Immunological responsiveness of neonatal A/J mice to isotypic determinants of syngeneic IgE.

We have previously shown that adult A/J mice produce high titers of anti-IgE with isotypic or idiotypic specificities in response to challenge with a conjugate of KLH with syngeneic monoclonal IgE. Thus, B cells that can synthesize anti-IgE are present in the mice. Adult mice are unresponsive to unconjugated IgE in CFA, suggesting that tolerance exists at the level of T cells. The present study shows that neonatal mice produce anti-IgE antibodies in response to unconjugated IgE in CFA, but that this capacity is lost after the age of 2-3 wk. The loss of responsiveness corresponds closely with the appearance of detectable IgE in serum, suggesting that the IgE may induce tolerance. The affinities of anti-IgE antibodies produced by neonatal mice fall in the range of values obtained with KLH-IgE in adult mice. Tolerance to unconjugated IgE in CFA can be induced in neonatal mice by administration of IgE in saline. In addition, the tolerant state can be induced by adoptive transfer of spleen cells from adult mice. The time-dependent acquisition of tolerance provides a useful model for studying mechanisms of tolerance and autoimmunity.

Passive transfer of the idiotypically suppressed state by serum from suppressed mice and transfer of suppression from mothers to offspring.

Mice that are suppressed with respect to an idiotype (CRIA) present in A/J anti-p-azophenylarsonate antibodies, hyperimmunized, and allowed to rest were previously found to possess high concentrations of suppressor T cells with anti-idiotypic receptors. We have now observed that the sera of such mice contain soluble factors that can selectively suppress the CRIA component of a humoral response when passively transferred to adult or neonatal recipients. When T cells from suppressed, hyperimmunized mice were transferred into female mice before mating, their offspring, upon immunization, produced anti-Ar antibodies that lacked CRIA. A state of idiotypic suppression was also produced in offspring when the mother was inoculated with serum from suppressed mice a few days before parturition. The results indicate that the suppressor factor is not an immunoglobulin.

Selective suppression of the major idiotypic component of an antihapten response by soluble T cell-derived factors with idiotypic or anti-idiotypic receptors.

Evidence is presented for the selective suppression of the major idiotypic component of the humoral response to the phenylarsonate hapten by soluble factors derived from T cells (TsF). The existence of TsF with anti-idiotypic receptors was also demonstrated. It was found that TsF with idiotypic and anti-idiotypic receptors coexist in cultures of spleen cells prepared from idiotypically suppressed, hyperimmunized mice. By gel filtration the molecular weight of each factor was found to be 50,000-100,000. Each is sensitive to trypsin and is bound to a column containing anti-H-2a antibodies. Evidence is discussed which suggests the possibility of mutual stimulation of suppressor T cells with idiotypic and anti-idiotypic receptors.

Quantitative investigations of idiotypic antibodies. IV. Inhibition by specific haptens of the reaction of anti-hapten antibody with its anti-idiotypic antibody.

Rabbit anti-idiotypic antibodies were prepared by injection of specifically purified anti-p-azobenzoate antibodies (D) from individual donor rabbits. Benzoate derivatives were found to be strong inhibitors of the reactions of D with anti-D antisera. There was a close correlation between the combining affinities of the benzoate derivatives used and their effectiveness as inhibitors. Compounds tested that are chemically unrelated to benzoate were ineffective. The results indicate either that the combining site of anti-benzoate antibody is part of an important idiotypic determinant, which is sterically blocked by hapten, or that the hapten induces a conformational change which alters idiotypic determinants not involving the active site. Such conformational changes, if they occur, must be restricted since hapten has little effect on the reactions of F(ab')(2) fragments of anti-benzoate antibodies with antisera directed to rabbit fragment Fab and no detectable effect on reactions with antibodies directed to allotypic determinants.

Isolation from individual A/J mice of anti-rho-azophenylarsonate antibodies bearing a cross-reactive idiotype.

Immuization of A/J mice with a KLH-p-azophenylarsonate conjugate induces the formation of antihapten antibodies, some of which share idiotypic specificity common to all recipients. The subpopulation carrying the idiotype generally comprises 20-70% of the total antibody content. Large quantities of antihapten antibody (occasionally over 100 mg) were obtained from individual mice through the induction of an ascites fluid. This facilitated isolation of antibodies with the cross-reactive idiotype by isoelectric focusing. Most of this subpopulation has pI values between 6.65 and 6.95 and essentially all is of the IgG1 subclass. Two peaks, near pI 6.7 and 6.9, were frequently observed. Upon refocusing, the protein artifact of the procedure, but indicates microheterogeneity. The antibodies in the two peaks were found to be idiotypically identical by measurements of cross-inhibition. Preliminary studies have indicated that it is feasible to initiate investigations of primary structure with antibodies from individual inbred mice.

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.

Quantitative investigations of idiotypic antibodies. 3. Persistence and variations of idiotypic specificities during the course of immunization.

Changes and persistence of idiotypic specificities of specifically purified rabbit anti-p-azobenzoate antibodies were studied by quantitative methods. In each rabbit idiotypes identified 2 months after the start of immunization were still present in comparable concentrations 2 months later. After month 4, they were replaced by new and unrelated specificities; the changes were abrupt in two rabbits and gradual in the third, and were associated with an increase in the average affinity for specific hapten. In two surviving rabbits the new sets of specificities persisted in part for at least 1 yr. Quantitative changes occurred during this period, and the antibody preparation used as immunogen reacted most effectively with the homologous anti-D serum. The antibody population present at month 17 (D(17)) in one rabbit was deficient in idiotypic specificities present in D(8) and lacked specificities present in D(2), indicating the presence in D(17) of a third group of specificities. The percentage of the antibody population from each rabbit reactive with homologous anti-idiotypic serum was greater at month 8 than at month 2, suggesting a decrease in heterogeneity. Since the donor rabbits were challenged repeatedly with antigen, it appears that, after month 8, a portion of the antigen was utilized to stimulate existing cell lines and a portion to initiate new clones. Precipitation of anti-p-azobenzoate antibodies removed idiotypic specificities, indicating that they were not present on the anti-bovine gamma-globulin antibodies in the same sera.

Distribution of a cross-reactive idiotypic specificity in inbred strains of mice.

The expression of an idiotype characteristic of anti-p-azophenylarsonate antibodies of all A/J mice was explored in F(1) progeny, in other inbred strains, and in congenic mice. Of the strains tested only those closely related to A/J produced antibodies with the cross-reactive idiotype (CRI). None of the mice synthesized intermediate levels of CRI. No relationship between H-2 type and idiotype was noted. Congenic mice with a strain A background but a different H-2 type produced CRI in amounts quantitatively equivalent to those of strain A mice. Conversely, the presence of the H-2 genotype of strain A on an unrelated background was not associated with the formation of CRI. Nearly all F(1) progeny of strain A mice formed CRI, indicating that failure of the other (non-A) parental strain to produce CRI is not attributable to the presence of a gene controlling the synthesis of a suppressor of CRI. NZB mice, which have the same heavy chain allotype as strain A, but are unrelated in origin, failed to produce CRI, although allotype has been shown to be linked to idiotype in congenic strains.

Effect of idiotype-specific suppressor T cells on primary and secondary responses.

Previous reports have shown that suppression of idiotype can be adoptively transferred by T cells, or by rosettes containing T cells with anti-idiotypic receptors, from an idiotypically suppressed, syngeneic mouse. The present data indicate that secondary B cells are highly resistant to such suppression. Priming recipients to the relevant hapten, p-azophenylarsonate, 6 days or 4 mo before the adoptive transfer prevented suppression. This was independent of the carrier used for the hapten group during priming or subsequent immunization, suggesting that resistance to suppression is attributable to secondary cells with specificity for the hapten. The effect of suppressor T cells could also be overcome by mixing them with specifically purified B cells having receptors for the hapten group before the adoptive transfer. Adoptive transfer of the suppressed state by specifically purified B cells from suppressed, hyperimmunized animals confirmed our previous finding that the suppression of idiotype can also be caused by B cells lacking idiotypic receptors, evidently through a mechanism involving clonal dominance. Possible mechanisms of idiotypic suppression by T cells are discussed.

Inhibition of an IgE response by secondary B cells of a different isotype.

We found that the synthesis of IgE anti-Ars antibodies is strongly inhibited by the presence of secondary non-IgE-producing cells that are specific for the Ars hapten. Such B cells can be induced by inoculation of a protein-Ars conjugate in CFA. The effect is seen after inoculation of OVA-Ars in CFA followed by KLH-Ars in alum, or, more convincingly, after adoptive transfer of B cells induced by antigen in CFA. Dose-response data indicated that inhibition can be effected by B cells containing noninhibitory numbers of contaminating T cells. Possible synergistic effects of carrier-specific regulatory T cells were ruled out by using a different protein carrier for immunization of donor and recipient mice. The effect was shown to be specific for the hapten used for immunization of donor mice.

Quantitative investigations of idiotypic antibodies. V. Factors affecting the persistence and replacement of clones of antibody-producing cells.

The effect of challenge by antigen on persistence of clones of antibody-producing cells and on the induction of new clones was investigated through quantitative measurements of idiotypic specificities. In each of nine rabbits idiotypic specificities present in the earliest bleedings were completely replaced after a few months; subsequent changes occurred much more slowly. On a quantitative basis the population of molecules used as immunogen always reacted most effectively with the homologous anti-idiotypic antiserum. Little effect of increased antigen dose on the rate of change of idiotype was observed. Even large amounts of antigen administered every 2 wk caused only gradual changes in idiotypic specificities. This was attributed either to more effective capture of antigen by memory cells, as compared to precursor cells, or to the induction of tolerance in those clones that were not expressed. In two of three rabbits on a monthly injection schedule, the idiotypic specificities identified underwent very slow changes over a period as long as 17 months. Changes occurred more rapidly when antigen was administered every 2 wk. In each of four rabbits investigated, all idiotypic specificities identified before a 5 month rest period were still present afterwards, indicating the survival of essentially all clones of antibody-producing cells during that interval. Quantitative inhibition data indicated that some new clones of cells were initiated.

Quantitative investigations of idiotypic antibodies. II. Nonprecipitating antibodies.

Idiotypic antibodies were investigated quantitatively by a method of indirect precipitation, which utilizes labeled F(ab')(2) fragments of specifically purified antibenzoate antibody from the donor, anti-antibody, and an antiglobulin reagent. The contribution of allotypic and hidden determinants to these reactions was excluded. Greater fractions of an idiotypic antibody population are precipitated by this method, as compared to direct precipitation, and in two instances large proportions of idiotypic antibodies were detected in populations which failed to form precipitates by double diffusion in agar gel. The greater sensitivity of the indirect method was attributed to its capacity to detect molecules bearing a small number of antigenic determinants. Extensive studies of cross-reactions, carried out by an inhibition technique, failed to reveal any strong reactions of anti-idiotypic antibodies with heterologous antibenzoate antibody preparations, heterologous sera, or IgG, although a few weak cross-reactions were noted. One definite cross-reaction was observed by a direct binding measurement with heterologous antiserum. Antisera prepared in more than one recipient against a single donor preparation reacted with identical or overlapping subpopulations of the donor molecules. Instances in which two recipient antisera reacted with different proportions of the molecules of a single donor provided evidence for the existence of more than one idiotypic antibody population in the antibenzoate antibody of an individual rabbit.

Significance of bivalence of antibody in viral neutralization.

The role of bivalence of antibody in its capacity to neutralize virus was studied with rabbit antibodies to the bacteriophage, phiX174. Univalent Fab or Fab' fragments of IgG isolated from antiviral antisera obtained early in the immunization schedule had virtually no activity compared to that of the intact IgG. When the antibodies were isolated from antisera of the same rabbits several months later, the univalent fragments and IgG were essentially equal in activity. The results are interpreted on the basis that an IgG molecule, because of its bivalence, has a higher effective combining affinity (avidity) than a univalent fragment. After prolonged immunization, however, the affinity of univalent antibody becomes sufficiently high that it exceeds a threshold value, above which further increase in affinity, through bivalence, is no longer significant. The results could explain the variability in relative effectiveness of univalent antibodies observed in previous studies. These data, and the fact that F(ab')(2) fragments from either "early" or "late" antisera were as effective as IgG, indicate that fragment Fc is not a significant factor in neutralization. No differences in dissociation from the virus of univalent antibody from early and late antisera could be demonstrated by dilution at temperatures up to 47 degrees C. The attachment at sites of neutralization on the virus appears to be functionally almost irreversible in this system.

Quantitative investigations of idiotypic antibodies. VI. Idiotypic specificity as a potential genetic marker for the variable regions of mouse immunoglobulin polypeptide chains.

Antisera were prepared in rabbits against anti-p-azobenzoate antibodies of an A/J and a BALB/c mouse and anti-p-azophenylarsonate antibodies of an A/J mouse. After appropriate absorption the antisera reacted with the anti-hapten antibody of the donor mouse but, by sensitive quantitative tests, not at all with other components of the hyperimmune serum or with preimmune serum of the donor mouse. The absorbed antiserum therefore appeared to be specific for idiotypic determinants. Nearly all idiotypic specificities identified in the serum of the donor were also present in the serum of other mice of the same strain, immunized against the same hapten group, but not in mice immunized with a different hapten. In each case the antibodies of the donor mouse reacted most effectively on a weight basis with antiidiotypic antiserum. Cross-reactions were observed among different strains of mice but homologous anti-bodies reacted most effectively with antiidiotypic antisera. C57/BL and DBA antisera contained very low concentrations of specificities present in the A/J and BALB/c antibody populations; antibodies of A/J and BALB/c antisera are more closely related to one another. The results indicate that idiotypic specificity may provide a genetic marker for the variable regions of immunoglobulin polypeptide chains.

Requirements for prolonged suppression of an idiotypic specificity in adult mice.

The appearance of an idiotypic specificity, present in anti-p-azophenylarsonate (anti-Ar) antibodies of all immunized A/J mice, ran be suppressed in adult mice by prior administration of an IgG fraction of rabbit antiidiotypic (anti-D) antiserum; anti-Ar antibodies arise but are of different idiotype. Prolonged suppression was observed in earlier experiments, but antigen was first administered to adult mice only 2 wk or 9 wk after anti-D antibodies; subsequent escape from idiotypic suppression could have been masked by the capture of antigen by large numbers of memory cells having receptors of a different idiotype. In the present experiments antigen was first administered at intervals up to 22 wk after the antiidiotypic antibody. Suppression was maintained for 6 wk in all mice and for 5 mo in about half the mice tested. It thus appears that suppression of idiotype is less reversible if antigen is administered soon after the antiidiotypic antibody. The data suggest that escape from suppression is attributable to the generation of new precursor cells rather than to reactivation of suppressed cells. The minimum dosage of antiidiotypic IgG required for effective suppression was about 2 mg. The subcutaneous or intraperitoneal routes of inoculation of antiidiotypic IgG were equally effective. When antiidiotypic antibody was administered 3 days after antigen no suppressive effects were observed. There was partial suppression when antiidiotypic antibody was injected on the same day as the antigen. Fab' and F(ab')(2) fragments of antiidiotypic IgG had no suppressive effect. Quantitative measurements revealed no significant differences among control and suppressed mice with respect to total concentration of precipitable anti-Ar antibodies produced.

Frequency of occurrence of idiotypes associated with anti-p-azophenylarsonate antibodies arising in mice immunologically suppressed with respect to a cross-reactive idiotype.

Inoculation of rabbit anti-idiotypic (anti-id) antibodies suppresses the subsequent appearance of a cross-reactive idiotype (CRI) associated with the anti-p-azophenylarsonate (anti-Ar) antibodies of A/J mice. Such suppressed mice produce normal concentrations of anti-Ar antibodies which lack the CRI, but against which anti-id antisera can be prepared. The anti-Ar antibodies of an individual, suppressed mouse do not in general share idiotype with anti-Ar antibodies of other A/J mice, either suppressed or nonsuppressed. The present experiments were undertaken to quantitate several "private idiotypes" in a large number of hyperimmunized A/J mice. Anti-Ar antibodies of three mice, suppressed for the CRI, were labeled with 125I and subjected to isoelectric focusing. Four single peaks, that were over 90% reactive with autologous antiid, were randomly selected for use as ligands in a radioimmunoassay, and ascitic fluids containing anti-Ar antibodies from 181 A/J mice were tested as inhibitors. Two of the four idiotypes could not be detected in any mouse other than the donor. The concentration of the idiotype was less than 1 part in 1,250 to less than 1 part in 25,000 of the anti-Ar antibody population; these are minimum values. A third idiotype was detected in 3 of the 181 mice, but at very low concentrations. The fourth idiotype was present in 28% of the mice, again at a low concentration. The data support the existence of a very large repertoire of anti-Ar antibodies in the A/J strain and are consistent with a process of random somatic mutation for generating diversity in hypervariable regions. It is proposed that the cross-reactive idiotype may be controlled by a germ line gene or a gene related to a germ line gene through a small number of somatic mutations; and that the idiotypes that were not detectable in other mice were the products of genes that had undergone extensive mutations, with a low probability of recurrence in other mice.

Complete inhibition of the expression of an idiotype by a mechanism of B-cell dominance.

Mice of the C.AL-20 strain, which express genes controlling CH regions of the AL/N strain on a BALB/c background, normally synthesize antibodies to the p-azophenylarsonate group (anti-Ar antibodies) with an idiotype characteristic of the A strain. The synthesis of the idiotype, as quantitated by a sensitive assay, can be completely inhibited by the transfer of leukocytes from BALB/c mice producing anti-Ar antibodies, which lack the idiotype. A number of control experiments show that the inhibition is not attributable to suppressor T cells and that the synergistic action of such cells is not required. The results indicate that B-cell dominance, mediated by secondary cells, can completely prevent the expression of unprimed cells with receptors of the same specificity. It is uncertain whether this effect is due entirely to selective capture of antigen by the secondary cells, or whether some type of active suppression by B cells is involved.

Presence on idiotype-specific suppressor T cells of receptors that interact with molecules bearing the idiotype.

All A/J mice produce anti-p-azophenylarsonate (anti-Ar) antibodies, some of which share a cross-reactive idiotype. The idiotype can be suppressed by treatment with anti-idiotypic antiserum before immunization, although normal concentrations of anti-Ar antibodies are synthesized. We have previously reported that such suppressed mice, if hyperimmunized and then allowed to rest, contain up to 10% of splenic T cells which form rosettes with autologous RBC coated with Fab fragments of anti-Ar antibodies bearing the idiotype. Our present results indicate that the rosette-forming T cells include the idiotype-specific suppressor T-cell population. The suppressive activity is largely depleted by removal of the rosette-forming lymphocytes, and the rosettes themselves are highly suppressive. The data do not establish whether all of the idiotype-specific rosette-forming cells are suppressor cells. The system may provide a source of large numbers of suppressor cells for further study, and facilitate investigation of the mechanism of generation of idiotype-specific suppressor cells.

Structure of idiotopes associated with antiphenylarsonate antibodies expressing an intrastrain crossreactive idiotype.

We have explored the structural basis of idiotopes associated with the major idiotype (CRIA) of A/J anti-p-azobenzenearsonate antibodies, with emphasis on the regions of contact with anti-idiotypic antibody. The analysis was facilitated by a recent description of the three-demensional structure of the Fab portion of a CRIA-related antibody molecule. Direct binding measurements failed to reveal idiotopes associated exclusively with the L chain. However, the L chain participated in the formation of approximately 80% of the idiotopes recognized by polyclonal anti-Id. This indicates that multiple complementarity-determining regions (CDRs) participate in the formation of idiotopes. The affinity of anti-Id for CDRs on L chains must be appreciable but insufficient to permit direct binding (i.e., less than approximately 10(4) M-1). Approximately 20-35% of polyclonal anti-Id reacted with high affinity with H chains recombined with non-CRIA-related L chains. This interaction was found to involve the D region as well as one or both CDRs in the VH segment, again indicating the contribution of multiple CDRs. It is suggested that a typical idiotope may be similar in size to that of protein epitopes whose three-dimensional structures are known; such epitopes comprise a substantial fraction of the surface area occupied by the CDRs of an antibody. The expression of an idiotope recognized by the mAb AD8, which interacts with the VH segment, was found to be unaffected by major changes in the neighboring D and VL regions. This observation is relevant to efforts to predict three-dimensional structure from the amino acid sequence of CRIA+ molecules.

Primary structure of IgE monoclonal antibodies expressing an intrastrain crossreactive idiotype.

We have obtained amino acid sequences (by mRNA and amino acid sequencing) for two IgE kappa mAb that have specificity for the Ars hapten group and are related to the major idiotypic family, CRIA (crossreactive idiotype A), in the A strain of mouse. One mAb, SE20.2, fully expresses CRIA; the other, SE1.3, possesses some but not all of the characteristic idiotopes. Both IgE proteins contain VH and V kappa segments that are closely related to those associated with CRIA. The D segment of SE20.2 is also typical of CRIA+ mAb, but that of SE1.3 is one amino acid residue longer. Chain recombination experiments indicated that the L chain of SE1.3 is fully capable of supporting CRIA expression. Its deficiency with respect to idiotopes of CRIA was attributed to the extra amino acid in the D region and/or substitutions in the VH segment. A major objective was to ascertain the frequency of somatic mutations in IgE. For the VH segment (amino acids 1-98) of SE20.2, there are only three nucleotide differences and one uncertainty with respect to the nucleotide sequence of the germline gene associated with CRIA. A somewhat higher frequency of substitutions is present in the VH segment of SE1.3. The VK amino acid sequences of the IgE proteins are nearly identical to those of a prototype of the CRIA family, mAb R16.7. The results are discussed with reference to the mechanism of the IgM to IgE switch.