Angiotensin II (AII)-related idiotypic network. II. Heterogeneity and fine specificity of AII internal images analyzed with monoclonal antibodies

Although the structural basis of internal images borne by beta type monoclonal anti-idiotypic antibody (Ab2) begins to be elucidated, there is little information on the repertoire of epitopes which make up the internal images expressed by polyclonal Ab2. We addressed this question by using a two-way approach in the angiotensin II (AII)-related idiotypic network, a system characterized by common occurrence of internal images on rabbit Ab2. First, two sets of internal images were purified in parallel by affinity chromatography on Sepharose 4B covalently linked to either mAb 110 (S4B-110), a mAb specific for a phenylalanine requiring carboxy terminus epitope (Phe8) on AII, or mAb 133 (S4B-133), reactive with a more central epitope also expressed on Phe8 substituted peptide analogs. The respective eluates, EL1 110 and E11 133, exhibited only partially overlapping reactivity, as demonstrated by 1) a different pattern of inhibition by various AII peptide analogues of EL1 110 and E11 133 binding to the same anti-AII antibody (Ab1) (either the homologous polyclonal Ab1 102 or mAb 133), 2) and a distinct profile of EL1 110 and EL1 133 binding to 12 biotinylated monoclonal Ab1 identifying a variety of epitopes on AII. To analyze further the respective distribution of mAb 110 and mAb 133 defined epitopes on Ab2-beta molecules, Ab2 were submitted to sequential affinity chromatography on S4B-110 followed by S4B-133, and the fractionated internal images were characterized by the pattern of binding to the various monoclonal Ab1. It was thus possible to purify two Ab2-beta subpopulations that exclusively imaged the determinant identified by mAb 110 (ii 110) or that identified by mAb 133 (ii 133). A third subpopulation which was successively retained on S4B-110 and S4B-133 expressed both internal images (ii 110 + 133), and was additionally reactive with all the other monoclonal Ab1 tested. In any case, monoclonal Ab1 binding to the different sets of internal images was totally inhibited by an excess of AII. These results indicate that the repertoire of internal epitopes is similar to that of the nominal Ag, but is scattered over distinct subpopulations of Ab2-beta molecules that can be fractionated by affinity chromatography. Some of the latter seem to bear several epitopes and resemble the whole nominal Ag, whereas others appear to image only one determinant. Second, we raised 7 anti-anti-idiotypic mAb (monoclonal Ab3) against affinity-purified Ab2-beta and analyzed their fine specificity for AII.(ABSTRACT TRUNCATED AT 400 WORDS)     

Angiotensin II (AII)-related idiotypic network. III. Comparative analysis of idiotopes and paratopes borne by monoclonal antibodies raised against AII (AB1) and its internal image (AB3)

We have previously produced mAb against angiotensin II (AII), a phylogenetically conserved vasopressive octapeptide, and shown that they identify four distinct epitopes on the AII molecule. In addition we used internal image bearing polyclonal antiidiotypic antibodies raised against rabbit anti AII to produce mAb3. In this study we analyze the segregation of the idiotypic and paratopic repertoires of the mAb1 and mAb3. Analysis of mAb1 carried out with polyclonal Ab2 raised against the four distinct paratopes permitted classification of the mAb1 into four categories: (p+, id+) comprises antibodies with shared paratopic and idiotypic specificities: (p+, id-) is made up of antibodies that fail to express the Id defined by Ab2 raised against other antibodies pertaining to the same paratopic group; (p-, id+) includes antibodies that express cross-reactive Id on distinct paratopes; (p-, id-) refers to antibodies unrelated by their paratopes and Id mAb2 confirmed these results and showed expression of identical or closely related Id on clearly distinct paratopes. At the Ab3 level, using polyclonal Ab4, there was a higher degree of Id cross-reactivity between the two paratopes available. These data suggest that the parallel set concept may apply to the immune response to a natural peptidic Ag and its internal image. Comparison of idiotypic repertoires of mAb1 and mAb3 (using Ab2 and Ab4 antibodies) confirmed the lack of public Id and showed the predominance on mAb3 of "new" idiotypes absent from mAb1 molecules, as expected for internal image-induced antibodies. Cross-reactive idiotypes defined on mAb1 and conserved on mAb3 were expressed on the two paratopes defined at the Ab3 level. They were located on the H chain of the homologous paratope and required the association of H and L chains on the heterologous paratope. Our analysis suggests that, in the AII system, the idiotypic and paratopic repertoires segregate at least in part independently. The paratopic repertoire is limited to a small number of phylogenetically conserved specificities and may be encoded by germline genes. In contrast, the idiotypic repertoire is broader with respect to specificities, species, and localization on H and L chains. This extended diversity may be generated by somatic mutations or use of various combinations of H and L chains and/or V, D, J segments.     

The idiotypic network and the internal image: possible regulation of a germ-line network by paucigene encoded Ab2 (anti-idiotypic) antibodies in the GAT system.

Heavy and light chain variable regions from eight monoclonal Ab2 (anti-idiotypic) antibodies of the GAT antigen, a (Glu60 Ala30 Tyr10)n co-polymer, have been analyzed by direct mRNA sequencing. Three mAb2s were directed against private idiotopes and used various VH-D-JH and Vk-Jk combinations. By contrast, the five 'anti-public' mAb2 antibodies used a very restricted repertoire, including all gene segments. Interestingly, within their D regions, Glu-Glu-Tyr or Tyr-Tyr-Glu sequences were reminiscent of the original (GAT) antigen and may act as possible internal images. A striking observation was that two mAb2 antibodies shared the same V-D-J sequence although derived from separate fusions. As this D sequence, 33 nucleotides long, has not been described so far, it is suggested that it may be encoded by a new germ-line D gene, acting as a crucial regulatory element in a GAT germ-line idiotypic network. An alternative model that may lead to the construction of this D segment by 'odd' rearrangements from pre-existing already reported sequences is also presented.     

Behavior of the idiotypic network in conventional immune responses. II. Affinity and heterogeneity of idiotypic and anti-idiotypic antibodies following immunization with T-independent and T-dependent antigens.

The relative affinity and heterogeneity of affinity of idiotypic and anti-idiotypic antibodies in mice immunized with the T-independent antigen DNP-Ficoll and the T-dependent antigen DNP-HGG were measured by a plaque inhibition assay. Idiotypic plaque-forming cells (PFC) were detected by a conventional assay utilizing DNP-coated SRBC. Anti-idiotypic PFC were detected with SRBC coated with affinity-purified anti-DNP antibody of rabbit origin. It was found that both idiotypic and anti-idiotypic antibodies elicited by immunization with the T-independent antigen had lower affinity and were less heterogeneous than the corresponding antibodies originating in mice immunized with the T-dependent antigen. In addition, the affinity and heterogeneity values of the idiotypic antibodies were correlated with the affinity and heterogeneity values of the anti-idiotypic antibodies from the same mice. This finding indicates that idiotypic and anti-idiotypic antibodies mutually regulate each other, thus pointing to internal immunoregulatory effects of the idiotypic network with respect to these parameters.     

Parallel sets and the internal image of antigen within the idiotypic network

Antibodies carrying the internal image of antigens and antibodies with different specificities sharing idiotypes, which were called parallel sets, have been looked on as mere curiosities or odd by-products of antibody diversity. New findings supporting the existence of these antibodies in various experimental systems were presented. In addition, molecular studies of parallel sets in an arsonate system suggested that the genes encoding for these antibodies originate from germ line genes but use a different JH segment. The functions of these antibodies within the idiotype network were discussed.     

Studies of idiotypic antibodies: reactions of isologous and autologous anti-idiotypic antibodies with the same antibody preparations.

Outbred rabbits were immunized with keyhole limpet hemocyanin diazotized to p-aminophenyl-N-trimethylammonium chloride (TMA). Anti-TMA antibodies were specifically purified and the F(ab')2 fragments were isolated after pepsin digestion. Glutaraldehyde-polymerized anti-TMA F(ab')2 fragments were reinjected back into the same animal that synthesized the antibodies to elicit an autologous anti-idiotypic (AAI) antiserum and were also injected into an allotypically matched recipient to elicit isologous anti-idiotypic (IAI) antiserum. AAI and IAI antisera reactive with the same F(ab')2 preparation were compared by assaying the populations of anti-TMA molecules reactive with each antiserum. The assays showed that the AAI antiserum reacted with smaller populations of molecules than did the IAI antiserum. The population of molecules recognized by the AAI antiserum was contained within the larger population recognized by the IAI antiserum. Hapten inhibition studies showed that the differences in the populations of molecules recognized by each antiserum were not related to the hapten-binding properties of the idiotypic antibodies: in one case both AAI and IAI reactions were equally inhibited by hapten; in another, the AAI reaction was inhibited more than was the IAI; in another, the IAI reaction was inhibited more.     

Biological effects of anti-idiotypic antibodies on lymphocyte function: I. Analysis of the effects on B lymphocytes of combining site and framework-directed anti-T-15 idiotypic antibodies

Anti-idiotypic antibodies against TEPC-15 myeloma protein (BALB/c origin) were raised in allogeneic animals by immunization of A/J mice with the myeloma protein. The antibody activities were fractionated into two specificities by TEPC-15 immunoadsorbent affinity columns by elution with free hapten (phosphorylcholine, PC), followed by elution with acidic buffer (glycine- HCl, pH 2.3). Idiotype binding analysis indicated that the fraction eluted with hapten could be inhibited in its binding to TEPC-15 by free hapten (i.e., binding site-directed anti-idiotypic antibody), whereas the acid-eluted fraction could not (i.e., framework-directed anti-idiotypic antibody). When analyzed for their biological activities on PC-specific B lymphocytes producing T-15 idiotype-bearing antibodies, both anti-idiotypic antibody fractions had similar suppressive effects on the in vitro production of antiphosphorylcholine antibody in culture.     

Detection of in ovo derived idiotypic antibodies. I. A model for maternal-neonatal idiotype network studies.

Idiotypic vaccines appear to have many advantages over conventional vaccines. Maternal Id or anti-Id that are passively transferred to the fetus or neonate could provide another avenue for vaccination. Based on this premise we have investigated the transmission of idiotypic antibodies from dam to embryo by inoculating laying hens and analyzing their egg yolks for the presence of Id, anti-Id, and anti-anti-Id. The Ag chosen for these studies was BSA. After isolation and concentration BSA Id titers were approximately 256,000. The presence of anti-Id antibody in yolk samples is characterized by the ability of anti-Id to inhibit BSA binding to Id. The anti-Id extracted from yolks inhibited BSA binding to Id by 7 to 53%. Not all of the anti-Id present in samples was binding to a paratope-associated Id because 11 to 16% of the fluoresceinated anti-Id bound to Id-coated beads in the presence of excess BSA. Because a portion of the anti-Id antibodies were internal images of the BSA Id, they should be able to mimic Ag. This idea was confirmed when anti-Id inoculated in hens caused the synthesis of antibodies that would bind BSA and could be detected in an ELISA. Binding of anti-anti-Id to BSA-coated wells could be inhibited by preincubation of anti-anti-Id with BSA in solution. The chicken model provides a novel system to investigate maternal-fetal and maternal-neonatal interactions in the idiotypic network and the cellular mechanisms involved in the ontogeny of the Id network in neonates.     

Angiotensin I converting enzyme (kininase II) in isolated retinal microvessels.

Swine retinae were homogenized and fractions enriched in retinal microvasculature were prepared by techniques of selective sieving and centrifugation. The identity and purity of the preparations were investigated by phase contrast and electron microscopy. Angiotensin I converting enzyme (kininase II) was concentrated in the retinal microvessels. Metabolism of angiotensins and kinins in localized sites of the vasculature may contribute to local regulation of blood flow.     

Molecular analysis of immunoglobulin heavy chain genes coding for idiotypic and anti-idiotypic antibodies involved in B-B cellular interaction.

We recently reported that a unique B cell clone (B19-1d), specific for a cross-reactive idiotype (CRI) on MOPC104E myeloma protein (M104E), enhances Igh-restricted CRI+ antibody production. In this paper, we report the nucleotide sequences of immunoglobulin heavy chain variable regions (VH) of both M104E and B19-1d-derived hybridoma (HB19) antibodies. The sequence data revealed that both belong to the J558 germ line VH gene subfamily. Strikingly, not only the VH region, but also the leader sequences of M104E and HB19 are very similar to each other at 88% (VH) and 91% (leader) homology, but they use different D and J segments. The VH region sequence similarity is highest among the germ line VH gene sequences of the BALB/c J558 subfamily so far screened. Southern hybridization data, using 5'-noncoding regions of either M104E or HB19 genomic VH gene clones as probes, revealed that both VH genes are conserved in the M104E CRI producer strains of mice. Moreover, these probes show the restriction length polymorphism pattern of mouse VH genes in various strains. That the HB19 VH gene locates to the 5' upper arm of the M104E VH gene on the chromosome was suggested by Southern blot hybridization. Immunoglobulin VH gene restriction of idiotypic and antiidiotypic B-B cellular interaction is discussed from a molecular point of view.     

Perturbation of the idiotypic network. I. Induction with multiple alloantigen stimulation

The effect of hyperimmunization on the immune network with allostimulated syngeneic lymphocytes responding to different haplotypes was analyzed. Ten different haplotypes were used to stimulate syngeneic donor mice. Control mice were multiply immunized with incomplete Freund's adjuvant alone or with syngeneic mixed lymphocyte culture-generated lymphocytes. BALB/c mice were immunized consecutively with alloreactive blasts or allogeneically stimulated spleen cells at 10-day intervals. After a rest period of 2 months, the ratio of T helpers to T suppressors was determined by immunofluorescent staining. The functional network was probed by immunizing the mice with phosphorylcholine (PC) coupled to hemocyanin. The sera were analyzed for anti-PC antibodies and TEPC15 (T15) idiotypic expression. The results demonstrated (i) a decrease in the level of anti-PC antibody titer and T15 idiotypic expression; (ii) a decrease in the number of T helper cells and an increase in the number of T suppressor cells; (iii) a loss of PC epitope specificity; (iv) an increase of IgM antibodies expressing T15 without anti-PC specificity; and (v) an elevated level of preimmune lymphocyte proliferation and Ig secretion. These results reveal a functional network linkage in the regulation of alloreactivity and antigen response and show how repeated exposure to alloantigens can induce a perturbation of the idiotypic network controlling the response of a non-alloantigen-related BALB/c strain dominant idiotype (T15).     

Perturbation of the idiotypic network. II. Transfer of suppression to neonatal mice

The cellular mechanisms which are responsible for idiotype perturbation induced by repeated stimulation with either allogeneic mixed lymphocyte culture-generated syngeneic blasts or allogeneically stimulated syngeneic spleen cells were investigated as described in the preceding article. Using the splenic fragment culture system, the precursor frequencies of T and B cells for anti-phosphorylcholine (PC) antibodies and T15 idiotypic antibodies were determined in allogeneically challenged mice. Adoptive transfers of T cells to neonatal BALB/c mice induced suppression of the T15+ anti-PC responses. In addition, the effect of immunization with internal image-bearing anti-idiotopes on the level of anti-PC antibodies and T15 idiotype was determined. Results from this study demonstrated (i) a decrease in the precursor frequency in the PC-specific and idiotype-specific B cell repertoire; (ii) a decrease in the precursor frequency of T helper cells, which recognize idiotypes and anti-idiotypes; (iii) the possibility to transfer T15 suppression to neonatal mice; and (iv) the possibility to restore T15 dominance by anti-idiotypic antibody immunization. These data indicate that both the T and B compartments are involved in the maintenance of suppression induced by repeated exposure to alloantigen-sensitized syngeneic cells. Collectively these findings show that a nonspecific general suppression induced by allohyperimmunization can perturb the T15+ anti-PC response.     

Regulation of immune responses via genetically restricted cellular interactions. II. I-region-restricted cooperation between idiotypic and anti-idiotypic B lymphocytes

Immunization of BALB/c mice with MOPC-104E myeloma protein induced idiotype-specific enhancing cells which acted on anti-dextran antibody-producing cells. The enhancing cells have surface phenotypes of B cells. Using BALB/c H-2 congenic strains, it was found that the cooperation between anti-idiotypic-enhancing B lymphocytes and dextran-primed B lymphocytes was controlled by major histocompatibility gene complex. Here we have described the loci which restrict the successful cooperation between B lymphocytes, wherein it was revealed that the interaction was restricted to the I-A and I-E subregions in H-2k haplotype and the I-A subregion in H-2b haplotype. Utilizing several monoclonal antibodies specific for Ia antigens, it was revealed that the enhancing B lymphocyte activity was completely inhibited by the pretreatment of antibody-producing B cells with anti-Ia.7 in H-2d haplotype as well as H-2k, and with anti-I-A antibody in H-2b haplotype. The results suggest that the anti-idiotypic B-lymphocyte response to the self idiotype is under control of H-linked immune response (Ir) gene.     

Induction of an anti-hepatitis B surface antigen response in mice by noninternal image (Ab2 alpha) anti-idiotypic antibodies

Anti-idiotype antibodies to a mouse monoclonal antibody A-12 directed against HBsAg were produced in rabbits. The anti-Id consisted of an Ab-2 alpha preparation that did not display any detectable internal image activity. Immunization of BALB/c mice with the anti-Id (Ab-2 alpha) coupled to KLH induced an anti-HBs response without subsequent HBsAg exposure. No anti-HBs was detected in control groups of mice immunized with other rabbit anti-Id-KLH preparations. The anti-HBs containing sera from mice immunized with the Ab-2 alpha were able to inhibit the Id-anti-Id reaction, indicating that an Id-positive, anti-HBs response was induced. This idiotype is not normally expressed during the murine immune response to HBsAg and suggests that noninternal image anti-Id activates silent clones. This study, along with our previous results obtained with the use of internal image anti-Id, suggests that there is more than one Id network operational during the BALB/c murine immune response to HBsAg.     

Monoclonal antibodies to streptococcal group A carbohydrate. I. A dominant idiotypic determinant is located on Vk

In an effort to better define the antibody repertoire to streptococcal group A carbohydrate (GAC), somatic cell hybrids were prepared from A/J mice immunized with streptococcal vaccine. Most antibodies were IgG3K and IgMK, while 2 of 26 antibodies were lambda type. Each of the IgG3 antibodies had a distinct isoelectric point consistent with previous estimates of clonal repertoires of approximately 200. IEF analysis of the L chains, however, showed that about half of the antibodies produce a common L chain, called VK1GAC, previously identified in A/J anti-GAC serum antibodies. Additional support for the structural similarity of these L chains was gained by developing an idiotype antiserum to VK1GAC. All proteins with the common L chain spectrotype react strongly with anti-VK1GAC. Thus, it appears that anti-GAC antibodies are composed of H chains bearing a few VH regions pairing with a few L chains.     

Tumor-specific idiotype vaccines. II. Analysis of the tumor-related network response induced by the tumor and by internal image antigens (Ab2 beta)

In this study the tumor-specific immuneresponse induced by irradiated tumor cells (L1210/GZL) and by anti-idiotype antibodies was analyzed. The anti-idiotype antibodies (Ab2) were made against the paratope of a monoclonal antitumor antibody (11C1) that recognizes a tumor-associated antigen which cross-reacts with the mouse mammary tumor virus-encoded envelope glycoprotein 52. Two Ab2, 2F10 and 3A4, induced idiotypes expressed by the monoclonal antitumor antibodies 11C1 and 2B2. Cytotoxic T cells, generated by immunization with irradiated tumor cells, lyse 2F10 and 3A4 hybridoma cells. Furthermore, immunization with Ab2 induces tumor-specific cytotoxic T lymphocytes. The frequency of tumor-reactive cytotoxic T lymphocyte was found to be similar in mice immunized with Ab2 or irradiated tumor cells when examined at the precursor level. However, only 2F10 induces protective immunity against the growth of L1210/GZL tumor cells. The depletion of a L3T4+ T cell population from 2F10 immune mice was found to increase the effectiveness of transferred T cells to induce inhibition of tumor growth. The inability of 3A4 to induce antitumor immunity could be correlated with the presence of a population of Lyt2+ regulatory T cells. Collectively, these results demonstrate the existence of a regulatory network controlling the expression of effective tumor immunity. Our results demonstrate that selection of binding site-related Ab2 may not be a sufficient criteria for the development of an idiotype vaccine. A better understanding of the regulatory interactions induced by anti-idiotypes is needed for the design of effective antitumor immunotherapy.     

Production and isolation of rabbit anti-idiotypic antibodies directed against the human monocyte receptor for yeast beta-glucans.

beta-Glucan receptors are present on mammalian phagocytic cells and provide an important physiologic mechanism for opsonin-independent clearance of yeasts and fungi. To prepare an immunologic probe to human monocyte beta-glucan receptors, an approach was taken that focused on the ligand specificity of the receptors as expressed by an anti-Id. The algal beta-glucan laminarin was chemically coupled to protein carriers to prepare an immunogenic beta-glucan. Three mouse IgG2a mAb were raised against laminarin, and one, mAb OEA10, exhibited specificity for the soluble unit ligand yeast heptaglucoside and the ligands present on zymosan and glucan particles that are recognized by monocyte beta-glucan receptors. These findings prompted usage of mAb OEA10 as immunogen for the preparation of an anti-Id. The resulting rabbit antiserum was subjected to sequential immunoaffinity chromatography to purify anti-idiotypic antibodies. The final product contained only IgG by SDS-PAGE and was shown to be specific by its selectively blocking binding of 125I-mAb OEA10 to laminarin. Pretreatment of adherent monocytes with 0.4 micrograms/ml of the anti-Id reduced the numbers of monocytes ingesting zymosan and glucan particles by 64 and 43%, respectively, whereas ingestion of IgG-coated SRBC was unaffected by as much as 16 micrograms/ml of the anti-Id. Incubation of adherent monocytes with increasing amounts of 125I-anti-Id in the absence and presence of 40-fold molar excess unlabeled anti-Id revealed dose-dependent specific binding, which approached plateau levels with 1 microgram/ml of labeled anti-Id. Thus, the anti-Id binds to monocytes and displays functional characteristics of soluble beta-glucan ligands, indicating that some of the anti-idiotypic antibodies recognize epitopes within monocyte beta-glucan receptors.