Idiotype-specific T helper cells are required to induce idiotype-positive B memory cells to secrete antibody.

We have tested the proposition that induction of certain sets of B cell clones to produce antibody requires a signal from T helper cells that recognize idiotypic determinants expressed on Ig receptors of the relevant B cell clones. The approach is based on the analysis of T cell populations required to induce B cells to secrete anti-arsonate antibodies that are marked by a cross-reactive idiotype (CRId). crid+ anti-azophenyl arsonate (Ar) antibodies are produced in A/J strain mice after immunization with Ar keyhole limpet hemocyanin and represent 20--70% of the total anti-Ar antibody response. These studies indicate that antibody secretion by idiotype+ B memory cells requires two signals: one provided by "carrier"-specific Ly-1 cells, and a second delivered by idiotype-specific Ly-1 cells. Both signals are required for optimal induction of idiotype+ B memory clones.     

Idiotype suppression by maternal influence.

In BALB/c mice, antibodies to the alpha-(1-3) glucosidic linkage of some dextrans (Dex) carry the idiotype of the BALB/c myeloma protein J558. Both specific antibody and idiotype are inherited in a dominant fashion, linked to the immunoglobulin (Ig) (heavy chain) allotype Igla of BALB/c mice (Eur. J. Immunol. 1975. 5: 775). In F1 hybrid mice from the parent strains SJL and BALB/c, we were able to suppress the expression of anti-Dex antibodies by immunizing prospective SJL mothers to the J558 idiotype. The state of suppression in the progeny was ascertained by immunization with Dex, and tests for the following were carried out: (a) antibodies specific for Dex; (b) inhibition of such antibodies (if present) by antiidiotypic serum to protein J558; (c) presence of the J558 idiotype; and (d) concentration of lambda1 chains (which are associated with the 558 idiotype) in the serum. SJL mothers, once immunized, conferred suppression upon several successive litters, spanning a period of 4-5 months. Suppression in F1 progeny animals lasted for 16 weeks or more. Spleen cells from suppressed F1 mice which had neither been treated with Dex nor with J558 protein, were able to confer suppression to further F1 newborn mice.     

In vivo clearing of idiotypic antibodies with antiidiotypic antibodies and their derivatives

At immunolocalization of experimental tumors, idiotypic monoclonal antibodies, such as TS1 against cytokeratin 8, can be used to carry and deposit in vivo terapeutics in the tumor. These carriers also remain in the circulation and may cause negative side-effects in other tissues. In this report, several derivatives of the antiidiotypic antibody alphaTS1 were produced and tested for their clearing capacity of the idiotypic carrier antibody TS1. Intact monoclonal alphaTS1, scFv of a alphaTS1 and alphaTS1 Fab'2 and fragments were produced by recombinant technology or by cleavage with Ficin. The scFv was tailored by use of the variable domain genes of the light and heavy chain from the hybridoma clone in combination with a (Gly4Ser)3-linker, followed by expression in E. coli. When tested for clearing capacity, the intact divalent antiidiotypic IgG was found to be the most efficient. The divalent and the monovalent Fab fragment also demonstrated significant clearing, but lower than the intact antiidiotypic IgG. The alphaTS1 scFv antibody when injected separately was not found to clear the idiotype, but could do so when preincubated with the idiotype. Rapid excretion and in vivo instability of this low molecular weight antibody fragment may be the major reasons. Similar results were obtained when the system was reversed and the 131I-labeled antiidiotype IgG was cleared with the idiotype fragment. It is concluded that both intact antiidiotypic IgG, and Fab'2 fragments are able to clear the idiotypic antibodies. The experimental data support the conclusion that the Fc parts from both the idiotype and the antiidiotype may contribute to this elimination.     

Detection of autologous antiidiotypic antibody-forming cells by a modified enzyme-linked immunospot (ELISPOT).

We describe here the utilization of a modified enzyme-linked immunospot assay (ELISPOT) in order to detect an autologous antiidiotypic response in mice at the level of single antibody-forming cells (AFC). Severals assays have been routinely used to detect anti-Id producing cells; however, these approaches often produce contrasting data. We present results obtained with the modified ELISPOT, using as a model system the antiidiotypic response in mice after immunization with a vaccine from Streptococcus pneumoniae R36a, expressing the immunodominant epitope phosphorylcholine (PC). The response to PC is mediated by a large fraction of antibodies bearing the public idiotype T15. Mice of different genetics make up were immunized with a single injection of the vaccine. We observed that one mouse strain (D1.LP) out of three was able to mount a significant anti-T15 response during the primary anti-phosphorylcholine response. BALB/c and C57BL/6 mice did not produce significant levels of anti-T15 antibody following a single injection of the antigen. In contrast, BALB/c mice which were repeatedly stimulated showed a specific anti-Id response. Experimental controls were performed using either specific anti-T15 monoclonal antibodies (mAb) or splenocytes from mice immunized with TEPC15 myeloma protein in complete Freund's adjuvant.     

Functional relationship between T15 and J558 idiotypes in BALB/c mice.

In inbred strains of mice, antiphosphorylcholine (PC) and anti-alpha 1,3 dextran (DEX) antibodies are structurally distinct from each other and have been shown to exhibit noncross-reactive antigen binding and idiotypic specificities. However, the prototype anti-PC and anti-DEX antibodies, TEPC15 and J558, respectively, were shown to be connected via a common autoantiidiotypic monoclonal antibody isolated from newborn BALB/c mice. The capacity of various monoclonal anti-PC and anti-DEX antibodies as well as the antigens PC and DEX to modulate T15 and J558 idiotypes in BALB/c mice was tested by their administration to newborn mice. Anti-PC antibodies of the T15 idiotype injected into 2-4-day-old mice, at a time when T15+ anti-PC precursors develop in BALB/c mice, suppressed the anti-PC response of these mice at 6 weeks of age. Similarly, J558 antibodies injected into 8-12-day-old mice, at a time when J558 precursors normally develop, suppressed the response to DEX. As a further demonstration of this connectivity, the injection of J558 into 4-day-old mice led to a down modulation of T15 idiotype, whereas both T15 and a minor idiotype-expressing antibody M167 when injected into 8-12-day-old mice caused a reduction in expression of the J558 idiotype. As predicted from in vitro analysis, injection of anti-PC antibodies of the M167 idiotype 2 to 4 days after birth enhanced the subsequent response to PC. However, anti-PC antibodies expressing another minor M603 idiotype did not affect the PC response. The results parallel the in vitro enhancement of M167 antibodies but not M603 on T15 binding to antiidiotype in vitro. Similarly, anti-DEX antibodies expressing the M104E idiotype had no detectable effects on the capacity to respond to PC or DEX or on the expression of T15 and J558 idiotypes as adults. Exposure of newborn mice to PC led to a dramatic reduction in the response to DEX as adults, whereas exposure to DEX at this stage of development had no effect on response to PC as adults. Collectively, these observations provide evidence for a complex functional connectivity between T15 and J558 idiotype-bearing B cells during ontogeny and extend our previous observations that development of these idiotypes is regulated by idiotype-directed interactions between B cells or their immunoglobulin products.     

Effect of anti-idiotypic antibody on production of anti-DNA antibodies by splenocytes in lupus prone mice

It has been suggested that production of autoantibodies is regulated by idiotype-antiidiotype network. In this study, we examined modulatory effect of the antiidiotypic antibody on the synthesis of anti-DNA antibodies by New Zealand black/New Zealand white F1 mice (B/W F1) splenocytes. The antiidiotypic antibodies were prepared by immunization of a monoclonal anti-DNA antibody derived from B/W F1 to rabbits. The prepared antiidiotypic antibody had specificity to the antigen binding site of anti-DNA antibodies. B/W F1 splenocytes were adjusted to 1 X 10(6) cells/ml and cultured in 1.0 ml aliquots in the presence of varying concentrations of the antiidiotypic antibody for 48 hours. The cells were then washed three times, resuspended in RPMI1640 containing 10% fetal calf serum and cultured again. On days 3 and 7 of the culture, the supernatants were harvested and secretion of anti-DNA antibodies was measured by ELISA. Production of anti-DNA antibodies by B/W F1 splenocytes was suppressed by pretreatment with the antiidiotypic antibody. When the concentration of antiidiotypic antibody was 1 microgram/ml, anti-DNA activity of the supernatants decreased 50%, compared with control on day 3, but the effect was reduced on day 7. The treatment of antiidiotypic antibody did not affect the proliferation and viability of B/W F1 splenocytes. The results indicated that anti-DNA antibodies synthesis were regulated by idiotype-antiidiotype network and could be manipulated by the antiidiotypic antibody.     

Functional Relationship Between T15 and J558 Idiotypes in BALB/c Mice

In inbred strains of mice, antiphosphorylcholine (PC) and anti-α1,3 dextran (DEX). antibodies are structurally distinct from each other and have been shown to exhibit noncrossreactive antigen binding and idiotypic specificities. However, the prototype anti-PC and anti-DEX antibodies, TEPC15 and J558, respectively, were shown to be connected via a common autoantiidiotypic monoclonal antibody isolated from newborn BALB/c mice. The capacity of various monoclonal anti-PC and anti-DEX antibodies as well as the antigens PC and DEX to modulate T15 and J558 idiotypes in BALB/c mice was tested by their administration to newborn mice. Anti-PC antibodies of the .T15 idiotype injected into 2-4-day-old mice, at a time when T15 anti-PC precursors develop in BALB/c mice, suppressed the anti- PC response of these mice at 6 weeks of age. Similarly, J558 antibodies injected into 8-12-day-old mice, at a time when J558 precursors normally develop, suppressed the response to DEX. As a further demonstration of this connectivity, the injection of J558 into 4-day-old mice led to a down modulation of T15 idiotype, whereas both T15 and a minor idiotypeexpressing antibody M167 when injected into 8-12-day-old mice caused a reduction in expression of the J558 idiotype. As predicted from in vitro analysis, injection of anti-PC antibodies of the M167 idiotype 2 to 4 days after birth enhanced the subsequent response to PC. However, anti-PC antibodies expressing another minor M603 idiotype did not affect the PC. response. The results parallel the in vitro enhancement of M167 antibodies but not M603 on T15 binding to antiidiotype in vitro. Similarly, anti-DEX antibodies expressing the M104E idiotype had no detectable effects on the capacity to respond to PC or DEX or on the expression of T15 and J558 idiotypes as adults. Exposure of newborn mice to PC led to a dramatic reduction in the response to DEX as adults, whereas exposure to DEX at this stage of development had no effect on response to PC as adults. Collectively, these observations provide evidence for a complex functional connectivity between T15 and J558 idiotype-bearing B cells during ontogeny and extend our previous observations that development of these idiotypes is regulated by idiotype-directed interactions between B cells or their immunoglobulin products.     

Detection of antibody, idiotype, and anti-idiotype forming cells by in situ immunocytochemical staining

Methods are described for the immunocytochemical staining of cryostat sections of lymphoid tissue with enzyme conjugates of antigen, idiotype (Id) and anti-idiotype. Results established this as a useful approach, for simultaneously detecting Id and anti-Id antibody forming cells (AFC) in situ. As a model, the 5AF6 Id family associated with the BALB/c mouse antibody response against the p-azophenyl-arsonate (Ar) epitope was examined by two-color immunocytochemical staining, allowing the simultaneous detection of both Id+ and Id- anti-Ar AFC. Spleens from mice secondarily immunized with Ar antigen but not normal mice contained anti-Id AFC stained with the 5AF6 Id but not with another immunoglobulin of the same isotype. A sequential staining method was developed which allowed the detection of both Id and anti-Id AFC in the same tissue, thus providing a means of examining Id and anti-Id antibody networks in intact lymphoid tissues.     

Structural studies on induced antibodies with defined idiotypic specificities--VIII. NH2-terminal amino acid sequence analysis of the heavy and light chain variable regions of monoclonal anti-para-azophenylarsonate antibodies from A/J mice differing with respect to a cross-reactive idiotype.

Monoclonal antibodies with specificity for the hapten p-azophenyl arsonate (Ar)^* were generated by somatic cell hybridization of A/J spleen cells and the non-secreting cell line, Sp2/0-Ag14, derived from a Balb/c myeloma. Of four hybridoma products examined, only one bore the previously described A/J anti-Ar cross-reactive idiotype (CRI). The heavy and light polypeptide chains of this CRI positive molecule along with one of of the CRI negative molecules were subjected to amino-terminal sequence analysis and compared with the induced CRI positive antibodies produced upon immunization of A/J mice. The results confirm the extremely restricted expression of V_H and V_L frameworks in the A/J anti-Ar population. Unexpectedly, when the hybridoma proteins were compared to the induced population of antibodies, from one to three amino acid substitutions were detected within the first framework segment (1–30) of each chain of both hybridoma products. The complete covalent structure of two monoclonal antibodies known to differ serologically only with respect to idiotype should prove useful in defining the exact structural basis of an idiotypic determinant.     

Idiotype suppression. I. Influence of the dose and of the effector functions of anti-idiotypic antibody on the production of an idiotype

Parameters that influence the effect of passively administered anti-idiotypic antibody on the expression of idiotype-positive and of idiotype-negative antibodies were investigated in adult A/J mice immunized with Group A streptococci. The results suggest: 1. idiotype-positive antibodies are eliminated from the spectrum of humoral antibodies as a consequence of the elimination of the idiotype-forming cell precursors. 2. The heterogeneity of the idiotype-negative compensatory response of individual suppressed mice is as restricted as that of the idiotype-positive normal response. The isoelectric spectra of idiotype-negative antibodies are individually specific, whereas the isoelectric spectra of idiotype-positive antibodies are individually specific, whereas the isoelectric spectra of idiotype-positive antibodies are shared between individual mice. 3. Suppression is transient in mice treated with low and with high doses of anti-idiotypic antibody, but is maintained in mice treated with intermediate doses. 4. Suppression is mediated by complement and macrophage fixing (guinea pig IgG₂) anti-idiotypic antibody only. IgG₁ anti-idiotypic antibody appears to have an enhancing effect on the production of the idiotype. The results are discussed with respect to possible regulatory functions of idiotype-anti-idiotype interactions in the immune response.     

Production of BALB/c Anti-Idiotypic Antibodies Against the BALB/c Myeloma Protein 315 Does Not Require an Intact Ligand-Binding Site

To determine whether the ligand-binding site of the BALB/c myeloma protein 315 is essential for the anti-idiotypic response in syngeneic animals, 17 BALB/c mice were immunized with M315 that had been affinity-labeled with bromo-acetyl-DNP-L-lysine (BADL). Essentially all the active sites of M315 were blocked by the affinity label. Fourteen mice produced antibodies that reacted with an idiotypic determinant localized in the Fv fragment of M315, but this idiotype was not part of the DNP-lysine-binding site, and it was absent from L315 and H315 chains. Two groups of BALB/c mice were immunized with nonaffinity-labeled M315, to determine whether the same idiotype was recognized with this immunogen. All animals in the group that received the most prolonged immunization produced antibodies that could be divided in two populations: about 75% were directed against the site-associated idiotype, and the rest reacted with the nonsite idiotype. The other group produced antibodies exclusively specific for the site. Thus, the site-associated idiotype of M315 is not essential for the antibody response of BALB/c mice against M315, and M315 carries at least two different idiotypes that can be recognized by B cells of syngeneic animals.     

The assessment of anti-idiopathic antibodies as effective immunoregulatory probes in vivo.

In order to increase our understanding of the potential to use anti-idiotypic antibodies as immune modulators in vivo, we extensively analysed influences induced by one such antibody (anti-Id-l) following its administration to animals of different ages, genetic backgrounds, and immunological histories. Id-l is an inter-strain idiotype associated with rat anti-Group A streptococcal carbohydrate antibodies. The intraperitoneal (i.p.) injection of anti-Id-l antibodies, prepared against Id-l+ antibodies from an HPR rat could effectively induce long-term idiotype suppression in all tested strains of rats, regardless of the age at the time of treatment, the RTl haplotype or IgG2c or k-chain allotype. Total anti-streptococcal antibodies were not suppressed by this treatment. Although long-term suppression could be induced at any age, the percentage of animals suppressed following neonatal injections was consistently less than that following adult injections of anti-idiotypic antibodies. In addition, neonatal injections of anti-Id-l or Id-l with anti-Id-l appeared to enhance Id-l production in a minority of the animals. Similar treatment of adult animals never increased Id-l synthesis, suggesting that cells associated with enhanced Id-l production in older animals are either refractory to activation-differentiation signals and/or are sequestered and no longer accessible by i.v. or i.p. routes of administration of the probe. Auto-anti-Id-l immunity induced by immunizing adult rats with heavy, light, F(ab')2 fragments or whole IgG molecules could also induce an Id-l suppressed state. We were not able to induce significant Id-l suppression if animals were immunized with antigen prior to the injection of anti-Id-l. There was evidence, however, that such treatment might lead in time to the development of some idiotype specific suppression.     

Structural studies on induced antibodies with defined idiotypic specificities—VIII.: NH2-terminal amino acid sequence analysis of the heavy and light chain variable regions of monoclonal anti-p-azophenylarsonate antibodies from A/J mice differing with respect to a cross-reactive idiotype

Monoclonal antibodies with specificity for the hapten p-azophenyl arsonate (Ar)^* were generated by somatic cell hybridization of A/J spleen cells and the non-secreting cell line, Sp2/0-Ag14, derived from a Balb/c myeloma. Of four hybridoma products examined, only one bore the previously described A/J anti-Ar cross-reactive idiotype (CRI). The heavy and light polypeptide chains of this CRI positive molecule along with one of of the CRI negative molecules were subjected to amino-terminal sequence analysis and compared with the induced CRI positive antibodies produced upon immunization of A/J mice. The results confirm the extremely restricted expression of V_H and V_L frameworks in the A/J anti-Ar population. Unexpectedly, when the hybridoma proteins were compared to the induced population of antibodies, from one to three amino acid substitutions were detected within the first framework segment (1–30) of each chain of both hybridoma products. The complete covalent structure of two monoclonal antibodies known to differ serologically only with respect to idiotype should prove useful in defining the exact structural basis of an idiotypic determinant.     

T-cell dependent response to immune complexes abrogates B-cell unresponsiveness to pneumococcal cell wall polysaccharide.

Although > 90% of B cells from M167 (mu, kappa) immunoglobulin transgenic (Tg) mice express surface immunoglobulin that binds phosphorylcholine (PC), we found that these mice are unresponsive to immunization with pneumococcal cell wall polysaccharide (PnC), a type II thymus-independent antigen that contains PC. However, when the PnC antigen was presented as a complex with TEPC-15 or McPC-603 antibodies (which are specific for PnC), a vigorous immune response occurred in which the Tg mice produced 10-50-fold more anti-PnC antibody than when immunized with antigen alone. Interestingly, MOPC-167, which expresses the VH and VL regions used to encode the transgene antibody, was found to be a relatively poor 'carrier' for PnC, eliciting a weak anti-PnC antibody response in M167 (mu, kappa) Tg mice. In vivo administration of anti-CD4 antibody dramatically reduced the response to TEPC-15/PnC complexes, suggesting that the response is mediated by immunoglobulin (idiotype)-dependent helper T cells. The results indicate that unresponsiveness to PnC is due not to tolerance of the transgenic B cells but rather to the lack of T-cell help resulting from T-cell tolerance to the transgene-encoded idiotype.     

Phosphorylcholine-binding hybridoma proteins of normal and idiotypically suppressed BALB/c mice. I. Characterization and idiotypic analysis

The immune response of BALB/c mice to phosphorylcholine (PC) is dominated by an antibody bearing the idiotype of the myeloma protein TEPC 15 (MP T15). Anti-PC antibody of a different idiotype can be elicited in BALB/c mice by neonatal suppression with an anti-T15 idiotypic antiserum (anti-Id-T15). In an attempt to further characterize the immune response to PC, spleen cells of normal and idiotypically suppressed mice have been fused with the myeloma line X63-Ag8, and hybrid lines secreting anti-PC antibodies have been isolated. The antibodies were characterized by two-dimensional gel electrophoresis and their affinity for Pneumococcus pneumoniae was measured. Using both an A/J and a rabbit anti-Id-T15 serum, we could show that 4 out of 5 hybridomas obtained from normal BALB/c spleen cells secreted antibodies which, within the limits of the analysis, were idiotypically and structurally indistinguishable from MP T15 (T15⁺). Hybridomas obtained from suppressed BALB/c mice, on the other hand, secreted anti-PC antibodies which differed idiotypically from MP T 15: of the six lines, two were negative for T15 idiotopes (T15^?), while four showed some cross-reactivity with the T15 idiotype (T15^cr). All six, however, showed structural differences in one or more immunoglobulin chains. Inhibition of anti-PC plaque-forming cells induced in neonatally suppressed mice revealed that the idiotypic spectrum of the hybridoma proteins was representative of clones arising under such conditions in BALB/c mice. The majority of the anti-PC antibodies in idiotypically suppressed mice still bear idiotopes resembling MP T15. Only a minority, less than 20%, are truly T15^?.     

Two structurally distinct and independently regulated idiotypic families associated with the A/J response to azophenylarsonate

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, Id^CR, 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 Id^CR-associated determinants revealed a second set of closely related molecules. The second idiotype family, Id^36–60, 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 Id^36–60 as Id^CR. A/J mice can be suppressed for subsequent expression of either idiotype by neonatal injection of anti-Id^CR or anti-Id^36–60 antisera. Suppression of Id^CR does not affect the production of Id^36–60 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.     

Idiotypic cross-reaction between MRL autoantibodies and a BALB/c myeloma

Y2, a monoclonal anti-Sm antibody of MRL origin, demonstrates an idiotype commonly expressed in autoimmune MRL mice, although not necessarily associated with anti-Sm activity. To identify non-anti-Sm antibodies with this common idiotype, a rabbit anti-Y2 anti-idiotypic antiserum was tested for activity with other MRL hybridoma products (HP) as well as BALB/c myelomas. Idiotypic cross-reactivity was thus demonstrated for Y2 and the product of the BALB/c fusing cell line 45.6TG1.7 (M45), an antibody without known antigen binding activity. This determinant was denoted the Y2-M45 idiotype and its presence in serum and other antibodies tested by an inhibition ELISA. The idiotype was demonstrated on some, but not all, MRL HP's tested and was not confined to antibodies of a unique specificity. The idiotype was also present in sera of some normal as well as autoimmune strains of mice with highest levels in two strains bearing the lpr gene. These results indicate that idiotypes of anti-Sm antibodies are not exclusive to the pathologic setting but may be found commonly expressed in mice with antibodies of a variety of binding activity.     

Anti-idiotype to monoclonal anti-swine SLA antibody detects a common idiotype shared by mouse anti-SLA sera and elicits an anti-SLA activity

Heterologous anti-idiotypic reagents have been prepared against a BALB/c anti-swine MHC (SLAd) monoclonal antibody (74-11-10) in order to test for possible interspecies idiotypic cross-reactions of anti-MHC antibodies. Using these reagents to examine xenoantisera produced in BALB/c mice immunized with swine SLAdd peripheral blood lymphocytes, all animals tested were found to express detectable levels of the 74-11-10 idiotype (Id). The Id could also be detected in one out of five BALB/c mice immunized with swine SLAcc PBL. Swine anti-SLAdd alloantibodies were also tested, but failed to show detectable levels of the 74-11-10 Id. The in vivo administration of anti-idiotypic reagents to BALB/c mice induced detectable levels of 74-11-10 Id positive antibodies that bound specifically to SLAdd PBL. Similar treatment of SLAgg swine (recombinant swine expressing the class I MHC molecules of c) with anti-idiotypic antibodies failed to induce anti-SLAd antibody activity. These results thus indicate that 74-11-10 represents a shared idiotype of BALB/c anti-SLAdd antibodies. The presence of 74-11-10 Id in one mouse immunized with SLAcc PBL suggests that the failure of pigs to express the 74-11-10 Id following treatment with anti-idiotypic antibodies may be the result of tolerance rather than absence of the relevant variable region gene(s).     

Biological and serological comparison of syngeneic and allogeneic anti-idiotypic antibodies

The majority of BALB/c antibodies with specificity for the (1→3)-glucosidic linkage of dextran B1355, fraction S (-dextran), share an idiotype crossreactive with the -dextran-binding BALB/c myeloma proteins J558 and MOPC104E. This crossreactive idiotype (IdX558) is denned by an allogeneic antiidiotypic antiserum raised in mice of strain A/HeJ against J558 myeloma protein (Carson & Weigert, 1973). Allogeneic anti-IdX558 antisera induce a long lasting complete suppression of the total anti--dextran immune response when given neonatally to BALB/c mice. Mice which recover from suppression fail to express the IdX558. In contrast, when BALB/c mice are immunized by use of J558 myeloma protein, no measurable effect on the subsequent total anti--dextran immune response or on the expression of IdX558 is found in mice making anti-J558 themselves nor in those mice which received the syngeneic anti-J558 idiotype antibody neonatally. In addition, no significant alteration of the anti--dextran response can be detected in BALB/c mice which were immunized against MOPC104E protein prior to -dextran immunization. This different suppressive capacity may be explained by different specificities of the allogeneic and syngeneic antiidiotype antisera. Hemagglutination and isoelectric focusing studies of the antisera show that BALB/c mice fail to produce antibodies against the crossreactive idiotype whereas in the allogeneic situation the anti-IdX558 is the overwhelming one. Syngeneic anti-idiotypic antibodies react only with the corresponding myeloma protein used for immunization, thus defining individual idiotypes (IdI558 and IdI104). IdI558 and IdI104 are expressed by every BALB/c mouse in response to -dextran but as a minor component of their idiotypic spectrum.     

The Influence of T Cells on the Immunoglobulin Repertoire and the Affinity Maturation of the Immune Response Against Dextran B512 in C57BL/6 Mice

A collection of hybridomas from C57BL/6 mice producing antibodies to dextran B512 was analysed and found to reflect the immune response in vivo with regard to immunoglobulin class expression, T cell dependency and antibody affinity. IgM-, IgG-3, and IgG-2b, and IgA-producing hybridomas were found. IgG3-producing hybridomas were obtained from nude mice, indicating T cell independent IgG3 synthesis. All monoclonal antibodies were of kappa light chain. A major anti-dextran idiotype was expressed in many monoclonals. Secondary immune responses to dextran were also suppressed at the hybridoma cell level. However, hybridomas from secondary responses produced antibodies expressing the major idiotype, suggesting that anti-idiotype mediated suppression was not responsible for the reduced secondary response. Most monoclonals belonged to the VHJ558 family, but the IgG3-producing hybridomas showed a preferential use of genes from the VHX24 family. All monoclonals were directed against internal structures of the dextran molecule. The affinity for dextran of the IgG antibodies produced in secondary immune responses was drastically increased, even when the mice were immunized with thymus-independent forms of dextran, indicating that T helper cells need not be involved in affinity maturation of the immune response.