Suppression of murine lupus nephritis by administration of an anti-idiotypic antibody to anti-DNA

The suppression of pathogenic antibodies to DNA in NZB/NZW f1 female mice was achieved by repeated inoculation of the mice with a monoclonal anti-idiotypic antibody (anti-Id). The anti-Id, an IgG1, kappa, was directed against a major cross-reactive idiotype (Id) on NZB/NZW IgG antibodies to DNA. One hundred micrograms of the anti-Id were inoculated i.p. every 2 wk, beginning at 6 wk of age (nondiseased mice--no circulating anti-DNA or proteinuria) or 20 wk of age (diseased mice--all with circulating anti-DNA, one-third with proteinuria). As controls, littermates received an IgG, kappa non-DNA-binding myeloma or no treatment. In the young mice, nephritis and anti-DNA antibodies appeared at the same time in all groups, and their circulating antibodies to DNA did not bear the target Id. In the older (20-wk-old) mice, survival was significantly prolonged because of delay in the onset of nephritis; the total quantities of antibodies to DNA were diminished, and the target Id, initially present on circulating IgG, was deleted. These benefits were transient; the suppression of antibodies was followed by the appearance of large quantities of anti-DNA that did not bear the major Id. Therefore, although administration of anti-Id was effective in reducing an undesirable antibody response after the target Id was present on circulating antibodies, the benefits were limited, probably by Id "switch" or by increased synthesis of pathogenic antibodies bearing a minor Id.     

Xid and normal mice express a light chain-associated cross-reactive idiotype in response to (T,G)-A--L and (T,G)-A--L-mBSA

Rabbit anti-idiotypic (Id) antibodies were prepared against purified ascites anti-(T,G)-A--L antibodies (TGB5) that had been absorbed to remove A--L-specific antibodies and were specific for (T,G)-side chain determinants. Purified rabbit anti-TGB5 Id antibodies detected an allotype-independent, light chain-associated cross-reactive Id expressed by the majority of individual mice immunized with (T,G)-A--L, (T,G)-A--L coupled to methylated bovine serum albumin (mBSA), or the linear terpolymer GAT. Primary and secondary monoclonal hybridoma protein (HP) antibodies from X/Xxid heterozygous (wild-type) mice immunized with (T,G)-A--L and/or (T,G)-A--L-mBSA were analyzed for isotypy and were grouped into eight antibody fine specificity sets defined by the patterns of direct binding to the antigens (T,G)-A--L, (Phe,G)-A--L, (T,G)-Pro--L, GT, and A--L. Analysis of these primary and secondary HP for TGB5 idiotypy showed a preferential expression of the TGB5 Id among GT+-binding HP (antibody fine specificity sets 1 through 3). All of the primary GT+-binding HP and the majority of secondary GT+-binding HP (sets 1 through 3) were TGB5 Id+. Most but not all of the TGB5 Id+ HP bound GAT. Of the side-chain-specific HP (sets 1 through 7), 78% of primary HP vs 49% of secondary HP bound GT. By these criteria, the primary HP response appears more restricted than the secondary HP response, consistent with the idea that Id diversification and antibody heterogeneity are regulated and selected events occurring during memory B cell generation. Although xid mice produce less antibody than wild-type mice to (T,G)-A--L, the TGB5 Id was produced early in the primary response by both xid and wild-type mice immunized with (T,G)-A--L or (T,G)-A--L-mBSA, and was maintained as a detectable Id in equivalent amounts in their secondary serum antibody responses. These results support the idea that distinct B cell subsets, including the xid B cell subset, share the same immunoglobulin gene repertoire.     

IL-4 and IL-10 are essential for immunosuppression induced by high molecular weight proteins from Ascaris suum

The extract from Ascaris suum worms (Asc) impairs Th1 and Th2 responses to a non-related antigen, i.e. ovalbumin (OVA). Its suppressive capacity is due to high molecular weight components present in a gel filtration fraction (PI). This fraction is able to elicit IL-4 and IL-10 secretion. Interestingly enough, it induces anti-PI non-anaphylactic IgG1 synthesis through the action of IL-12/IFN-gamma. Here, we investigated the down-regulation of the immune response to OVA by PI in IL-12, IFN-gamma, IL-4 or IL-10 C57BL/6 knockout mice immunized with OVA+PI in adjuvant. OVA-induced delayed-type hypersensitivity (DTH) reactions, secretion of IL-2 and IFN-gamma, and IgG1, IgG2c and IgE antibody production were suppressed by PI in wild-type mice, as well as in IL-12- or IFN-gamma-deficient mice. In contrast, PI had no effect on anti-OVA IgE production and DTH, and induced only a partial suppression of IgG1 and IFN-gamma in IL-10(-/-) mice. The experiments also showed that IL-4 was involved in the PI-induced suppression of IgG2c antibodies and IL-2 secretion. Finally, down-regulation of IFN-gamma was not seen in mice lacking both IL-4 and IL-10, i.e. IL-4(-/-) mice treated with anti-IL-10 antibodies before immunization. These results exclude the participation of IL-12 and IFN-gamma in PI-induced immunosuppression, and highlight the essential role of IL-10 in the suppression of OVA-specific Th2-related parameters, as well as the cooperation between IL-10 and IL-4 in the suppression of Th1-related parameters.     

Expression of a cross-reactive idiotope on naturally occurring murine antibodies against 3-fucosyllactosamine, levan, and dextran

We recently identified a cross-reactive Id (6C4) that is expressed on the H chain of many BALB/c mAb against the 3-fucosyllactosamine (3-FL) determinant, Gal(beta 1-4) (Fuc(alpha 1-3] GlcNAc-R. The VH segments of seven mAb that we recently sequenced are encoded by VH441, which also encodes VH segments of antibodies against galactan, levan, and dextran. To analyze the expression of the 6C4 Id on naturally occurring anti-carbohydrate antibodies, we isolated 6C4+ antibodies by affinity chromatography from pools of normal BALB/c serum. Approximately 20 to 30% of antibodies against 3-FL and levan, and all antibodies against dextran, were removed from the sera by passage over a column containing mAb 6C4. Absorption of the eluate with 3-FL beads removed anti-3-FL antibodies but not anti-dextran or anti-levan. The expression of a cross-reactive Id on naturally occurring antibodies against several carbohydrate Ag suggests that these antibodies may participate in an Id network. We also reported previously that BALB/c mice have naturally occurring anti-3-FL antibodies and respond well to immunization against this determinant, whereas C57BL/6 mice do neither. To examine the role of the Igh-C allotype in the regulation of the anti-3-FL response, we studied congenic strains of BALB/c and C57BL/6 mice. Both congenic strains produced anti-3-FL antibodies in response to immunization, but only C.B-20 mice exhibited naturally occurring antibodies. These data suggest that the naturally occurring and elicited antibody responses against 3-FL are differentially regulated.     

Vaccination with membrane-associated idiotype provides greater and more prolonged protection of animals from tumor challenge than the soluble form of idiotype

The purpose of this work was to compare the efficacy of immunizing mice with a soluble vs a cell-associated form of a tumor Ag. A murine B cell tumor (2C3), which displays an Id on its cell surface, grows progressively and gives rise to Id-negative tumor variants in nonimmunized animals. We previously reported that the tumor variants arise as a consequence of Id-specific T cell suppression of the Id+ tumor. The Id-specific effector T cells are CD4+, CD8-. Based upon these findings vaccination protocols have been designed and tested to determine whether the expansion of tumor-specific effector T cells would eliminate the Id+ tumors and prevent the subsequent generation of Id- tumor variants in vivo. MHC-restricted T cells typically recognize soluble Ag subsequent to modification by an APC, and APC may ultimately express a processed form of the Ag that is different from that expressed on the surface of the tumor cells. Based upon this assumption, the efficacy of immunizing mice with cell-associated 2C3 Id was compared to immunization with a soluble form of the same Id. Mice were immunized with either irradiated 2C3 cells or syngeneic spleen cells to which 2C3 protein was covalently linked. These immunization protocols provided a complete and lasting protection against a tumor challenge of up to 1 x 10(6) tumor cells. In contrast, most mice hyperimmunized with a soluble form of the Id did not survive this level of tumor challenge in spite of the production of significant levels of anti-Id antibodies. Mice immunized with the soluble form of the Id, which did survive, produced slowly progressing tumors expressing a 1000-fold less of the marker Id. These results illustrate the importance of understanding and properly exploiting a host's natural response to a tumor-specific Ag when designing effective immunization protocols for cancer therapy.     

IgA isotype-restricted idiotypes associated with T15 Id+ PC antibodies

Idiotypes are believed to be due to the structural conformation of the variable region of immunoglobulins (Ig). We have found an idiotype (C3-24) that requires both variable and constant regions of the heavy chain to be expressed. C3-24 Id is associated with both the T15 variable region from anti-phosphorylcholine (PC) antibodies and the constant region for the alpha-heavy chain. High titer anti-PC serum from a variety of inbred strains of different Ig haplotypes failed to express C3-24 Id. However, when IgA but not IgG or IgM fractions were isolated from a pool of anti-PC serum from BALB/c mice, more than 70% of the molecules expressed C3-24 Id. The high frequency of the expression of C3-24 Id in IgA anti-PC hybridoma proteins from mice of different Ig haplotypes and in the IgA fraction of normal anti-PC antibodies from BALB/c and presumably other strains of mice suggests that idiotypic determinants produced by the three-dimensional product of VH and CH regions may not be unusual.     

Rapid changes in the regulatory potential of autologous anti-idiotopic T cells during an antigen-driven primary response

The antibody response of C57BL/6 strain mice to Streptococcus pneumoniae R36a (Pn) is dominated by the T15 idiotype, but the responding cells appear to be idiotypically heterogeneous, in that individual antibody plaque-forming cells (PFC) may express some but not all idiotopes (Id) of the T15 complex. The presence of these distinct Id on the PFC was detected by a plaque-inhibition assay with three different monoclonal anti-Id antibodies, designated AB1-2, MaId5-4, and B36-82. A periodic change in the expression of AB1-2 and MaId5-4 Id was observed during primary (IgM) antibody response to Pn in the spleen. Those two Id were poorly expressed in the log phase of the response between day 2 and day 4 after immunization (few PFC in the spleen bore the Id), but they became detectable on the majority of PFC at the peak of the response, day 5 to day 7. The proportion of the Id-(AB1-2 or MaId5-4) positive PFC declined, again at day 10 after immunization. In contrast, the B36-82 Id was expressed on greater than or equal to 80% PFC throughout the entire primary response. The possibility that the apparent changes in the Pn-reactive cell populations are regulated by autologous anti-Id T cells was tested in vitro. Normal, unimmunized B cells were cultured with Pn, either alone or in the presence of syngeneic T cells isolated from the spleen of mice at the appropriate intervals after immunization: day 2 (T2), day 5 (T5), and days 10 to 14 (T10 to T14); T cells from unimmunized donors (T0) served as a control. The specific response after 4 days in culture was determined in regard to the total PFC as well as the proportion of PFC expressing the Id. Pn-stimulated B cells, alone or with the control T0 cells, produced moderate, variable levels of AB1-2+ and MaId5-4+ PFC. The expression of these two Id in the assay cultures was suppressed by addition of either T2 cells or T10-14 cells, but it was enhanced if T5 cells were added. However, these various T cell populations did not differ in their effect on the total PFC response. Also, the proportion of PFC bearing the third Id, B36-82 was high, and it was not consistently influenced by the added T cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.     

Molecular analysis of heavy and light chains used by primary and secondary anti-(T,G)-A--L antibodies produced by normal and xid mice

The primary (1 degree) antibody response to (T,G)-A--L shows limited heterogeneity, consisting mostly of side chain-specific antibodies that bind GT and that express the TGB5 idiotype (Id). The secondary (2 degrees) response is very diverse: antibodies that bind the backbone A--L constitute a third of the response, and a high proportion of the side chain-specific antibodies do not bind GT and are TGB5 Id-. To provide a molecular basis for understanding this difference in repertoire expression, we analyzed the Ig genes used by heavy and light chains of 1 degree and 2 degrees side chain-specific anti-(T,G)-A--L hybridoma antibodies (HP). Southern blot restriction analysis and nucleotide sequence analysis of the expressed genes used by three TGB5 Id+ 2 degrees HP showed usage of three different VH genes in two VH gene families (36-60 and J558), different D segments, and two different Vk1 genes (the Vk1A and Vk1C subgroups). Thus, antibody heterogeneity in the 2 degrees response is contributed by combinatorial diversity of distinct germ-line genes. Nucleotide sequence analysis of the expressed genes used by TGB5 Id+ 1 degree HP showed use of highly homologous VH genes in the J558 VH gene family and highly homologous Vk1A genes. The majority of TGB5 Id+ 1 degree HP from different donors gave similar heavy and similar light chain gene rearrangements by Southern blot restriction analysis, after correction for known or potential J region differences. The combined nucleotide sequence and Southern blot restriction analysis data suggest that most 1 degree B cells use the same or very similar VH and Vk genes, i.e., the 1 degree response is paucigenic. Different D segments were used by the TGB5 Id+ 1 degree and 2 degrees HP that were sequenced, and there was no apparent correlation between TGB5 idiotypy and VH, D gene, or JH gene usage. However, all TGB5 Id+ HP sequenced used highly homologous genes from the Vk1 group. Expression of a Vk1 light chain correlates with, but is not sufficient for, TGB5 idiotypy, because one GT-binding, TGB5 Id- HP was found to use a Vk1C subgroup light chain. By Southern blot and nucleotide sequence analysis, the Vk genes used by two TGB5 Id+ 2 degrees HP from xid mice are highly homologous, if not identical to the Vk1A gene(s) used by 1 degree and 2 degrees Id+ HP from wild-type mice.     

Idiotype restriction in murine lupus; high frequency of three public idiotypes on serum IgG in nephritic NZB/NZW F1 mice

Antibodies to self-antigens are characteristic of several human and murine autoimmune diseases. Subsets of those autoantibodies cause organ damage in some instances, such as IgG antibodies to DNA in human and murine systemic lupus erythematosus (SLE). Our experiments in the NZB/NZW F1 (BW) female mouse model of SLE were designed to define idiotypic (Id) structures on antibodies to DNA in attempts to distinguish pathogens from nonpathogens within the anti-DNA population. Two important findings emerged. First, the number of public Id expressed became relatively restricted as the mice aged, with three such Id (IdX, IdGN1 and IdGN2) dominating and accounting for 30 to 95% of the total serum IgG in all individual nephritic mice studied, and 81 to 86% of the total IgG in serum pools from 30-wk-old nephritic mice. Second, IdGN1 and IdGN2 constituted approximately 50% of the IgG deposited in glomeruli of nephritic mice; IdX was present in negligible quantities in glomeruli, whereas it was usually the most frequent Id in BW serum. These latter findings suggested that pathogens and nonpathogens can be distinguished by their idiotypy in this animal model. The finding of relative Id restriction suggests the occurrence of an idiotypic "spreading" phenomenon, in which a regulatory process appears as BW mice age that results in repeated selection and expansion of this small number of Id, one group of which, the IdGN, is pathogenic. This process was further suggested in experiments in which IdX was suppressed by administration of anti-IdX; the "escape" antibodies to DNA appearing after suppression of IdX were composed largely of IdGN1 and IdGN2, without a major contribution from Id-negative mutants. Defining the basis of this Id spreading or restriction phenomenon may provide important information regarding the pathogenesis of this autoimmune disease.     

Regulation of idiotope expression. III. H-2 influences the magnitude and the idiotypy of a T-independent antibody response in mice of certain genetic backgrounds

Antibody response to the phosphocholine (PC) epitope on Streptococcus pneumoniae R36a (Pn), a T-independent Ag type 2, was studied in H-2 congenic mouse strains. The PC-specific antibody plaque-forming cells (PFC) were enumerated in the spleen at various intervals after the primary Pn injection, and the proportion of PFC that produced antibody expressing the AB1-2 idiotope (Id) was determined by using the corresponding monoclonal anti-Id. AB1-2 is a cross-reactive Id, detectable on germline-encoded PC antibody of the T15 family, and on most, but not all, somatic variants of that antibody. The specific PFC responses in BALB/c (H-2d) and BALB.B (H-2b) strains were of comparable magnitude and most, if not all, PFC were ABl-1 Id-positive (AB1-2+). This was not the case in the responses of the B10D2 (H-2d) vs C57BL/10 (H-2b) strains and the D1.C (H-2d) vs D1.LP (H-2b) strains (on DBA/1 background). In each of these pairs, the H-2d mice were high responders, and the response was dominated by AB1-2 Id (greater than or equal to 80% AB1-2+ PFC at the peak, on day 5). The H-2b mice were low responders, and only a minor proportion of PFC (less than or equal to 30%) were AB1-2+; an increase of AB1-2+ was seen later in the response (d.10). The results of PFC assays were confirmed by measuring the PC-binding antibody and AB1-2 Id in the sera of D1.C and D1.LP mice immunized repeatedly with Pn. Moreover, D1.LP mice that had very low levels of AB1-2 Id had higher serum levels of antibody expressing two other T15 Id, B36-82, and B24-44. The B36-82 and B24-44 Id have been previously found on somatic variants of PC antibody expressed independently of the Ab1-2 Id. The concentrations of these two Id in D1.LP mice after repeated immunization approached those in D1.C. These results indicate that 1) the H-2 allelism may have a significant effect on TID antibody response in mice of a certain genetic background, but not in the BALB/c; and 2) the idiotypic repertoire of the response may be influenced by H-2 at the level of clonal variants of PC-reactive cells.     

The polyclonal and antigen-specific IgE and IgG subclass response of mice injected with ovalbumin in alum or complete Freund's adjuvant

BALB/c mice were injected ip with 1 microgram ovalbumin (OVA) in alum or complete Freund's adjuvant (cFA) and the changes of the IgE and IgG subclass serum levels and isotypes of the anti-OVA specific antibodies determined by radioimmunoassays. By Day 10, OVA in alum had induced a 5- to 10-fold increase of the IgE serum level and an initial decrease of the IgG subclass levels which subsequently increased to two to threefold over the preinjection level. OVA in cFA induced a gradual twofold increase of the IgE serum level, a rapid fourfold increase of the IgG2a level occurring by Day 7, and a gradual two to threefold increase of the other IgG subclasses. Over 90% of the anti-OVA antibodies were of the IgGl isotype with both adjuvants; OVA in alum induced slightly more IgGl anti-OVA antibodies than cFA. In contrast, the OVA in alum injected mice formed significantly more (5- to 10-fold) IgE anti-OVA antibodies than the cFA-injected mice. OVA in alum also induced a large nonspecific increase of the IgE serum level because only approximately 40% of the increase observed on Day 14 was absorbable with OVA, whereas approximately 90% the IgE increase in cFA injected mice was absorbable with OVA. The data demonstrate that mice form mainly IgGl and IgE antibodies to OVA irrespective of the adjuvant. The low specific and lack of nonspecific IgE formation by mice injected with OVA in cFA may be the result of cFA-induced interferon-gamma (IFN-gamma) production because IFN-gamma has been shown to stimulate IgG2a and inhibit IgE secretion in vitro.     

Intratracheal and intranasal immunization with ovalbumin conjugated withBacillus firmus as a carrier in mice

Inactivated Bacillus firmus (BF), G+ nonpathogenic bacterium of the external environment, was coupled to ovalbumin (OVA) and used in immunization experiments as antigen carrier. Balb/c mice were immunized thrice intra-tracheally and intra-nasally with conjugates of OVA and BF. Surprisingly, administration of OVA-BF conjugates inhibited anti-OVA IgG response in both sera and mucosal secretions if compared to an exposure to OVA alone. The suppression of antigen-specific antibody production was accompanied by promotion of TH1 phenotype.     

Influence of the immunoglobulin heavy chain locus on expression of the VK1GAC light chain

The VK1GAC light chain represents the dominant V kappa structure employed in the antibody response of A/J mice to streptococcal group A carbohydrate ( GAC ). Two anti-idiotypic antisera, anti- Id5 and anti- Id20 , with specificity for the VK1GAC light chain were used to examine anti- GAC antibody responses in a series of inbred mouse strains that differ at the heavy chain constant region ( IgCH ) allotype locus. Both idiotypes were expressed in normal and immune sera from mice of most IgCH allotypes, except IgCHb (C57BL/6J) and IgCHf (CE/J). C57BL/6J mice expressed Id5 , but not Id20 , whereas CE/J mice did not express either idiotype. Testing of recombinant inbred strains between BALB/c and C57BL/6 indicated that the pattern of idiotype expression did not correlate with IgCH allotype. The C X B recombinants expressed all three idiotype patterns that were observed in the panel of inbred strains. Testing of allotype congenic mice between BALB/c and C57BL/6 showed that CB.20 and BC.8 mice were Id20 -, whereas BAB-14 mice were Id20 +, indicating that both VH and background (V kappa or regulatory) loci must be derived from BALB/c to obtain Id20 expression. The difference in the frequency of idiotype expression observed between BALB/c and BAB-14 mice indicates that the IgCH locus may exert a quantitative influence on the expression of this light chain. To examine the Id20 -, Id5 + antibodies of C57BL/6 mice, anti- GAC hybridomas were prepared. Of 16 C57BL/6-derived anti- GAC monoclonal antibodies, six were reactive with anti- Id5 and not with anti- Id20 . Isoelectric focusing of the purified kappa light chains from three of these antibodies revealed two distinct spectrotypes that co-migrated with the two known VK1GAC spectrotypes observed with A/J anti- GAC light chains. Idiotypic analysis of in vitro recombinants between the heavy and light chains of A/J and C57BL/6 monoclonal antibodies demonstrated that the C57BL/6 light chains were idiotypically similar to A/J light chains when they were free in solution or paired with A/J heavy chains. These results demonstrate that C57BL/6 mice can express a light chain that is very similar, if not identical, to the VK1GAC light chain, although the light chain is expressed in lower frequency and is paired with a distinct VH structure, which can mask expression of one of the VK1GAC idiotypes. These effects on V kappa expression map to at least three genetic loci: VH, CH, and an unlinked locus.     

A public idiotypic determinant is present on spontaneous cationic IgG antibodies to DNA from mice of unrelated lupus-prone strains

A monoclonal anti-idiotypic antibody (anti-Id) to a public idiotype (Id) present on spontaneous IgG antibodies to DNA from NZB/NZW F1 mice recognized similar determinants on polyclonal and monoclonal IgG anti-DNA antibodies from mice of the unrelated MRL/lpr and BXSB strains. Incubation of the anti-Id with four of five monoclonal Id in solid phase inhibited their ability to bind DNA; however, different Id+ antibodies recognized different epitopes within the DNA molecule. Therefore, the public Id was located close to the antigen-binding regions but did not comprise all of those regions. Analysis of multiple polyclonal and monoclonal antibodies to DNA showed the Id on all subclasses of IgG. However, antibodies bearing the Id carried a neutral or cationic charge (10 of 10 monoclonals with pI greater than 7 were Id+); the presence of the Id on anionic IgG (pI less than or equal to 7) was infrequent (one of 21 serums, one of eight monoclonal antibodies). Therefore, IgG autoantibodies to DNA are constructed from closely related public idiotypes in several mouse strains that spontaneously develop lupus, and that Id is restricted to antibodies with a pI of 7 or greater.     

"Nonspecific" immunoenhancing T cells in tumor-bearing mice include anti-idiotypic subsets

Splenocytes from DBA/2 mice inoculated 3 wk earlier with syngeneic P815 mastocytoma tumor cells produce increased numbers of antibody plaque-forming cells (PFC) when stimulated with either sheep red blood cells (SRBC) or phosphorylcholine (PC) on Streptococcus pneumoniae R36a in vitro. The nature of this nonspecific hyperreactivity was investigated in mixed cultures of purified splenic T and B cells. The addition of T cells from P815 tumor-bearing mice (TP815) into the cultures of normal B cells produced a significant enhancement of the PFC response to both SRBC and PC, when compared with the effect of normal T cells added to control cultures. The idiotypic profile of the enhanced anti-PC response was studied by a PFC-inhibition assay with monoclonal antibodies against two distinct idiotopic determinants (Id) of the T15 family. Normal B cells produced greater than 90% of T15 Id-positive (Id+) PFC. Addition of normal T cells diminished the proportion of T15 Id+ PFC to approximately 60%, whereas the rest of PFC were Id-. Addition of the immunoenhancing TP815 cells into the normal B cells cultures elevated the number of both T15 Id+ and Id- PFC responses, proportionally. However, when TP815 cells were first incubated on T15 protein-coated dishes and the non-adherent fraction was added to B cell cultures, the anti-PC PFC response remained enhanced but consisted of predominently T15 Id- PFC. These observations suggest that the early stage of P815 tumor growth activates various populations of specific helper/amplifier T cells including subsets with anti-idiotypic activity and that the generalized increase of antibody response to various antigens in tumor-bearing mice may be regarded as a polyclonal activation of specific T cells.     

Idiotypic properties of the murine anti-arsonate antibody response: B- and T-cell influences

In a previous report characterizing the arsonate (ABA)-specific plaque-forming cell (PFC) responses of A/J mice induced by ABA-KLH, two interesting characteristics of the idiotypic (Id) profile were noted: (1) an apparent Id selectivity in the isotype switch since the earliest appearing IgG PFC in the primary response were significantly more "cross-reactive Id" (CRI)-dominant than the IgM PFC population, and, (2) a temporal waning of CRI dominance with time among IgG PFC, from 75-100% CRI+ PFC to about 25-45% CRI+ PFC in secondary responses. Experiments were performed to determine whether these effects are largely attributable to T or to B cells. Mice were immunized with a T-independent (TI) form of ABA (ABA-Brucella abortus) and apparent Id selectivity was observed; the earliest IgG PFC averaged 75% CRI+ while IgM PFC were only 39% CRI+. Due to the TI nature of the Ag, this provides suggestive, but not conclusive, evidence that the Id asymmetry in the isotype switch may be attributable to the direct interaction of Ag with B cells. Other studies addressed the temporal shift in CRI dominance. First, it was found that preexposure of mice to either KLH or to ABA (on an irrelevant carrier) resulted in diminished CRI dominance in subsequent "primary" responses to ABA-KLH. Secondly, adoptive transfer experiments with B and T cells from virgin mice (Bv, Tv) or ABA-KLH-primed mice (Bp, Tp) showed that recipients of Bv + Tp or Bp + Tv generated anti-ABA PFC responses with intermediate CRI levels. The Tv cells had some preferential tendency to activate CRI+ clones in the Bp population. The results demonstrate that CRI levels are jointly determined by the immune status of both B and T cells. A simple model is offered which accounts for early Id dominance and its gradual decline and has as its central postulate the assumption that CRI+ B cells in the virgin ABA-specific repertoire have an affinity advantage over CRI- clones.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.     

In vitro manipulation of human anti-DNA antibody production by anti-idiotypic antibodies conjugated with neocarzinostatin

Anti-DNA Id, 0-81, consist of 5 to 51% of Id in human anti-ssDNA antibodies; NE-1-Id shares 2 to 20% of those in anti-dsDNA antibodies. Thus, both 0-81-Id and NE-1-Id are of the cross-reactive Id that are commonly present among anti-DNA antibodies. In order to manipulate the production of anti-DNA antibodies by human PBL, we used mouse antiidiotypic mAb or those conjugated with a cytotoxic agent, neocarzinostatin. Treatment with the conjugates caused profound suppression of anti-ssDNA and anti-dsDNA antibody synthesis related to 0-81- and NE-1-Id. This was attributed to the specific killing of the clones bearing anti-DNA Id among the lymphocytes, evidenced by the indirect rosette formation tests. The Id-mediated suppression was not solely due to selective elimination of Id-positive B cells, because 50 to 92% of anti-DNA antibodies were suppressed by treatment with the conjugates. This was supported by flow cytometry analysis that showed a decrease of anti-Id-reactive cells when T cells were treated with the conjugates. This method, then, will permit an analysis of the question as to whether T cells reactive to anti-idiotypic antibodies might participate in the regulatory mechanism for anti-DNA production and, in addition, may lead to a new therapy for SLE.     

Delayed-type hypersensitivity to hen egg-white lysozyme. I. The surface phenotypes of suppressor T cells induced by intravenous injection of lysozyme-modified spleen cells, and their molecular interactions

Suppressor T cells (Ts) induced by lysozyme-modified syngeneic lymphocytes were characterized. Hen egg-white lysozyme (HEL)-specific delayed-type hypersensitivity (DTH) was suppressed when HEL-induced Ts were transferred into naive mice. These HEL-induced Ts had surface markers of both Thy-1 antigen, and I-J gene products. The suppression of HEL-specific DTH was greatly increased, when these Ts had been enriched with HEL-coated petri dishes. Isolated anti-HEL antibodies from B10.BR or A/Sn mice were inoculated into rabbits to induce anti-cross-reactive idiotype (CRI) antibodies. The rabbit antisera were extensively absorbed with normal B10.BR or A/Sn immunoglobulins (Igs) and MOPC 104E ascites Igs to render them idiotype (Id) specific. Using these anti-CRI antibodies, we observed that these Ts possessed Id receptors on their cell surface. Results of both fluorescence techniques and cytotoxicity tests revealed that about 10% of the enriched T cells containing these Ts were Id positive. Moreover, these enriched T cells were substantially killed by anti-I-J antiserum plus complement. However, this killing was completely blocked by HEL antigen. These results suggest that both Id receptors and I-J gene products might be forming the same molecular complexes or might coexist in the vicinity of the molecule.