Major Idiotype Research Articles

Deregulation of idiotype expression. Induction of tolerance in an anti-idiotypic response.

The induction of tolerance in an anti-idiotypic response was attempted by in vivo exposure to excess idiotype. Monoclonal immunoglobulin from the anti-p-azobenzenearsonate (ABA) hybridoma R16.7 was used as a representative of cross-reactive idiotype-positive (CRI+) antibodies because this hybridoma protein (HP) shares one or more closely related public idiotypic determinants with the serum CRI in A/J mice. Immunologic unresponsiveness was established by a single injection of the R16.7 idiotype and persisted for at least 6 wk. The level of circulating anti-idiotypic antibodies in tolerized A/J mice was significantly depressed after immunogenic challenge with eigher antigen, ABA-keyhole limpet hemocyanin (KLH), or the idiotype R16.7 HP. Experimental depletion of anti-idiotypic antibodies by tolerization allowed assessment of immunoregulation within this altered idiotype-anti-idiotype network. Deregulation of idiotype expression in tolerized mice challenged with ABA-KLH was manifest with up to 96% of the anti-ABA antibodies cross-reacting with the R16.7 idiotype. This selective enhancement of a major idiotype was accomplished without substantial alteration of the level of the overall anti-hapten response. Both the unresponsiveness established in anti-idiotypic antibody-producing cells and the enhanced synthesis in idiotype-producing cells were stable upon adoptive transfer into lethally irradiated, syngeneic recipients. Finally, previous immunization with the antigen ABA-KLH interfered with the induction of unresponsiveness to the idiotype. This interference is presumed to be mediated by prior activation of anti-idiotypic cells and/or antibody because injection of antigen with tolerogenic idiotype did not abrogate tolerance induction.

Deposition of idiotype-anti-idiotype immune complexes in renal glomeruli after polyclonal B cell activation.

We investigated the possible role of idiotypic interactions in the pathogenesis of the glomerular lesions observed in mice undergoing polyclonal B cell activation. BALB/c mice were studied for the presence of renal deposits of T15 idiotype-anti-T15 idiotype-immune complexes (IC) after injection of bacterial lipopolysaccharides (LPS). The T15 idiotype is the major idiotype of BALB/c mice anti-phosphorylcholine (PC) antibodies, which are cross-reactive with the idiotype of the TEPC-15 myeloma protein. This model was used because T15 idiotype-anti-T15 idiotype IC have been detected in the circulation of BALB/c mice after polyclonal B cell activation. First, an idiotype-specific immunofluorescence technique allowed us to detect T15 idiotype-bearing immunoglobulins in glomeruli from day 6 to day 28 after LPS injection. Second, fluorescein isothiocyanate-conjugated TEPC-15 myeloma protein was found to localize in the glomeruli after in vivo injection 18 d after LPS administration. This renal localization was shown to be idiotype-specific and could be quantified in a trace-labeling experiment. Third, kidney-deposited immunoglobulins of mice injected with LPS were eluted, radiolabeled, and analyzed by radioimmunoassay. Both T15 idiotype-bearing immunoglobulins and anti-T15 idiotype antibodies were detected in the eluates, providing further evidence for a renal deposition of T15 idiotype-anti-T15 idiotype IC. Polyclonal B cell activation is likely to result in a simultaneous triggering of many idiotypic clones and of corresponding anti-idiotypic clones represented in the B cell repertoire. This could lead to the formation of a variety of idiotype-anti-idiotype IC that could participate in the development of glomerular lesions.

Induction of T15-specific suppressor T cells in mice with the CBA/N defect by neonatal injection with anti-T15 idiotype antibody.

Mice with the CBA/N defect (xid) are unresponsive to phosphorylcholine (PC), To determine whether idiotype-specific suppressor T cells can also be generated in these defective mice, defective (CBA/N X BALB/c)F1 male and nondefective (CBA/N X BALB/c)F1 female or (BALB/c X CBA/N)F1 male mice were neonatally injected with antibodies specific for the major idiotype of anti-PC antibody, i.e., anti-TEPC-15 idiotype (T15id) antibody. Suppressor cell activity was examined by co-culturing spleen cells from neonatally treated F1 mice with spleen cells of normal nondefective F1 mice in the presence of antigen. Spleen cells from defective (CBA/NM X BALB/c)F1 mice treated with anti-T15id antibody demonstrated a level of suppressor activity (greater than 83% suppression) comparable to that of similarly treated nondefective F1 mice. This suppression was specific for the T15id of anti-PC response, and a Lyt-1-2+-bearing T cell population appeared to be responsible for the active suppression. These suppressor T cells recognized T15 but not PC, based on a functional absorption test. These results indicate that the CBA/N defects, including the deficiency in the anti-PC response by B lymphocytes and a possible T cell defect, do not influence the generation of T15id-specific suppressor T cells by neonatal injection with anti-T15id antibody.

The immune response to ferredoxin: Characterization of a major idiotype in serum using monoclonal antibody derived by cell fusion

Ferredoxin (Fd) is a low mol. wt protein (6000 d) isolated from Clostridium pasteurianum. This antigen possesses two non-cross-reactive antigenic determinants and engenders a restricted antibody response in selected strains of mice. Immunochemical studies of Fd have shown that antibody responses are confined to two sequences of between five and seven amino acids in extent located at the NH 2- and COOH-termini of the molecule. Serum antibodies from responder strains of mice bind these epitopes in proportions which are regulated by genes mapping in the Ir-region of the H-2 complex. A hybrid cell line secreting monoclonal Fd-binding antibody has been isolated from an immune mouse through fusion with the SP2/0 myeloma cell line. The resulting antibody binds to a single determinant located at the NH 2-terminal of the molecule. An anti-idiotype antibody to this monoclonal antibody was raised in rabbits. After appropriate absorptions, its specificity for the paratopic regions of the hybridoma antibody was established by demonstrating its displacement from reaction with the idiotype by Fd. Analysis of the distribution of the hybridoma idiotype in serum antibodies from congenic mouse strains indicates that it is a major idiotype expressed in different inbred strains sharing identity at the Igh-1 locus.

Mechanisms of idiotype suppression. IV. Functional neutralization in mixtures of idiotype-specific suppressor and hapten-specific suppressor T cells.

Specific tolerance to phosphorylcholine (PC) can be induced in BALB/c mice by neonatal injection with either pneumococcal C-polysaccharide (PnC) or anti-TEPC 15 idiotype (T15Id) antibody specific for the major idiotype (Id) of anti-PC antibody. Spleen cells from these tolerant mice exhibited T cell-mediated active suppression of anti-PC response when they were co-cultured with normal spleen cells. Suppressor cells from the PnC-injected mice appeared to bear either Lyt-1 or Lyt-2 antigens, whereas suppressor cells from anti-Id-treated mice expressed Lyt-2 antigens. Analyses of the specific receptors of these suppressor T cells, based on either adherence to PC and T15-coated petri dishes or cytolysis by rabbit anti-T15Id and monoclonal IgM anti-PC antibody with complement, revealed that receptors of PnC-induced suppressor T cells recognize PC, whereas receptors of anti-Id-induced suppressor T cells react with the T15Id. The possible interaction of the two different types of suppressor T cells was examined by co-culturing normal spleen cells with mixtures of the different suppressor cell types in various cell ratios in the presence of the T-independent PC-antigen, R36a. A brief incubation of anti-Id-induced, T15Id-specific suppressor T cells with PnC-induced, hapten-specific, and T15Id-bearing suppressor T cells resulted in complete cancellation of their suppressor function. These results suggest that idiotype network regulation may also occur among suppressor T cell population.

Relationship of idiotypes of the anti-p-azophenylarsonate antibodies of A/J and BALB/c mice.

It has previously been shown that A/J anti-Ar antibodies contain 2 different families of cross-reactive idiotypes, referred to as the major and minor idiotypes populations. The present report shows that the minor A/J idiotype is related to a major idiotype of BALB/c anti-Ar antibodies. Anti-idiotype directed against the minor A/J idiotype binds 5 to 10% of A/J anti-Ar but an average of about 40% of BALB/c anti-Ar. This BALB/c population corresponds to the major BALB/c anti-Ar idiotype. For individual BALB/c anti-Ar preparations the maximum percentages of antibody bound by anti-id directed to A/J or BALB/c anti-Ar are very similar. Anti-id reactive with the minor A/J idiotypic population suppressed the formation of the BALB/c major idiotype when injected into BALB/c mice. Adsorption experiments showed that only about one-third of the minor A/J population is related to the BALB/c idiotype and that the expression of this idiotype is highly variable in individual A/J sera. Several types of evidence, obtained with hybridoma products expressing the major A/J idiotype, revealed no detectable relationship between the major A/J and BALB/c anti-Ar idiotypes.

Regulation of clones responding to α 1 → 3 dextran: I. Individual variation in the expression of idiotypes

Balb/c mice were immunized with dextran B1355S and assayed for serum antibodies and direct plaque-forming cells. The earliest detectable anti-dextran response occurred in 2-week-old animals. Anti-idiotypic antisera against MOPC-104E and J558 were raised in A/He mice and rendered individual idiotype specific by cross-absorption. When the amounts of MOPC-104E and J558 idiotypes in immune sera and the PFC response of individual mice were analyzed, the ratio of both idiotypes were found highly variable in all tested animals. This observed individual variability in the expression of two major idiotypes in the Balb/c response to dextran B1355S provides an experimental basis for investigating the mechanisms regulating idiotype expression.

Characterization of the NPa idiotype through the analysis of monoclonal BALB/c anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies.

Spleen cells from BALB/c mice were fused with the nonsecreting myeloma line X63.Ag8.6.5.3 seven days after immunization with NP-CG (4-hydroxy-3-nitrophenyl) acetyl-chicken gamma-globulin. The hybrid cell lines obtained were analyzed for heavy and light chain distribution, fine specificity, and idiotype. The majority of monoclonal antibodies possessed either gamma 1 or mu chains. The distribution of L chains among these antibodies was approximately half lambda and half kappa . Thirteen monoclonal antibodies were grown as ascites tumors in mice. Examination of their fine specificity patterns showed that all of the lambda antibodies are heteroclitic and have similar fine specificity patterns. Five of the seven kappa antibodies are also heteroclitic, but their fine specificity patterns are more heterogeneous than those of the lambda antibodies. Polyspecific anti-idiotypic sera directed against pooled primary serum antibody (R a-NPa) or against individual monoclonals were used for idiotypic characterization of the monoclonal antibodies. The Ra-NP bound all of the lambda antibodies but none of the kappa antibodies suggesting that the kappa antibodies may be much more heterogeneous and were therefore not recognized in the presence of the more homogeneous lambda antibodies. Further idiotypic analysis demonstrated that the lambda antibodies, although no two are identical, are a very homogeneous group of antibodies which cross-react with one another but not with the kappa antibodies. Some, but not all, of the kappa antibodies cross-react with each other although none are cross-reactive with the lambda antibodies. Because the lambda-associated idiotype is recognized by the R a-NPa and its characteristics are similar to that of the C57BL/6 major idiotype (NPb), it is referred to as NPa. There may be a second major idiotype associated with at least some of the kappa antibodies.

Compensation for idiotype suppression. I. Acquirement of ability to compensate for TEPC-15 idiotype suppression in mice during the early neonatal period.

Neonatal injection of BALB/c mice with antibodies specific for the idiotype of TEPC-15 myeloma protein (T15id), which is serologically identical to the major idiotype of anti-phosphorylcholine (PC) antibody, renders the recipients completely unresponsive to PC. C57BL/6, (BALB/c X C57BL/6)F1 and C.B20 mice, similarly treated with anti-T15id antibody, also displayed tolerance to PC although they were relatively more resistant (8-13%) than BALB/c mice (less than 2% control response). When anti-T15id antibody was injected into 15-day-old neonates, the resistance to the tolerance in C57BL/6 and C.B20 mice was much more apparent (up to 80% of the control response) in contrast to that in BALB/c mice, which was not significant. Adoptive transfer of spleen cells from idiotypically suppressed BALB/c mice into 20-day-old, normal C. B20 mice resulted in suppression of T15id but not in tolerance to PC, due to increased production of non-T15id-bearing anti-PC antibody. These results suggest that the ability of clonal compensation for T15id suppression is acquired during early life (2-10 days), under the influence of gene(s) associated or linked with the Igh.

Genetic control of the immune response to the L‐Glu60‐L‐Ala30‐L‐Tyr10 (GAT) terpolymer V. Three types of idiotypic specificities on BALB/c anti‐GAT antibodies

Three types of idiotypic specificities compose the major idiotype of anti-poly (L-Glu60-L-Ala30-L-Tyr10) (GAT) antibodies from BALB/c mice (idiotype termed GAT-715). Assays have been designed to analyzed and study the distribution of these specificities. The highly conserved idiotypic specificity (h.c.GAT) has been assayed by the binding of serum 715-7A4 to radiolabeled rat anti-GAT antibodies. Guinea pig and mouse anti-GAT antisera all express the same h.c.GAT specificity. The public specificity (p.GAT) has been shown to be present in an identical form in all anti-GAT antisera from all strains of mice studied. The assay used for p.GAT was the binding of serum 715-7A4 to C57BL/6 anti-GAT antibodies that express only p.GAT. Finally, the strain-restricted specificity s.r.GAT has also been investigated by radioimmunoassay; this specificity is expressed only by strains BALB/c, BALB/b, BUB/J, DBA/2, DBA/1 and ATL. This expression is independent of known allotypic markers. However, the expression of the s.r.GAT specificity of BALB/c mice follows the genetic distribution of VH genes of BALB/c origin indicating that s.r.GAT can be considered as a genetic marker of some VH gene(s) involved in the specific immune response to the GAT terpolymer.

Antigen-specific molecules from murine T lymphocytes and T cell hybridomas

Strain A/J mice immunized with azobenzenearsonate-isologous IgG conjugates made strong antigen-specific suppressor T cell responses. The ABA-specific T cells‡ were enriched by affinity purification and an average of 59% of such cells expressed the major idiotype (CRI) associated with anti-ABA specificity in A/J mice. The cellular proteins of affinity enriched, ABA-specific T cells were biosynthetically radiolabelled and ABA-binding molecules from cell lysates were purified by affinity chromatography. SDS-PAGE analysis of T cell-derived ABA-binding molecules revealed a major, invariant component with an apparent mol. wt of about 92,000 daltons (p92). This protein is susceptible to proteolytic degradation as its recovery from cell lysates required the presence of protease inhibitors. Serological analyses of p92 failed to reveal classical immunoglobulin heavy or light chain determinants or Ia markers. Evidence is presented that this class of molecules possesses suppressor regulatory activity, probably in conjunction with a noncovalently associated smaller polypeptide chain. Affinity-enriched ABA-specific suppressor T cells were hybridized with the AKR tumor line BW 5147. Ten hydridomas were obtained which formed rosettes with ABA-SRBC. Rosette formation was inhibitable with soluble ABA-protein conjugates. Three of the lines stained for the CRI in an immunofluorescence assay. Culture supernatants from these lines were tested for modulation of an in vitro primary anti-ABA response and exerted a strong antigen-specific suppressor activity. Thus, hybridoma cell lines have been obtained which express an ABA-specific receptor and secrete antigen-specific regulatory factors. They therefore offer great promise as sources of sufficient material for chemical characterization of antigen specific T cell molecules.

Two major idiotypes in mouse anti-(4-hydroxy-3-nitro-phenyl)acetyl (NP) antibodies are controlled by "allelic" genes.

Anti-NP [(4-hydroxy-3-nitro-phenyl)acetyl)] antibodies from the primary response of BALB/c mice contain a major idiotype NP-a that is controlled by an allotype-linked gene VHNPa. A new strain distribution pattern for a VH marker was discovered: strains BALB/c, RF, DBA, RIII and SM were positive, and strains C57BL/6, SJL, CBA, A/J, CE, AKR and NZB were negative for the idiotype NP-a. NP-a idiotype chared many characteristics with previously described idiotype NP-b and, therefore, allelism was suggested between them. Both idiotypes have lambda light chains, both seem to be conservative in evolution, both are predominant early in the antibody response. Later during the response, antibodies become increasingly idiotype-negative while at the same time, homogeneous isoelectric focusing patterns become very heterogeneous. The study of VH recombinant strains supported the concept of allelism because in every case studied, the idiotype were mutually exclusive. (C57BL/6 X BALB/c)F1 mice expressed idiotypes NP-a and NP-b regularly and in approximately equal amounts.

Genetic control of the immune response to the terpolymer l-glutamic acid 60- l-alanine 30- l-tyrosine 10 (GAT)—IV : Heterogeneity of idiotype GAT-715

A major idiotype GAT-715 was defined in mice by a rabbit anti-idiotypic antiserum raised against BALB/c anti-poly (Glu 60-A!a 30-Tyr 10) (GAT) antibodies. Corresponding idiotypic specificities were found to be present on anti-GAT antibodies from responder and non responder mice regardless of their allotypic markers. Some of the idiotypic specificities that we have termed highly conserved idiotypic specificities are expressed by rat and guinea pig after immunization with GAT. In this paper we report experiments that show that idiotype GAT-715 can be further analysed and subdivided into public and private idiotypic specificities. All mice tested express the public specificities that represent 80% of idiotype GAT-715. Some strains also express private specificities; no genetic linkage is observed between the inheritance of heavy chain allotypic markers and private specificities. In addition, public and highly conserved specificities have been compared. A structural model for GAT-715 idiotype is discussed.

Neonatal expression of a cross-reactive idiotype associated with anti-phenylarsonate antibodies in strain A mice.

AbstractData are presented which indicate that B cells expressing the cross‐reactive idiotype associated with anti‐p‐azophenylarsonate antibodies of A/J or allotype‐congenic C.AL‐20 mice are present in neonatal mice. Data were obtained by direct immunization of neonatal mice or by adoptive transfer of cells from neonatal C.AL‐20 mice into idiotype‐negative BALB/c recipients. These findings, together with those previously reported for antibodies to phosphocholine in BALB/c mice, support the generalization that major cross‐reactive idiotypes may usually be found early in the life‐span of a mouse. The contrast between the frequency of appearance of the major cross‐reactive idiotypes and “private” idiotypes with the same antigen‐binding specificity, which are found after immunological suppression of the major idiotype, is consistent with the possibility that cross‐reactive idiotypes are the product of germ line genes or minor somatic variants of germ line genes while private idiotypes arise through somatic diversification. Alternatively, regulatory mechanisms may account for the striking difference. The present results are therefore consistent with the early expression of germ line genes controlling the major idiotype or of corresponding regulatory genes. The average concentration of idiotype, per unit weight of anti‐phenylarsonate antibody, is somewhat lower in neonatal than in adult A/J mice indicating that the major idiotype does not account for all of the antibody produced in response to this hapten in the neonate.

Production of auto-anti-idiotypic antibody during the normal immune response to TNP-ficoll. I. Occurrence in AKR/J and BALB/c mice of hapten-augmentable, anti-TNP plaque-forming cells and their accelerated appearance in recipients of immune spleen cells

Attempts were made to elucidate the cause of the downward regulation of the splenic plaque-forming cell (PFC) response in AKR/J and BALB/c mice between days 4 and 7 after a single intravenous injection of 2,4,6,trinitrophenyl- lys-Ficoll(TNP-F). AKR/J spleen cells, taken 7 d after injection of TNP-F, were transferred, together with TNP-F, into normal AKR/J mice. The day-3 or - 4 PFC response of the recipients was much lower than that of recipients of normal cells. However, the suppression was only apparent because the presence of 10(-8)-10(-7) M 2,4,6-trinitrophenyl-epsilon-amino-n-caproic acid (TNP- EACA) (or 10(-7)-10(-6) M 2,4,-dinitrophenyl-epsilon-amino-n-caproic acid) in the PFC assay caused a dramatic increase in observed PFC, averaging 298 percent on day 3 and 122 percent on day 4. Recipients of normal cells showed no such hapten-augmentable PFC. T-depleted immune spleen cells did not cause any apparent suppression of the response to TNP-F, but hapten-augmentable PFC in recipient spleens were again prevalent. Suppression of the PFC response, as well as hapten-augmentable PFC, were seen after transfer of immune serum. It was postulated that hapten augmentation of PFC was caused by displacement of auto-anti-idiotypic antibody from the surface of blocked antibody- synthesizing cells. Further studies showed that such hapten-augmentable PFC occurred in the spleens of a large percentage of both AKR/J and BALB/c mice examined after day 4 of the primary response to TNP-F. Thus, it was hypothesized that the downward regulation of the magnitude and, possibly, also of the heterogeneity of the splenic-PFC response was due to an auto-antibody response to one or more major idiotypes of the anti-TNP response.