mixture was then injected into fresh rabbits, which were boosted with similar mixtures after 3 and 6 weeks. Blood samples were removed 10 days after each injection. Such sera, when used together with complement, inhibited specifically the stimulation of CBA cells by C57BL/6 antigens in MLC and the CBA anti-C57BL/6 killer cells.     

Protection Against a Syngeneic Mammary Adenocarcinoma by Administration of Idiotypic and Anti-Idiotypic Antibodies

A hybridoma secreting a monoclonal antibody (mAb) with specificity for tumor-associated cell surface antigens of a transplantable murine mammary adenocarcinoma (SMC-168) was prepared by fusion of syngeneic C3H/He spleen cells with SP2 myeloma cells. Mice which were pretreated with this mAb (C–73) were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168 cells when compared to controls. The treated nice developed tumor-specific cell-mediated immunity, measured by leukocyte adherence inhibition (LAI), which was equal to that of mice immunized with live tumor cells. The IgG fraction from serum of mice receiving mAb C-73 contained antibodies which would bind to that mAb suggesting the presence of anti-idiotypic antibodies (anti-Id). This binding could be partially inhibited by a soluble l-butanol cell surface extract of SMC-168. Rabbits were immunized with mAb C-73 to produce a polyclonal anti-Id. The purified and absorbed IgG fraction of this serum would bind only to mAb C-73 and not to other mAbs of the same isotype or normal C3H/HeN IgG. Binding of the rabbit anti-Id to mAb C-73 could be partially inhibited by soluble tumor-associated antigen extracted from SMC-168. Mice immunized with this polyclonal anti-Id vaccine developed tumor-specific cell-mediated immunity and were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168.     

Protection Against a Syngeneic Mammary Adenocarcinoma by Administration of Idiotypic and Anti-Idiotypic Antibodies

A hybridoma secreting a monoclonal antibody (mAb) with specificity for tumor-associated cell surface antigens of a transplantable murine mammary adenocarcinoma (SMC-168) was prepared by fusion of syngeneic C3H/He spleen cells with SP2 myeloma cells. Mice which were pretreated with this mAb (C-73) were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168 cells when compared to controls. The treated nice developed tumor-specific cell-mediated immunity, measured by leukocyte adherence inhibition (LAI), which was equal to that of mice immunized with live tumor cells. The IgG fraction from serum of mice receiving mAb C-73 contained antibodies which would bind to that mAb suggesting the presence of anti-idiotypic antibodies (anti-Id). This binding could be partially inhibited by a soluble l-butanol cell surface extract of SMC-168. Rabbits were immunized with mAb C-73 to produce a polyclonal anti-Id. The purified and absorbed IgG fraction of this serum would bind only to mAb C-73 and not to other mAbs of the same isotype or normal C3H/HeN IgG. Binding of the rabbit anti-Id to mAb C-73 could be partially inhibited by soluble tumor-associated antigen extracted from SMC-168. Mice immunized with this polyclonal anti-Id vaccine developed tumor-specific cell-mediated immunity and were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168.     

Phenotypic and Functional Changes of Tumour Cells from Patients Treated with Monoclonal Anti-Idiotypic Antibodies

In this paper data are presented indicating that immunotherapy with monoclonal anti-idiotypic antibodies (MoAb anti-id) can provoke different responses in the B-cell tumour concerned. With respect to the course of disease during and after immunotherapy, the in vitro findings may very well explain the in vivo observations in the two patients (D.E.F., B.O.R.) with B-cell chronic lymphocytic leukaemia (B-CLL) who were treated with MoAb anti-id. After initial tumour reduction, there was a recurrence of tumour cells with altered functional and phenotypic properties. In both cases the recurring tumour cells still expressed the same idiotype. In one patient (D.E.F.) the phenotypic changes (a surface Ig change from IgM, IgG, IgA, and IgD to weakly positive IgM and IgD) and functional changes (a 10-fold increase in [3H]thymidine uptake and a decreased idiotype secretion in vitro), together with the in vivo findings with respect to the course of disease--at relapse an impressive tumour regrowth rate with constant serum idiotype level--suggest that immunoselection might have taken place favouring the survival and relapse of a less mature, more aggressive tumour cell population with a lower idiotype expression. In the second patient (B.O.R.), the phenotypic changes (an isotype change from IgM and IgD to IgM with the loss of IgD, and a gradual decrease in expression of CD19 and CD24) and functional changes (a 10-fold increase of idiotype secretion in vitro), together with the in vivo finding that the serum idiotype level had increased 25-fold compared with the preimmunotherapy serum level with comparable tumour load, strongly suggest an immunotherapy-induced differentiation of the malignant B cell. We also describe an increased expression of CD74, detected by MoAb BoM22, on the recurring tumour cells of patient B.O.R., whereas the expression of HLA-DP, -DQ and -DR did not change. The significance of this finding is unclear.     

Normal Human Cord Blood B Cells can Produce High Affinity IgG Antibodies to dsDNA that are Recognized by Cord Blood-Derived Anti-Idiotypic Antibodies

It is possible to identify, in Epstein-Barr virus-transformed normal human cord blood B cell populations, cells present at a low frequency that produce IgG antibodies specific for dsDNA. By cloning out these B cells as immortalized monoclonal cell lines, it could be shown that the antibodies were the products of CD5 positive B cells. Two monoclonal anti-dsDNA antibodies were derived from cell lines T52 and A7 and these were further characterized as anionic (pI ～ 6.4) IgG4k antibodies that bound with affinities of 7.18 × 10⁹ 1/mol and 3.28 × 10⁹ 1/mol, respectively, to dsDNA but did not bind to ssDNA. These affinities were similar to those of polyclonal IgG anti-dsDNA antibodies from lupus patients, which ranged from 1 × 10⁹-8.9 × 10¹⁰ 1/mol. Both T52 and A7 monoclonal anti-dsDNA antibodies were recognized by cord blood-derived IgM antibodies. These IgM antibodies were not rheumatoid factors but bound to the F(ab')2 of A7 and T52 while failing to recognize T50, which is an autologous IgG4k monoclonal antibody without specificity for dsDNA. A cloned B cell line A24 generated from the same cord blood sample as A7 produced an IgM monoclonal antibody that bound to the heavy chains of T52 and A7, but not T50 on Western blot and inhibited the binding of these antibodies to dsDNA. A7 and T52 competitively inhibited each other in their binding to the anti-idiotype A24, and A24 inhibited the binding to dsDNA of some polyclonal IgG anti-dsDNA antibodies purified from sera of lupus patients. The level of inhibition of binding of these antibodies to dsDNA was directly proportional to the levels of expression of the idiotype recognized by A24 on these antibodies. The normal human cord blood, therefore, may contain cells that form an idiotype/anti-idiotype network in which the idiotype is expressed on IgG antibodies with specificity for dsDNA and the anti-idiotype is an IgM antibody that binds to a heavy chain idiotope in such a way as to interfere with its interaction with dsDNA. The presence of a similar idiotype on some polyclonal anti-dsDNA antibodies in lupus that are similarly inhibitable by the cord blood-derived anti-idiotype raises the possibility that this network may persist in later life and perhaps become dysfunctional in systemic lupus erythematosus.     

Further Characterization of Internal Image-Bearing Anti-Idiotypic Antibodies: Specific Binding to Immunoglobulin Receptors on Murine Hybridoma Cells Secreting Antibodies to Hepatitis B Surface Antigen.

The further characterization of internal image anti-idiotypic antibodies (anti-Id) that represent a potential alternative vaccine candidate for type B viral hepatitis is described. The anti-Id preparation contains an internal image component or related epitope that mimics hepatitis B surface antigen (HBsAg) and binds to murine hybridoma cells that secrete antibodies to HBsAg (anti-HBs). This binding to anti-HBs-secreting hybridomas was partially inhibited by intact HBsAg particles and was associated with the expression of an interspecies idiotype. Immunoprecipitation studies demonstrated that the anti-Id bound to immunoglobulin molecules expressed on the surface of the hybridoma cells. These data suggest that internal image anti-Id, which induces an in vivo antibody response by antigenic mimicry in the absence of HBsAg, binds to anti-HBs molecules on the surface of cells actively secreting anti-HBs. The possible mechanism for internal image anti-Id-based antibody vaccines that mimic the overall conformation of antigens associated with infectious agents is discussed.     

B-Cell and T-Cell Epitopes in Anti-factor VIII Immune Responses

Adequate hemostasis is achieved for many hemophilia A patients by infusion of plasma-derived or recombinant factor VIII (FVIII), but unfortunately, a significant subset of patients develop an immune response in which anti-FVIII antibodies, referred to clinically as “inhibitors,” interfere with its procoagulant activity. Inhibitors are the subset of anti-FVIII antibodies that bind to surfaces on FVIII (B-cell epitopes) that are important for its proper functioning in coagulation. Less antigenic FVIII molecules may be designed by identifying and then modifying the amino acid sequences of inhibitor B-cell epitopes. Conversely, characterization of these epitopes can yield important information regarding functionally important surfaces on FVIII. The production of inhibitor antibodies is driven by T cells. T cells recognize FVIII as foreign when FVIII-derived peptides bind to major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells. The class II–peptide complexes must then be recognized by T-cell receptors (TCRs). T-cell stimulation requires sustained association of antigen-presenting cells and T cells through formation of a class II–peptide–TCR complex, and peptide sequences that mediate this association are termed “T-cell epitopes.” MHC class II tetramers that bind FVIII-derived peptides and recognize antigen-specific TCRs are proving useful in the characterization of human leukocyte antigen-restricted T-cell responses to FVIII.