Purification of functional, determinant-specific, idiotype-bearing murine T cells.

Strain A/J mice immunized with azobenzenearsonate (ABA)-mouse IgG conjugates develop suppression for anti-trinitrophenyl(TNP) responses to doubly conjugated (ABA,TNP) proteins. This suppression is specific for the ABA epitope and is mediated by T cells in cell transfer experiments. ABA-binding T cells from suppressed animals were purified by a two-stage procedure in which B cells were removed from spleen cell populations by adherence to plastic surfaces coated with anti-mouse Ig antibody, followed by binding the nonadherent population (more 95 percent Thy-1-positive) to surfaces coated with ABA-protein conjugates. Approximately 90 percent of the cells recovered by temperature-dependent elution from the ABA plates (similar to 2 percent of the spleen cells) bound antigen immediately afterward, and up to 50 percent of the cells bound anti-cross-reactive idiotype antibody. On the other hand, the nonadherent T-cell population was completely negative in the antigen- binding and idiotype assays. Another distinguishing feature of the two T-cell populations was that 78 percent of the adherent cells, but only 2 percent of the nonadherent cells, were Ia positive, although the specific I-region marker(s) expressed on the cells was not identified. The biological function of the antigen-binding T cells was investigated using a standard cell transfer protocol. Suppressor cells were enriched in the adherent population by a factor of at least 25, establishing that functional, epitope-specific, idiotype-bearing T cells can be significantly purified by this procedure. Note Added in Proof. We have recently isolated two types of ABA-binding molecules biosynthetically labeled with (35)S-methionine from NP-40 lysates of purified antigen-specific T cells. The molecules were purified by adsorption onto an ABA-Sepharose immunoadsorbent followed by elution with 9 M urea. Autoradiograms of SDS-PAGE of the eluates revealed components with tool wt of approximately 60,000 and 33,000 dahons. These molecules were not present in eluates from a bovine IgG-Sepharose control immunoadsorbent and thus represent specific ABA-binding products synthesized by T cells.

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.

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.

Idiotype profile of an immune response. I. Contrasts in idiotypic dominance between primary and secondary responses and between IgM and IgG plaque-forming cells.

The primary response of A/J mice to p-azobenzenearsonate-keyhole limpet hemocyanin (ABA-KLH) was investigated. A day-by-day analysis at the plaque- forming cell (PFC) level was performed, with inhibition by anti-cross- reactive idiotype (CRI) serum to determine percentage of CRI(+) PFC. A regular pattern in the dynamics of Id (idiotype) dominance was observed. Just as in the NP-b and NP-a systems (9, 12), the major Id (CRI) is more dominant in primary than in secondary or hyperimmune responses. This trend is more apparent in IgG PFC which are generally 80-95 percent CRI(+) at day 10 in the primary response but only 30-40 percent CRI(+) at day 10 in secondary or hyperimmune responses. A somewhat different pattern is seen with IgM PFC. These may reach a peak of 85 percent CRI(+) in the primary response, but secondary or hyperimmune IgM PFC, which are lower in numbers than IgG PFC, remain high in CRI content at approximately 70 percent. The PFC data on extent of id dominance in secondary or hyperimmune responses is fully compatible with previously reported serological data by others. Analysis of IgG PFC by hapten inhibition indicated that heterogeneity was in the order secondary PFC {greater than} primary PFC {greater than} hybridoma AK-2.2 PFC with H(75)/H(25) values of 22.9, 6.2, and 2.7, respectively; where H(75) and H(25) are the hapten concentrations required to give 75 percent and 25 percent of inhibition of PFC, respectively. Hapten inhibition data also suggested that secondary IgG PFC were 10 times higher in median binding avidity for ABA-L-tyrosine than primary IgG PFC. The kinetic analysis strongly indicated that CRI(+) IgM PFC were preferentially switched to IgG PFC in the primary response. In both studies, the CRI content of the earliest-appearing IgG PFC was significantly higher than that of IgM PFC on that day. For example, in one case IgM PFC were 60 percent CRI + on day 6 whereas IgG PFC were 100 percent CRI(+). The high Id dominance and selective isotype switching may have either a B or a T cell basis.

Parallel evolution of antibody variable regions by somatic processes: consecutive shared somatic alterations in VH genes expressed by independently generated hybridomas apparently acquired by point mutation and selection rather than by gene conversion.

We identified, in independently generated hybridoma antibodies, blocks of shared somatic alterations comprising four consecutive amino acid replacements in the CDR2s of their heavy chain variable regions. We found that the nucleotide sequences encoding the shared replacements differed slightly. In addition, we performed genomic cloning and sequencing analyses that indicate that no genomic sequence could encode the block of shared replacements in any one of the antibodies and thus directly serve as a donor by a recombinational process. Finally, in a survey of other somatically mutated versions of the same heavy chain variable gene, we found several examples containing one, two, or three of the shared CDR2 mutations in various combinations. We conclude that the shared somatic alterations were acquired by several independent events. This result, and the fact that the antibodies containing the four shared mutations were elicited in response to the same antigen and are encoded by the same VH and VK gene segments, suggests that an intense selection pressure has fixed the shared replacements by favoring the clonal expansion of B cells producing antibodies that contain them. The basis of this selection pressure is addressed elsewhere (Parhami-Seren, B., L. J. Wysocki, M. N. Margolies, and J. Sharon, manuscript submitted for publication).

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.

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.

Antigen- and receptor-driven regulatory mechanisms. II. Induction of suppressor T cells with idiotype-coupled syngeneic spleen cells.

Anti-p-azobenzenearsonate (ABA) antibodies, coupled covalently to normal syngeneic spleen cells and then given intravenously to normal animals, were found to be potent tolerogens for delayed-type hypersensitivity (DTH) to ABA. The ability of the antibody-coupled cells to induce tolerance was determined to be a result of the cross-reactive idiotype (CRI+) fraction of the antibodies, because anti-ABA antibodies lacking the CRI+ components when coupled to spleen cells were unable to cause any significant inhibition. Furthermore, genetic analysis revealed that the ability of CRI-coupled cells to inhibit ABA-specific DTH is linked to Igh-1 heavy chain allotype, in as much animals which possess heavy chain allotypes similar to that of A/J were sensitive to this inhibition. Adoptive transfer experiments provided evidence that CRI-coupled cells induce suppressor cells, and spleen cells or thymocytes from animals received CRI-coupled cells were able to transfer suppression to naive recipients. In addition, treatment with anti-Thy1.2 serum plus complement completely abrogated their ability to transfer suppression. Thus, this active suppression is a T-cell-dependent phenomenon. In investigating the specificity of these suppressor T cells, it was found that they functioned in an antigen-specific manner and were unable to suppress the development of DTH to an unrelated hapten 2,4-dinitro-1-fluorobenzene.

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.

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.

Regulation of azophenylarsonate-specific repertoire expression. 1. Frequency of cross-reactive idiotype-positive B cells in A/J and BALB/c mice.

A large proportion of p-azophenylarsonate (ARS)-specific antibodies from A/J mice share a cross-reactive idiotype (CRIA) that comprises a family of closely related but nonidentical clonotypes. I determined that only 2.6 % (7 out of 267) A/J ARS-specific monoclonal antibodies generated in the splenic focus system possess the predominant CRIA. Because ARS-specific B cells are present at a frequency of 1/68,000 B cells, the frequency of the entire idiotype family is 1 per 2.8 X 10(6) splenic B cells. Thus, there is a striking discrepancy between the representation of this idiotype at the clonal precursor cell level and the serum antibody response. In addition, BALB/c mice have the potential to generate CRIA-positive precursor cells within their nonimmune repertoire. When A/J mice are immunized with ARS-protein conjugates, the serum antibody response and precursor cell population are both dominated by CRIA. The frequency of CRIA-positive B cells increases over 100-fold after immunization, whereas CRIA-negative precursor cells may initially decrease, followed by a later rise in frequency. Finally, although ARS-specific precursor cells are present in high frequency at birth, CRIA-positive monoclonal anti-ARS antibodies are not observed during the early neonatal period. These data provide evidence to suggest that complex regulatory networks influence precursor cell and serum antibody expression.

I-J-restricted interactions in the generation of azobenzenearsonate-specific suppressor T cells.

The genetic restrictions of the activation of third-order suppressor cells (Ts3) were studied in mice, using two different types of anti-azobenzenearsonate (ABA)-immune responses, namely delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) generation. Ts2 cells were induced in several different strains of mice by injecting monoclonal T hybridoma molecules or first-order suppressor factors (TsF1) originating in A/J (H-2a, Igh-1e) mice and then testing the TsF2 molecules derived from these Ts2 in A/J and A.By (H-2b, Igh-1e) or (A/J X A.By)F1 (H-2a/b, Igh-1e) and (C57Bl/6 X A/J)F1 (H-2b/a, Igh-1e) mice. It was shown that the activity of TsF2 was restricted to the I-J of the strain in which Ts2 was induced. By genetic analysis, restriction was shown to be due to the requirement of H-2 identity between ABA-coupled cells used for Ts3 activation and the strain of the TsF2 origin. Moreover, by using H-2-congenic ABA-coupled cells, we were also able to precisely map and demonstrate that ABA-coupled cells I-J identical to TsF2 induced in various strains were necessary for effective suppression to occur. This selective activation of Ts3 suggested the existence of I-J-related antigen presentation for suppression as the counterpart of I-A or I-A-I-E-restricted antigen presentation for positive immune responses.

Antigen- and receptor-driven regulatory mechanisms. VII. H-2-restricted anti-idiotypic suppressor factor from efferent suppressor T cells.

Azobenzenearsonate (ABA)-specific T cell-derived suppressor factor (TsF1) from A/J mice was used to induced second-order suppressor T cells (Ts2). Comparison of suppressor T cells induced by antigen (Ts1) with Ts2 induced by TsF1 revealed that Ts1 were afferent suppressors active only when given at the time of antigen priming, and not thereafter, whereas Ts2 could act when transferred at any time up to 1 d before antigen challenge for a delayed-type hypersensitivity response. This was true even when the recipient could be shown to be fully immune before transfer of Ts2, thus defining these cells as efferent suppressors. The anti-idiotypic specificity of the Ts2 was demonstrated by the ability of Ts to bind to idiotype (cross-reactive idiotype [CRI])-coated Petri dishes. A soluble extract from Ts2 (TsF2) was also capable of mediating efferent suppression that was functionally antigen- (ABA) specific. Comparison of TsF1 with this new factor, TsF2, revealed that both lack Ig-constant-region determinants, possess H-2-coded determinants, and show specific binding (to ABA and to CRI+-Ig, respectively). TsF1 acts in strains that differ with respect to H-2 and background genes, whereas TsF2 shows H-2- and non-H-2-linked genetic restrictions. This existence of H-2 restriction of TsF2 activity suggests that the apparent discrepancies in studies of H-2 restriction of TsF may be a result of the analysis of two separate classes of TsF, only one of which shows genetically restricted activity, thus unifying several models of suppressor cell activity.

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.

T cell hybrids with arsonate specificity. I. Initial characterization of antigen-specific T cell products that bear a cross-reactive idiotype and determinants encoded by the murine major histocompatibility complex.

T cell hybrids have been constructed between the BW5147 thymoma cell line and A/J splenocytes from mice suppressed with the p-azophenylarsonte hapten. Three independently derived cloned lines have been chracterized. Each secretes or sheds a 62,000-dalton antigen-specific product bound by rabbit antisera directed against the arsonate cross-reactive idiotype. In addition, each of the antien-specific molecules contains determinants encoded within the I region of the murine major histocompatibility complex. Peptide mapping analysis indictes that, whereas these molecules are remarkably similar, each is individually distinct in primary structure. The availability of cloned T cell lines that produce antigen-specific idiotype-positive I region-containing products should facilitate a more thorough dissection of the interrelationships of T-B interctions in the arsonate idiotypic system.

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.

Two distinct mechanisms regulate the in vivo generation of cytotoxic T cells.

Treatment of responder cells with monoclonal anti-Ly-1,2 antibodies plus complement in vitro completely eliminated their ability to generate azobenzenearsonate (ABA)-specific cytolytic T lymphocytes (CTL). However, addition of the concanavalin A-stimulated supernatants of rat spleen cells (Con A-Sup) can fully reconstitute the response. Therefore, Lyt-1,2-bearing T cells are required for the generation of ABA-specific CTL, and such requirement can be replaced by factors present in the Con A- sup. Suppressor T cells (Ts), when adoptively transferred into naive recipients, will inhibit the in vivo priming of CTL. This inhibition can also be reversed by in vitro addition of Con A-Sup. furthermore, mice serving as donors of Ts also show profound unresponsiveness when primed and restimulated in vitro. In contrast to the Ts-mediated inhibition, in vitro addition of Con A-Sup was unable to abolish the unresponsiveness observed in these cultures. Thus, we identified two unresponsive states in a hapten-specific killing system that differ in their ability to be reconstituted by Con A-Sup.

Contact sensitivity to azobenzenearsonate and its inhibition after interaction of sensitized cells with antigen-conjugated cells.

Painting mice on the skin with the diazonium salt of p-arsanilic acid elicited two types of T cell activity. One was restricted by the I region of the major histocompatibility complex and was responsible for the transfer of azobenzenearsonate (ABA) sensitivity to naive mice. The other was H-2K restricted and could be demonstrated by its ability to interact specifically with ABA-coupled cells in vitro and to inhibit nonspecifically the transfer of sensitivity by cells sensitized either to ABA or to another antigen. Free antigen, or antibody directed against the cross-reactive idiotype on the anti-ABA antibodies of A/J mice, could inhibit the H-2K-restricted suppressive activity induced in the ABA immune A/J cells.

Antigen- and receptor-driven regulatory mechanisms. VIII. Suppression of idiotype-negative, p-azobenzenearsonate-specific T cells results from the interaction of an anti-idiotypic second-order T suppressor cell with a cross-reactive-idiotype-positive, p-azobenzenearsonate-primed T cell target.

The suppressor pathway that regulates the T cell response to p-azobenzenearsonate (ABA)-coupled cells has been studied. It has been found that the ability of anti-idiotypic second-order T suppressor cells (Ts2) to inhibit T cell-dependent delayed-type hypersensitivity (DTH) responses depended upon the presence of cross-reactive-idiotype (CRI)-bearing T cells present in ABA-primed mice. This suppressor T cell subset, termed Ts2, so exists with CRI-negative T cells that mediate DTH in vivo. It appears that antigen-activated CRI+ Ts3 require signals from the anti-CRI Ts2 subset to suppress DTH reactions in an idiotype-nonspecific manner. The relevance of these observations to a comprehensive scheme of T and B cell regulation is discussed.

T cell development in B cell-deficient mice. II. Serological characterization of suppressor T cell factors (TsF1) produced in normal mice and in mice treated chronically with rabbit anti-mouse IgM antibodies.

Serological analysis of idiotypic specificities present in azobenzenearsonate (ABA)-specific first-order suppressor T cell factors (TsF1) from C.AL-20 and BALB/c mice revealed a significant difference between TsF from these two strains of mice. The idiotypic composition of TsF1 from BALB/c mice appears to be more heterogeneous, and at least two different fractions can be readily identified. One bears the characteristic BALB/c-associated CRI(C) (crossreactive idiotype) determinants, and the other is non-CRI(C)-bearing. Analysis of ABA-specific TsF1 from animals lacking B cells uncovered a fundamental change in the expression of their idiotypic specificities. TsF from rabbit anti-mouse IgM (anti-mu)-treated C.AL-20 mice failed to express the characteristic CRI(A) determinants. Instead, they express CRI(C) specificities. Similarly, TsF1 from anti-mu-treated BALB/c mice did not express their characteristic CRI(C) specificities, but rather express CRI(A) determinants. These experiments provide strong evidence that the Igh restriction specificity of TsF is dictated by the particular idiotypic specificities expressed. They also clearly demonstrate that B cells and their products play an important role in establishing the idiotypic composition and repertoire of suppressor T cells.