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We have used a well-defined idiotypic system, the cross-reactive idiotype of A strain (CRIA) (Ab1) idiotype generated in A/J mice injected with arsonate coupled to keyhole limpet hemocyanin (ARS-KLH), to determine the frequency of precursors for auto-anti-idiotypic antibodies (auto-Ab2) in naive and immunized A/J mice by limiting dilution analysis after polyclonal activation by lipopolysaccharide. In naive animals, the precursor frequencies of auto-Ab2 B cells were below the limit of sensitivity of the technique in the majority of A/J mice, and could be detected in only 20% of the animals. Upon immunization with ARS-KLH, a large increase in auto-Ab2 precursor frequency was observed. This shift in frequency was not found when A/J mice were injected with KLH alone, or when BALB/c mice, which do not express the CRIA idiotype, were injected with ARS-KLH. To study the functional role of the auto-Ab2 B cells, we injected neonatal A/J mice with polyclonal rabbit Ab3 antibodies directed against a recurrent idiotype of auto-Ab2. Thereafter, these mice were injected with ARS-KLH. Although the anti-arsonate response level was normal, the CRIA Ab1 expression was reduced tenfold. Thus, the suppression of auto-Ab2 affects Ab1 dominance. We further show that the presence of maternal Ab1 can strongly modify the immune response of the offspring by inducing higher levels of the idiotype after immunization. Furthermore, IgM anti-arsonate antibodies were detected before immunization with antigen. From these data, we conclude that the affinity of antigen alone cannot explain the dominance of CRIA. Network selection is important in the shaping of the available repertoire.

We have compared by functional assays the relative preference among Ia molecules for their ability to present tyrosine-azobenzenearsonate (ABA-Tyr) to T cells. Immunization of B10.BR mice (IAk, IEk) with ABA-Tyr resulted in the induction of IAk-restricted T cells only. Immunization of B10.A(5R) mice (IAb, IEb/k) gave only IEb/k-restricted T cell clones even though IAb-restricted responses could be induced in C57BL/6 mice (IAb). These results indicated that IAk was preferred over IEk and IEb/k was preferred over IAb for presentation of ABA-Tyr. A comparison between IAk and IEb/k made by immunizing [B10.BR x B10.A(5R)]F1 mice (IAk, IEk, IAb, IEb/k), showed that IEb/k was favored over IAk. No IAb- or IEk-restricted response was seen. Further attempts were made to compare Ia preference for ABA-Tyr presentation by competitive inhibition assays. It could be shown that the presence of IEb/k molecules on an accessory cell interfered with the ability of IAb molecules on the same cell to present ABA-Tyr to an IAb-restricted T cell clone by direct competition. Such a competition was not observed between IAk and IEk. Finally, it could be shown that addition of ABA-Tyr inhibited the presentation of moth cytochrome-c peptide (81-103) by IEb/k but did not influence its presentation by IEk. From these functional studies we suggest that the binding affinity of ABA-Tyr with the Ia molecules will fall in the order: IEb/k greater than IAk greater than IAb greater than IEk.

At least two distinct subpopulations of A/J anti-azophenylarsonate (Ars) antibodies can be defined on the basis of serological cross-reactivity and sequence homology of heavy and light chains. The most frequently occurring idiotype family, IdCR, was initially identified by a rabbit antiserum and is present only in A/J mice. Examination of several randomly selected hybridoma proteins which failed to express IdCR-associated determinants revealed a second set of closely related molecules. The second idiotype family, Id36-60, is recognized by a rabbit antiserum prepared against the hybridoma protein 36-60. It is present in practically all A/J and BALB/c, but not B10. A Ars immune sera. On average, A/J immune sera contain approximately one-fifth as much Id36-60 as IdCR. A/J mice can be suppressed for subsequent expression of either idiotype by neonatal injection of anti-IdCR or anti-Id36-60 antisera. Suppression of IdCR does not affect the production of Id36-60 and vice versa. Together, these idiotype systems provide two independent markers for monitoring network regulation of the anti-Ars antibody response in A/J mice.

The distribution of idiotype-positive plaques in mice of various strains injected with T-independent (TI) and T-dependent conjugates of p-azobenzenearsonate (Ars) has been investigated. With maturation of the responses there is a progressive loss of dominance of the major cross-reactive idiotype (CRI) in A/J and C.AL-20 mice. The extremes are represented by the predominantly CRI+ IgM plaque response to a single injection of the TI antigen, Ars-coupled Brucella organisms (Ars-Br), and the low frequency of CRI+ plaques in the polyisotypic response elicited by Ars-KLH (keyhole limpet hemocyanin) in adjuvant. The secondary response to Ars-Br was characterized by an intermediate display of idiotype-positive plaques in IgM, the IgG subclasses and IgA. Detailed analyses of the distribution of CRI+ plaques between isotypes in 60 mice hyperimmunized with Ars-KLH revealed that the expression of idiotype in any of the IgG subclasses and IgA was constant and independent. This is interpreted to indicate random assembly of VH with C gamma and C alpha genes during the switch. BALB/c and C3H mice also express a predominant idiotype during primary and secondary responses to Ars-Br as detected by our heterologous anti-A/J CRI serum. These strains only very rarely maintain the idiotype after hyperimmunization with Ars-KLH. We conclude that hyperimmune responses which probably involve multiple modes of regulation give a poor indication of the germ-line V gene repertoire.

Immunization of GT (random copolymer of L-glutamic acid51-L-tyrosine49) nonresponder animals with p-azobenzenearsonate (ABA) GT conjugates elicits an antibody response to both ABA and GT epitopes which is induced by ABA-specific T helper cells. Expression of these hapten-specific helpers is under the control of an I region gene which also regulates the proliferative T cell response to ABA. Conversion of the unresponsive phenotype to GT is, therefore, dependent on the ABA Ir gene and escapes the influence of the GT-specific I region-controlled suppressive pathway. Studies on the influence of ABA/polymer coupling ratio on T and B cell responses suggest that ABA-specific T cells, like conventional carrier-specific helpers, require linked interactions with B lymphocytes to provide helper signals. GAT (terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10) nonresponder animals immunized with ABA-GAT conjugates also develop an antibody response to ABA which is induced by ABA-specific T helper cells. Comparison of antibody affinity, specificity, isotypes and idiotypes in different mouse strains demonstrates that hapten-specific helper cells stimulate antibody responses to ABA which are qualitatively similar to those induced by GAT-specific helpers. However, ABA-specific helper cells do not permit the conversion of the I region gene-controlled nonresponder phenotype to GAT. The data suggests that high ABA density, which is required for optimal ABA help expression, extinguishes the immunogenicity of GAT determinants at both T and B cell levels.

The anti-p-azophenylarsonate antibodies of A/J mice express a major intrastrain cross-reactive idiotype (CRIA). Using a sensitive radioimmunoassay we were unable to detect significant quantities of molecules bearing CRIA in nonimmune A/J sera. The upper limit is 140 ng/ml. Possible reasons for variability of such data in the published literature are discussed.

Immunization of the A strain of mice with the hapten p-azophenylarsonate (Ars) results in an immune response in which approximately 50% of the anti-Ars antibodies share cross-reactive idiotypic determinants (IdCR). A gene or genes linked to the heavy chain constant region locus is required for the production of this idiotype. The expressed VH gene from a hybridoma cell line which expresses the IdCR has been cloned. DNA hybridization studies utilizing the VH gene have revealed that there are many related genes in both idiotype-producing and idiotype-nonproducing strains of mice. However, under stringent hybridization conditions, only a single band of 6.4 kb is present in Eco R1-digested A strain DNA. Strains of mice which are phenotypically idiotype-negative either lack this band completely or possess a much weaker one at this position. Utilizing DNA from Igh recombinant strains of mice, it has been shown that the VH locus controlling idiotype expression contains the structural gene information for the idiotype-positive heavy chains. It has also been shown that DNA at this locus appears to be sufficient for the production of the cross-reactive idiotype. Utilizing a DNA probe derived from regions flanking the structural gene has confirmed the relatedness of V genes in a variety of mouse strains and revealed a significant degree of polymorphism at the Igh locus.

The amount of natural anti-DNP-cap, anti-NIP-cap or anti-ABA-TYR in human IgM was measured by using immunoabsorbents and hapten elution. We found that 1.0% of pooled human IgM is specific for DNP-cap, 0.2% for NIP-cap and 0.02% for ABA-TYR. Haptens NIP and DNP were found to be cross-reactive when tested with natural antibodies even though they were not cross-reactive when tested with immune antibodies.

The T cell repertoire of BALB/c mice contains clones capable of recognizing p-azobenzenearsonate (ABA)-tyrosine (Tyr) in association with both I-A and I-E-encoded class II molecules. Immunization of BALB/c animals with ABA-GAT (terpolymer of L-Glu60-L-Ala30-L-Tyr10) or ABA-GLT (terpolymer of L-Glu51-L-Lys34-L-Tyr15) instead of ABA-Tyr reduces the secondary proliferative response to ABA-Tyr in vitro. Limiting dilution experiments indicate that this situation corresponds to the recruitment of fewer ABA-specific T cells in vivo. The same experiments, performed in A.TH mice, which are nonresponder to both GAT and GLT, demonstrate that the number of ABA-specific T cells stimulated in vivo with ABA conjugates depends on the Ir gene-controlled immunogenicity of the carrier rather than on the ABA epitope density on the immunogen. Although GAT is preferentially recognized in association with A, and GLT with E, ABA-GAT and ABA-GLT stimulate both A and E-restricted ABA T cells in vivo and in vitro. The ABA-Tyr-specific T cell repertoire is not qualitatively affected by the carrier. This demonstrates that the inhibition of hapten-specific T cell expression upon immunization with ABA conjugates does not result from a competition between hapten and carrier-specific T cells for epitope recognition in association with the same Ia molecule on antigen-presenting cells.

A nucleic acid probe specific for heavy chains bearing the cross-reactive idiotype (Id) associated with the anti-p-azophenylarsonate response of strain A mice has been prepared. Analysis of arsonate-binding Id+ hybridoma cell lines has revealed that all of them contain the same germ-line VH gene rearranged to the JH2 segment. An Id+ hybridoma which is unable to bind arsonate utilized the same VH gene, but it has apparently rearranged to the JH4 segment. Id- cell lines contain other rearranged VH genes. Analysis of DNa of strain A mice revealed that there is apparently only one germ line gene that can give rise to Id+ heavy chains. Since the Id is expressed as a large collection (greater than 50) of related but nonidentical heavy chain sequences, we conclude that their diversity is the result of a somatic mutation process. Analysis of a single hybridoma cell line (45-59) reveals that somatic mutation can operate on an Id-encoding gene and result in an antigen-binding molecule that has lost all of its Id determinants. Further analysis of the genome of strain A mice has revealed the presence of germ-line genes differing from the Id-encoding gene by at least 8 base pairs. These genes, however, apparently do not contribute to the anti-arsonate Id response.

Dinitrophenylated (DNP) conjugates of Ficoll, hydroxyethyl starch, levan, dextran, type 3 pneumococcal capsular polysaccharide SIII, pectin, alginic and hyaluronic acid at various epitope densities were tested for their capacity to inhibit secondary IgG anti-DNP antibody responses using an in vivo transfer system. Some aminobenzylarsonate conjugates were examined similarly. Conjugates of the acidic polysaccharides were markedly more effective than those of uncharged polysaccharides. Conjugated with high epitope densities were particularly tolerogenic. DNP conjugates of Ficoll and hydroxyethyl starch elicited prolonged IgM responses over a wide dose range, and there are indications in the literature that conjugates of dextran do likewise. All the polysaccharide conjugates, labeled with radioiodine, persisted in the body for long periods (half-lives 8.5--63 days), and all were cleared rapidly from, but remained detectable in the blood for many days. Gross tissue distributions varied markedly from one conjugate to another, and autoradiography revealed unexpected differences in cellular localization. Despite substantial retention in the liver, some uncharged polysaccharides were localized only in parenchymal and not in Kupffer cells. In the spleen, only the acidic polysaccharides were predominantly localized in red pulp macrophages, and the neural polysaccharides were detectable exclusively, and in the case of Ficoll, hydroxyethyl starch and some dextrans very intensely in macrophages of the marginal zone of the white pulp. It is suggested that retention of thymus-independent antigens in marginal-zone macrophages favors, whereas trapping in red-pulp macrophages diminishes immunogenicity, and that tolerogenic effectiveness depends upon a balance between tolerization of B cells by free antigen and stimulation by antigen presented by marginal-zone macrophages. The distinction between functionally different macrophages in the spleen is elaborated in the following report.

Subcutaneous immunization of CBA/N or (CBA/N X BALB/c)F1 (NBF1) male mice, which were defective in phosphorylcholine (PC)-specific humoral antibody response, with PC-conjugated syngeneic spleen cells induced a PC-specific delayed-type hypersensitivity (DTH) response, while intravenous administration of the same cells induced PC-specific suppressor T cells for the DTH response. Treatment of PC-specific effector or suppressor T cells for the DTH response induced in NBF1 male mice with anti-T15 idiotypic antibody inactivated effector or suppressor functions of these T cells implying that PC-specific T cells in NBF1 mice expressed T15 idiotypic determinants on their surface. Enrichment of PC-specific suppressor T cells was also shown by employing anti-T15 antibody-coated dishes.

A small bifunctional antigen (4-hydroxy-5-iodo-3-nitrophenyl)acetyl-epsilon-aminocaproyl-L-tyrosine-azobenzene-p-arsonate [NIP-cap-TYR(ABA)] was found to induce fair humoral antibody formation against NIP-cap but very little anti-ABA-TYR. This was observed in rats and guinea pigs. Prior immunization with ABA-TYR, either as such or coupled to dodecanoylated bovine serum albumin (lipid-BSA), primed rats for an enhanced anti-NIP response to NIP-cap-TYR(ABA). An attempt to encourage rats to produce anti-ABA-TYR in response to the bifunctional antigen by priming them with NIP-cap-lipid-BSA failed. Priming with ABA-TYR was dose-dependent. An injection of 1.5-15 nanomoles per rat primed for an increased production of anti-NIP while 150 nanomoles did not. Adult thymectomized x-irradiated rats had a poor anti-NIP response to the bifunctional antigen if they were reconstituted with T-enriched lymphoid cells from control mice, but a good response if reconstituted with similar cells from ABA-TYR-primed syngeneic rats.

The sera of immunized A/J mice contain low but detectable levels of immunoglobulin, bearing previously described cross-reactive idiotypic (CRI) determinants diagnostic of the strain A anti-p-azophenylarsonate (Ars) response. Such molecules from nonimmune sera cannot be adsorbed onto affinity columns coupled to a high density with Ars. After extensive immunization with Ars-coupled proteins, Ars-nonbinding CRI is found at the same low level, even though substantial Ars-binding CRI appears. On the other hand, immunization with a monoclonal rat anti-CRI, which was originally raised against Ars-binding CRI, elicits high concentrations of Ars-binding as well as Ars-nonbinding CRI immunoglobulin. Three hybridoma proteins produced from an A/J mouse immunized with the rat anti-CRI react with all tested anti-idiotypic sera from three species of animals, but show no reactivity toward Ars in several different assays. One hybridoma protein from the same fusion demonstrates Ars-binding capacity.

The antigen-binding selectivity of 2 sets of anti-DNA antibodies from autoimmune mice and from normal mice was examined. Eighteen affinity-purified anti-DNA auto-antibodies from MRL-lpr/lpr mice were examined for binding to the haptens azobenzenearsonate, phosphorylcholine, (4-hydroxy-3-nitrophenyl)acetyl and (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP). Five of these autoantibodies bound to NIP-protein conjugates. In contrast, none of 12 monoclonal antibodies to single-stranded DNA or left-handed Z-DNA induced by immunization of BALB/c and C57BL/6 mice with nucleic acid antigens reacted with the tested haptens. In a reciprocal test of the relationship between anti-DNA and anti-NIP binding, we examined 24 monoclonal antibodies to NIP, from various strains of mice, for binding to DNA. One such antibody from a BALB/c mouse also bound to DNA. These results are discussed in the context of the mechanisms underlying autoantibody hyperproduction.

When mice of the A/J strain were immunized with azobenzenearsonate (ABA) directly coupled to a protein, they produced antibodies that share an inherited cross-reactive idiotype (CRI). In the antigenic determinant that induces CRI, ABA is very probably coupled to tyrosine, and the structure that protrudes from the polypeptide backbone has two benzene rings and a molecular weight of 419. When this same structure is separated from the polypeptide backbone by a spacer of 99 daltons, it induces a different inherited idiotype (ABA-HOP-e) in the same strain of mice. Our data suggest that antibodies with the idiotype CRI recognize the terminal benzene ring and the azo group, but do not fit closely around the second ring structure. Antibodies with the idiotype ABA-HOP-e have fine specificity for both benzene rings. Both idiotypes are inherited and both are linked to genes controlling constant regions of the heavy chains.