Major Cross-reactive Idiotype Research Articles

Expression of the major cross-reactive idiotype in a primary anti-azobenzenearsonate response.

We have studied the occurrence of IgM plaque-forming cells secreting the cross-reactive idiotype (CRI) characteristic of the anti-azobenzenearsonate antibody responses in individual mice of different strains after one injection of the T-independent antigen, p-azobenzenearsonate-Brucella. Under these conditions of stimulation we find that idiotype is not unique to the Igh-1e and Igh-1d allotypes, but is expressed prominently in Igh-1a and Igh-1j strains and to a lesser but significant extent in Igh-1c and Igh-1b strains. We confirmed previous work that idiotype expression in mice hyperimmunized with protein conjugates of azobenzenearsonate is mainly restricted to mice of the Igh-1e allotype, but we find the display to be less marked than in mice given a single injection of the Brucella conjugate. We conclude that the B cell repertoire is inadequately revealed by analysis of serum antibodies of hyperimmune mice. We suggest the apparent correlation of the anti-azobenzenearsonate idiotype to allotype may be influenced by the activity of heavy chain regulatory genes, rather than the presence or absence of structural genes coding for the major CRI. We cannot exclude the possibility that the previously designated CRI-negative strains may express a cross-reactive determinant that is not necessarily a product of the CRI gene family.

Antigen and receptor-driven regulatory mechanisms: IX. T cell-T cell interaction in the generation of first-order idiotype-bearing suppressor T cells

We have studied the influence of allogeneic effects directed at specific intra-I region gene products on the stimulation of suppressor cells. Intravenous administration of low doses (1.26 m M) of azobenzenearsonate (ABA)-derivatized spleen cells, but not ABA-protein conjugates, induces a population of thymus-derived suppressor cells (pre-Ts) which can be activated by an I-J-specific allogeneic effect. After the allogeneic effect such Ts express their antigen-specific suppressive capacity. These cells were found to be Lyt 1 + 2− and I-J+ by antiserum plus complement treatment. In A/J mice, which express a major cross-reactive idiotype to ABA, these pre-Ts also bear idiotypic determinants on the cell surface. Evidence is presented which shows that the pre-Ts are allotype but not H-2 restricted in their ability to transfer suppression to other strains. The pre-Ts are also able to suppress the afferent (sensitization) but not the efferent (elicitation) limb of the immune response. The cell(s) which induce the allogeneic effect were found to be radioresistant (1500 R), Lyt 1 + 2− T cells. These results indicate that a population of Lyt 1 I-J+ cells unable to suppress Lyt 1 DTH reactions can be induced to do so by specific allogeneic effects mediated by an Lyt 1 population.

The relation of idiotype expression to isotype and allotype in the anti‐p‐azobenzenearsonate response

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.

Monoclonal ant-idiotypic antibodies reactive with a highly conserved determinant on A/J serum anti-para-azophenylarsonate antibodies.

Previous reports have shown the A/J anti-para-azophenylarsonate (anti-Ar) antibodies that share a major cross-reactive idiotype (CRI) comprise a family of closely related but nonidentical molecules. Serological studies with CRI+ monoclonal anti-Ar antibodies have suggested the presence of a conserved idiotypic determinant within the family. The present study utilized monoclonal ant-idiotypic determinant within the family. The present study utilized monoclonal anti-idiotypic antibodies to define further the nature of the conserved idiotypic determinant. It was found that 8 of 10 CRI+ monoclonal antibodies possess an idiotypic determinant reactive with each of three monoclonal anti-idiotypic antibodies. In addition, approximately 60% of CRI+ serum anti-Ar antibodies reacted with one of the monoclonal anti-idiotypic preparations. The monoclonal anti-idiotypic antibodies react with an idiotope in the region of the hapten-binding site, as indicated by the ability of free haptens to inhibit idiotype-anti-idiotype interactions. Finally, two of three monoclonal anti-idiotypic antibodies suppressed the subsequent production of CRE+ serum anti-Ar antibodies when administered before antigen, without significantly affecting the total anti-Ar response.

Properties of a minor cross-reactive idiotype associated with anti-p-azophenylarsonate antibodies of A/J mice.

Experiments are described that further characterize an A/J anti-phenylarsonate (anti-Ar) antibody population, which is idiotypically cross-reactive within the strain but differs from the family of antibodies expressing the major cross-reactive idiotype (CRI). This population has been designated as expressing a "minor" cross-reactive idiotype. Chain recombination studies with hybridoma products (HP) revealed that 2 of 3 HP expressing minor idiotypic determinants contain L chains that are idiotypically closely related to L chains in anti-Ar antibodies bearing the major CRI. The results indicate the expression of the same or very similar L chains in antibodies that differ in idiotype. The minor idiotypic population in serum was investigated by separating antibodies with minor and major idiotypic determinants by use of an immunoadsorbent containing anti-idiotype directed to an HP expressing the major CRI. Studies with the nonadherent, minor idiotypic population showed that such molecules constitute about 8 to 10% of the anti-Ar antibodies in a pool of serum but that individual sera vary markedly in their content of anti-Ar antibodies bearing minor idiotypes. Preinoculation of mice with conventional anti-idiotypic antiserum before immunization with KLH-Ar was found to suppress the appearance of the minor idiotypes as well as the major CRI upon subsequent immunization. The suppression of the minor idiotypic population is presumably mediated by the small fraction of rabbit anti-id antibodies directed against such molecules.

Regulation of expression of a family of cross-reactive idiotypes.

It has recently been shown that anti-p-azophenylarsonate antibodies of A/J mice, which share a common idiotype, are not homogeneous but actually comprise a family of idiotypically related molecules. To obtain additional insight into the degree of relatedness of antibodies within this family, we have determined whether inoculation of any idiotype-positive hybridoma product, conjugated to thymocytes, can activate a regulatory mechanism capable of controlling the entire cross-reactive idiotypic response. In addition anti-idiotypic antibodies were prepared against individual hybridoma products, and their suppressive effects on the idiotypic component of the humoral response were determined. Inoculation of each of the idiotype-positive hybridoma products or anti-idiotypic antisera caused virtually complete suppression of the idiotypic component of the immune response. Furthermore, it was shown that the state of suppression induced by the inoculation of a hybridoma products could be adoptively transferred with T cells to mildly irradiated syngeneic recipients. The results indicate that there are sufficient structural similarities among the family of antibodies comprising the major cross-reactive idiotype to permit their recognition by the same set of elements involved in idiotypic regulation.

Role and strain distribution of genes controlling light chains needed for the expression of an intrastrain cross-reactive idiotype.

Several strains of mice were tested for their capacity to provide immunoglobulin L chains required for the expression of the major cross-reactive idiotype (CRIA) associated with p-azophenylarsonate-specific antibodies of strain A mice. To facilitate testing, mice were bred that were homozygous for Igh-Ce and Lyt-2a, 3a, i.e., they possessed genes controlling H chains but not L chains required for expression of the CRIA. Such male mice were mated to females of various strains and their offspring were tested; expression of CRIA indicated the presence in the female parent of genes controlling the appropriate L chains. All females bearing the Lyt-2a, 3b or Lyt-2b, 3b genotype yielded offspring, most of which were CRIA+, whereas all the offspring of females that were Lyt-2a, 3a were CRIA-. The female parents included mice of several strains that are congenic for Lyt-2a, 3a, Lyt-2b, 3b or Lyt-2a, 3b, thus demonstrating very close linkage between the Lyt loci and the expression of CRIA. In addition, doubly congenic strains of mice with the heavy chain allotype of the CRIA+ AL/N strain and the Lyt-2a, 3a genotype on a BALB/c background failed to express CRIA. The data provide further evidence for the similarity of repertoires of L chains in Lyt-3b mice of various strains. When genes were present controlling A/J H chains and L chains of C57BL/6 or BALB/c origin, the quantitative expression of CRIA was only slightly lower than that observed in A/J mice. Mice possessing genes controlling the H or L chains required for CRIA expression, but not both, did not express CRIA but synthesized Ar-specific antibodies which contained low but significant concentrations of the idiotype-associated chain.

Presence of highly conserved idiotypic determinants in a family of antibodies that constitute an intrastrain cross-reactive idiotype.

It has been shown that A/J anti-p-azophenylarsonate antibodies that share a major cross-reactive idiotype (CRI) comprise a family of closely related, but nonidentical, molecules. Our results demonstrate that 12 of 14 monoclonal hybridoma products that express the CRI have in common at least one highly conserved idiotypic determinant. It is proposed that this reflects conservation of a portion of the amino acid sequence, presumably in hypervariable regions. That the conserved determinant(s0 are located in the region of the hapten-binding site is indicated by the ability of haptens to inhibit idiotype-anti-idiotype interactions involving the conserved, or public determinants.

Antigen- and receptor-driven regulatory mechanisms. IV. Idiotype-bearing I-J+ suppressor T cell factors induce second-order suppressor T cells which express anti-idiotypic receptors.

Administration of azobenzenearsonate (ABA)-coupled syngeneic spleen cells intravenously to A/J mice leads to the generation of suppressor T cells (Ts1) which exhibit specific binding to ABA-bovine serum albumin (BSA)-coated dishes. These Ts1 share idiotypic determinants with the major cross-reactive idiotype (CRI) of the anti-ABA antibodies of A/J mice, and also produce a soluble suppressor factor (TsF) bearing CRI and I-J subregion-coded determinants. Injection of this TsF into naive A/J mice elicits a second set of specific suppressor cells (Ts2) which are not lysed by anti-CRI antibody plus C, and which do not bind to ABA-BSA-coated dishes. However, in contrast with Ts1, these Ts2 do bind to plates bearing CRI+ anti-ABA immunoglobulin. Thus, Ts2 exhibit anti-idiotypic specificity. These data indicate that antigen elicits the production of a soluble T cell product bearing both variable portion of the Ig heavy chain (VH) and I-J subregion-coded determinants which serves to communicate between T cell subsets to establish an idiotype-anti-idiotype regulatory pathway.

Antigen- and receptor-driven regulatory mechanisms. I. Induction of suppressor T cells with anti-idiotypic antibodies.

Delayed-type hypersensitivity (DTH) to the azobenzenearsonate (ABA) hapten can be readily induced in A/J mice injecting ABA-coupled syngeneic spleen cells subcutaneously. To further characterize this T-cell-dependent immunological phenomenon, the effect of passively administered anti-cross-reactive idiotype common to anti-ABA antibodies of A/J mice (CRI) antibodies on the development of ABA-specific DTH was investigated. Animals given daily injections (of minute amounts) of anti-CRI antibodies subsequent to immunization with ABA-coupled cells show significant reduction of ABA specific responses. This inhibition is antigen specific and requires the intact immunoglobulin molecule, as F(ab')2 treatments were ineffective in suppressing the reaction. Investigations of the mechanism of the anti-CRI-induced suppression of ABA DTH revealed that the observed suppression is a result of the activation of suppressor cells. Spleen cells taken from animals which received anti-CRI antibodies were able to adoptively transfer suppression to naive recipients. This suppression was shown to be mediated by T cells, as anti-Thy1.2 plus complement completely abrogated the transfer of suppression. In addition, animals pretreated with low doses of cyclophosphamide were not suppressed by the administration of anti-CRI antibodies. The genetic restriction of anti-CRI-induced suppression was demonstrated. Antibodies to the major cross-reactive idiotype, (CRI) associated with anti-ABA antibodies in A/J mice were unable to suppress the development of DTH to ABA in BALB/c mice (H-2d, Igh-1a). Such antibodies were, however, fully active in suppressing ABA DTH in the allotype-congenic C.AL-20 strain which has an allotype (Igh-1d) similar to that of A/J (Igh-1e) on a BALB/c background, and which produces humoral antibodies with the CRI.

Suppression of idiotype and generation of suppressor T cells with idiotype-conjugated thymocytes.

Inoculation of A/J mice with syngeneic thymocytes conjugated with specifically purified A/J anti-phenylarsonate (anti-Ar) antibodies, selectively suppressed the subsequent synthesis of those anti-Ar antibodies which carry the major cross-reactive idiotype. High titers of anti-Ar antibodies were produced upon subsequent immunization but in most mice the idiotype was undetectable. Suppression similarly occurred in F1(A/J X BALB/c) and in C.AL-20 mice. Although some mice were suppressed when unconjugated antibody was injected, the suppressive effect was much more pronounced, particularly in the F1 and C.AL-20 recipients, when the antibody was coupled to thymocytes. The state of suppression could be adoptively transferred with T cells to mildly irradiated syngeneic recipients. A population enriched for B cells had little if any suppressive effect. There was no requirement for antigen in the generation of suppressors. Thymocytes conjugated with antibody did not induce idiotype-specific suppression in mice that had been recently challenged with antigen. Thymocytes from BALB/c and C57BL/10 mice were effective carriers for the anti-Ar antibodies, i.e., there was no evidence for H-2 restriction. The experiments demonstrate the feasibility of suppressing idiotype production and generating idiotype-specific suppressor T cells without the use of anti-idiotypic antibody or antigen.