Dimeric IVIG contains natural anti-Siglec-9 autoantibodies and their anti-idiotypes.

BACKGROUND: Intravenous immunoglobulin (IVIG) preparations are increasingly used for the treatment of autoimmune and chronic inflammatory diseases. Naturally occurring autoantibodies against Siglec-9 and Fas are thought to contribute to the anti-inflammatory effects of IVIG via cell death regulation of leukocytes and tissue cells. Dimeric IVIG fractions are suspected to contain idiotypic (Id)-anti-idiotypic complexes of antibodies, which might also include anti-Siglec-9 and anti-Fas autoantibodies. METHODS: Dimeric IVIG fractions were separated from monomeric IVIG by size-exclusion chromatography and remonomerized by low pH treatment. Binding studies of total, monomeric, and dimeric IVIG were performed using surface plasmon resonance and flow cytometry on primary human neutrophils. RESULTS: Anti-Siglec-9 and anti-Fas autoantibodies were contained in both monomeric and dimeric IVIG fractions, but anti-Siglec-9 antibodies were highly enriched in dimeric IVIG. The propensity to engage in dimer formation was paratope dependent. IVIG binding to Siglec-9 was specific and sialylation independent. Interestingly, we detected anti-idiotypic antibodies (anti-Ids) against anti-Siglec-9 autoantibodies in dimeric, but not in monomeric fractions of IVIG. CONCLUSIONS: Our study supports the concept that idiotype-anti-idiotype (Id-anti-Id) interactions contribute to the dimer formation in IVIG preparations. To our knowledge, this is the first description of Id-anti-Id dimers of death receptor-specific antibodies in IVIG. Such Id-anti-Id interactions might determine the activity of immunomodulatory antibodies present both in IVIG and the patient.

Idiotypic cross-reactivity between antibodies of different specificities.

Cross-idiotypic specificity has been demonstrated between antibody populations of different specificities using antibodies directed toward human sickle cell hemoglobin (HbS). A site-specific antibody directed toward the beta6-position of HbS, anti-Val, was used to elicit an anti-idiotypic response in rabbits. Using this anti-idiotypic serum, idiotypic cross-reactivity was demonstrated between antibody populations that bind to human adult hemoglobin (HbA). It was demonstrated that in the case of the goat antibodies, these idiotypically cross-reacting antibodies are directed towards the beta6-position of the hemoglobin molecule. However, they differ in their specificity, binding to this site on HbA, whereas anti-Val binds only to HbS. The sheep antibody populations directed toward HbS differ qualitatively from those of the goat. Using rabbit anti-idiotypic serum specific for sheep anti-Val, cross-reactivity could be demonstrated with antibodies directed toward the alpha-chain of the hemoglobin molecule, as well as the beta-chain. There was also a low level of cross-reactivity with antibodies from a goat immunized with HbA.

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

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

Idiotype variant cell populations in patients with B cell lymphoma.

Using isolated idiotype (Id) protein we generated panels of antibodies in two patients with follicular lymphoma, one of whom had never received prior chemo-or radiotherapy. Flow cytometry and frozen section tissue staining of tumor with these monoclonal antibodies (mAb) revealed multiple subpopulations within each tumor. Individual mAb stained between 7% and 83% of surface Ig+ cells in the tumor samples. These subpopulations were overlapping and no single antibody recognized all the tumor cells. However, combinations of antibodies seemed to capture total tumor in both cases. In some instances, the percentage of tumor stained by a single mAb varied over time, and differed between lymph nodes sampled at the same time. Because a single species of Id protein was used to generate mAb in each case, it appears that the antibodies were directed against idiotopes variably shared by different populations within each tumor, and this was confirmed by crossblocking studies. Tumor cells from one patient were fused to a nonsecreting heteromyeloma line K6H6/B5, and most of the resulting hybrids secreted Id protein. Four mAb were used to screen the Id proteins secreted by these hybrids, and 11 different variants (16 maximal) were found. Southern blot analysis of rearranged Ig genes was done in two hybrids and biopsy material. Identically rearranged light-chain genes were seen but it appeared as though extensive somatic variation had occurred in heavy chain genes. These studies indicate that: striking Id variation can exist at diagnosis in untreated patients, the percentage of tumor represented by an individual variant may change with time and may differ between tumor sampled from different anatomical locations, and somatic variation appears to be responsible for the observed heterogeneity. Although this degree of variation makes anti-Id antibody therapy more difficult, appropriate combinations of mAb should be more efficacious than single antibodies in such cases.

Loss of an individual idiotype on chemical modification. A strategy for assigning idiotypic determinants.

Two dextran-binding myeloma proteins, J558 and Hdex 24, which possess the same individual idiotype (IdI) were diazotized to low levels (1-3.3 groups per subunit) with 1-[14C]-p-aminobenzoate. Both proteins lost the IdI idiotype under these conditions with most of the label incorporated on the heavy chains of each protein. When the diazotization ws carried out in the presence of the hapten 1-O-methyl-alpha-D-glucopyranoside the loss of idiotypic reactivity could be prevented for J558 but not for Hdex 24. Under these conditions most of the label was incorporated on the light chains of J558, but on the heavy chains of Hdex 24. For J558, these results show that a major determinant of the individual idiotype is within the hypervariable positions of the heavy chain. For Hdex 24 the determinant being modified is on the heavy chain but not involved in hapten binding. These results are consistent with previous work showing that J558 and Hdex 24 differ in amino acid sequence in the D and the J segments of the heavy chain and offer an alternative and complementary strategy for assigning idiotypic determinants.

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.

Idiotypic determinants on T-cell subpopulations.

Killer T cells with specificity for major histocompatibility antigens have been shown in mice and rats to display idiotypic receptors allowing the lysis of such cells at the effector phase by anti-idiotypic antibodies and complement. A comparison was made between idiotypes displayed by Lyt-1-2+3+ and Lyt-1+2-3- T blasts, generated in the same mixed leucocyte culture (MLC), across an entire H-2 locus barrier. This was done by absorption of anti-idiotypic antibodies with respective T blasts, followed by estimation of the ability of the absorbed antiserum to inhibit MLC or killer T-cell function. Further, the capacity of Lyt-purified, MLC-generated T blasts to provoke specific unresponsiveness via anti-idiotypic immunity in syngeneic recipients was analyzed. Taken together, the results demonstrate that Lyt-1+2-3- T blasts responsible for the major part of MLC proliferation have distincly different idiotypes from those on the Lyt 1-2+3+ killer T cells. That the idiotypes on the killer T-cell presursors can serve as triggering sites for induction of effector T-cell function was then suggested by experiments with Lyt-1-2+3+-purified, normal T cells as precursor cells in vitro. The fact that autoanti-idiotypic antibodies may circumvent the need for helper Lyt-1+2-3- T cells in the generation of allospecific killer T cells indicates that the former cells may normally function partly via such anti-idiotypic reactions.

Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains.

Evidence was obtained that both the WA and BLA crossidiotype (XId) groups are conformational antigens requiring both L and H chains and that with heat denaturation the antigens that define the XIds and antigen-binding activity are lost in parallel. In contrast, the primary structure-dependent crossreactive idiotype (CRI), PSL2, which is only weakly detected on native Wa and Bla monoclonal rheumatoid factors (mRFs), became prominently detected on the heated Wa and Bla mRFs. Heat denaturation may provide a simple method for distinguishing Ids determined by conformational antigen from primary structure-dependent Ids. In addition to heat denaturation, some acid conditions commonly used for preparation of RFs were also found to cause marked loss of Id antigen. The finding of PSL2-CRI on Bla mRF indicates that this Id is not unique to the WA XId.

Idiotype-anti-idiotype network. II. Activation of silent clones by treatment at birth with idiotypes is associated with the expansion of idiotype-specific helper T cells.

BALB/c mice immunized with bacterial levan (BL) produce a vigorous antibody response that fails to include antibodies expressing the idiotype of the beta 2 leads to 6 fructosan-binding myeloma protein ABPC48 (A48). Treatment of newborn BALB/c mice at 1 d of age with 0.1-10 microgram of either the A48 myeloma protein or monoclonal proteins that share idiotopes with the A48 family, followed by immunization with BL 2-4 wk later, produces an anti-BL response that is dominated by the A48Id. Various degrees of activation of the A48Id BL response were observed by injecting mice with A48 monoclonal protein only up until 3 wk of age. Activation of the A48Id clones by treating with A48 monoclonal protein was ineffective in mice who were older than 4 wk. Elicitation of an A48Id BL response required specific antigenic stimulation with either beta 2 leads to 6 or beta 2 leads to 1 fructosan epitopes, because it does not occur after injection with TNP-Ficoll in spite of the A48 treatment. The expansion of A48Id clones in mice treated at birth with A48 monoclonal protein is associated with an increase in A48Id-specific helper T cells. The binding specificity of these cells was demonstrated by infusing them into nu/nu BALB/c mice and observing that they rendered help that enalbed the animal to mount an anti-TNP response after immunization only with A48-TNP, but not with MOPC384-TNP conjugates. The helper activity of these cells is sensitive to the effects of treatment with anti-Lyt-1.2 antibodies plus complement. A predominantly A48Id BL-specific response can be transferred into lethally irradiated mice by infusing them with purified T and B cells from A48-treated mice. The transfer of this response can be ablated by treating the T cells with anti-Lyt-1.2 antibodies plus complement. These results indicate that A48Id-specific helper cells possess the ability to select the A48Id-bearing B cell precursors for expression, thus exerting a fine-tuning effect on the idiotypic expression of the anti-BL repertoire. We propose that this idiotype-induced idiotype response, which can be, in principal, induced by idiotypes provided by the mother, plays an important role in the expansion of precursors of antibody-forming cells during embryonic as well as postnatal life.

Fine idiotype analysis of B cell precursors in the T-dependent and T-independent responses to alpha 1-3 dextran in BALB/c mice.

In this study BALB/c B cell precursors responsive to the T-independent (TI) type 2 (TI-2) antigen, dextran B1355S (DEX), and the T-dependent (TD) derivative, dextran-Limulus hemocyanin (DEX-Hy) were examined for isotype and idiotope expression using the splenic focus assay. The predominant isotype detected in the TI assay was IgM, while IgA was the predominant isotype expressed in the TD assay. There was also a fourfold increase in the number of foci secreting more than one isotype in the TD assay vs. the TI assay without an overall change in anti-DEX precursor frequency, suggesting that carrier-primed T cells enhance the expression of non-IgM isotypes possibly by increasing the frequency of isotype switching by individual B cell precursors. A panel of distinct monoclonal antiidiotype antibodies (MAIDs) was then used to examine idiotope expression by antibodies secreted in splenic foci responding to DEX and DEX-Hy. This analysis revealed considerable diversity in the idiotope profiles expressed by all isotypes tested. There appeared to be no differences in idiotope diversity among the various isotypes. A similar diversity of idiotope profiles was obtained from both TI and TD splenic foci, indicating that a comparable degree of diversity was associated with the antibodies generated by TI and TD precursors. Idiotype analysis of IgM-IgA-secreting foci with a panel of monoclonal antiidiotope antibodies revealed slight idiotypic differences between the two isotypes secreted in the same focus in about half the cases. These results suggest that somatic variation occurs during the antigen-driven maturation of B cell precursors, within the 15-d time frame of the splenic focus assay, and may be associated with isotype switching.

Molecular characteristics of antibodies bearing an anti-DNA-associated idiotype.

Anti-double-stranded DNA antibodies are the hallmark of the disease systemic lupus erythematosus and are believed to contribute to pathogenesis. While a large number of anti-DNA antibodies from mice with lupus-like syndromes have been characterized and their variable region genes sequenced, few human anti-DNA antibodies have been reported. We describe here the variable region gene sequences of eight antibodies produced by Epstein-Barr virus (EBV)-transformed B cells that bear the 3I idiotype, an idiotype expressed on anti-DNA antibodies and present in high titer in patients with systemic lupus. The comparison of these antibodies to the light chains of 3I+ myeloma proteins and serum antibodies reveals that EBV transformation yields B cells producing antibodies representative of the expressed antibody repertoire. The analysis of nucleotide and amino acid sequences of these antibodies suggests the first complementarity determining region of the light chain may be important in DNA binding and that paradigms previously generated to account for DNA binding require modification. The understanding of the molecular genetics of the anti-DNA response requires a more complete description of the immunoglobulin germ line repertoire, but data reported here suggest that somatic diversification is a characteristic of the anti-DNA response.

ABPC 48 cross-reactive idiotopes in BALB/c mice. Natural and levan-induced expression.

Using monoclonal antiidiotypic antibodies, we developed a sensitive binding assay that detects molecules with one or with two idiotopes of the ABPC48 idiotype. ABPC48 cross-reactive idiotypes were thus shown to be present in substantial amounts in sera of nonimmunized mice. Levan binding sites are found on these idiotypes. During the life time of the mice, the natural anti-levan titer increases while ABPC48 idiotypic expression remains constant, suggesting different controls for these two activities. On the other hand, ABPC48 cross-reactive idiotypes participate--as minor components--in the response that follows a deliberate immunization with bacterial levan. This induction process is likely to reflect the selection of idiotopes expressed by the B cell clones preactivated in sera of nonimmunized mice rather than the activation of silent clones. We suggest that a similar situation might explain the reported emergence of ABPC48 idiotypes in animals primed with antiidiotypic antibodies and subsequently stimulated with levan.

Generation of immunoglobulin heavy chain diversity subsequent to cell surface immunoglobulin expression in the avian bursa of Fabricius.

The avian bursa of Fabricius represents a site for the generation of antibody diversity. Transfer of neonatal bursal cells into cyclophosphamide-treated neonatal chickens results in reconstitution of recipient bursae with donor-derived bursal stem cells. These stem cells express cell surface IgM and, under conditions of limiting donor bursal stem cell numbers, each reconstituted bursal follicle is colonized by a single precursor cell. The expression of an Ig VH idiotype, CVH-1, was found to be heterogenous within such clonal follicles. The diversity generated within the bursa is subsequently found within the peripheral B cell compartment. Thus, the generation of functional Ig H chain diversity is shown to occur subsequent to Ig H chain rearrangement and expression.

Extracellular idiotypic immunoglobulin arising from human leukemic B lymphocytes.

The peripheral blood lymphocytes of nine out of nine patients with typical surface Ig-positive chronic lymphocytic leukemia but no paraprotein visible on serum electrophoresis have been shown by radioimmunoassay to export small amounts of pentameric IgM during culture (in the range of 2.4-7.2 ng/10(7) cells per h); three out of nine also exported monomeric IgD (0.7-1.4 ng/10(7) cells per h). Immunoglobulin turned over on the cell surface did not appear to contribute to material in the culture fluid, except possibly as vesicle-bound Ig. In three cases, which included two of the IgD producers, anti-idiotypic antibody raised against the cell surface Fab mu was used to demonstrate the idiotypic nature of the exported Ig. Anti-idiotypic antibody was also used to measure levels of idiotypic Ig in the sera of these three patients as a proportion of the total Ig. Total serum IgM was depressed in all three patients, and the idiotypic IgM represented 43%, 65%, and 96% of the IgM. The findings suggest that in typical chronic lymphocytic leukemia involving B lymphocytes, the export of a small amount of idiotypic Ig by the neoplastic cells in a common or even usual occurrence.

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

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

Regulatory idiotypes. T helper cells recognize a shared VH idiotope on phosphorylcholine-specific antibodies.

Priming of BALB/c mice with phosphorylcholine-hemocyanin (PC-Hy) induces T helper cells that are detected in splenic fragment cultures responding to immunization with trinitrophenylated PC-binding myeloma proteins, TEPC 15 (TNP-T15) and MOPC 167 (TNP-M167). Trinitrophenylation did not alter the binding site, idiotype, or isotype of the antibodies as demonstrated by binding studies. To assay idiotype-recognizing helper cells, Ly-2.2-depleted T cells from PC-Hy-primed donor mice were transferred to syngeneic athymic mice. Splenic anti-trinitrophenol fragment cultures were prepared from the nude recipients, and the response to TNP-T15 and TNP-M167 was measured by enzyme-linked immunosorbent assay. The number of responding fragments is dependent on the number of transferred primed T cells. The homing efficiency of 51Cr-labeled helper cells into the spleen of nude recipients was determined. The frequencies of T helper cells taken from PC-Hy-primed donors required for a B cell response to TNP-T15 or TNP-M167 were indistinguishable. The fine specificity of the anti-PC idiotype-recognizing T helper cells was studied by adding hapten (PC) or unconjugated myeloma proteins to fragment cultures as inhibitors at the time of immunization. PC and PC-bovine serum albumin, as well as T15 and M167, inhibited the helper function in vitro. Furthermore, free heavy chains of T15 and M167 partially inhibited T help, but free light chains of both idiotypes had no effect. These findings collectively show that T helper cells, induced by priming with antigen, recognize a shared idiotypic determination on T15 and M167 that is part of the PC binding site. The heavy chains of T15 and M167 appears to be the major structural component of this determinant. Evidently, T helper cells can recognize a shared determinant that is present on idiotypically different myeloma proteins. This determinant appears to be conserved throughout evolutionary and somatic mutations. The role of this shared, binding site-related idiotypic determinant as a regulatory idiotype in T-B cell interaction is discussed.

Immunization of mice against African trypanosomiasis using anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised against three protective monoclonal antibodies, each with specificity for the variable antigen type (VAT) of a clone of Trypanosoma rhodesiense. The IgG1 fractions of each were pooled and administered to BALB/c mice 3-4 wk before homologous challenge. The course of primary parasitemia was altered in 19 of 30 anti-Id-treated animals. The immunity was manifested as either: (a) complete protection, (b) reduced parasitemia, or (c) selection against parasites bearing the original VAT. The three idiotypes (Id) were found in variable levels in serum during the course of infection in control animals. However, in all anti-Id-treated mice that displayed immunity, one Id in particular (7H11) was detectable much earlier in infection and in higher levels than in control mice or anti-Id-treated, nonimmune mice. Six of nine mice treated with the anti-7H11 Id alone also displayed immunity, manifested in this case exclusively as selection against parasites bearing the original VAT. The effect was again associated with the more rapid appearance of the Id after infection. Specificity of the anti-Id-induced immunity was supported by the failure of anti-7H11 Id treatment to alter the course of infection with a heterologous clone of T. rhodesiense. To our knowledge, this is the first report of the antigen-independent induction of antimicrobial immunity using anti-Id antibodies.

Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline.

5-15% of lymphocytes in the peritoneums of normal adult B10.H-2aH-4bp/Wts (2a4b) mice are CD5+ (Ly-1) B cells that recognize phosphatidyl choline (PtC), a phospholipid component of all mammalian cells. We produced a set of IgM-secreting hybridomas from the peritoneal cells of normal, adult 2a4b mice. We found that this set of hybridomas shows a similarly high frequency of antibodies specific for PtC (21 of 86) that also react with bromelain-treated mouse erythrocytes. Restriction fragment analysis of Ig gene rearrangements and analysis of expressed Ig idiotypes reveal that these cells use a restricted set of variable region genes to generate the PtC-specific antibodies. The Ig genes used by the PtC-specific hybridomas appear to be the same as those found in the PtC-specific Ly-1 B cell lymphomas, CH27 and CH34.

Uniform high frequency expression of autoantibody-associated crossreactive idiotypes in the primary B cell follicles of human fetal spleen.

At 23 wk of gestation, the fetal spleen contains follicles of lymphocytes that coexpress B cell differentiation antigens, surface Ig, and the 67-kD pan-T lymphocyte antigen, CD5 (Leu-1). Such cells are thought to represent the normal equivalent cells of B chronic lymphocytic leukemia (CLL). This B cell leukemia is distinctive in that high proportions of patients have leukemic cells that express sIg bearing one or more crossreactive idiotypes (CRIs) that commonly are found on IgM autoantibodies. We performed immunohistochemical studies on fetal spleen at 23 wk of gestation using a panel of mAbs specific for autoantibody-associated CRIs. We find that high proportions (5-17%) of the lymphocytes within each follicle react with any one of the anti-CRI mAbs. Furthermore, there is little variation between primary follicles in the proportions of cells that express a particular CRI. Using a cocktail of four anti-CRI mAbs, we detect autoantibody-associated CRIs on approximately one-third of the lymphocytes within each of the primary B cell follicles. These data indicate that the many of the Igs produced during early B cell development may be structurally related to IgM autoantibodies and to Ig expressed in CLL and related CD5 B cell malignancies. Furthermore, these studies suggest that the repertoire of Ig V genes expressed in each primary B cell follicle may be representative of the total restricted Ig V gene repertoire expressed during early B cell ontogeny.

Selection of antigen-specific, idiotype-positive B cells in transgenic mice expressing a rearranged M167-mu heavy chain gene.

Flow cytometric analysis of antigen-specific, idiotype-positive (id+), B cell development in transgenic mice expressing a rearranged M167-mu gene shows that large numbers of phosphocholine (PC)-specific, M167-id+ B cells develop in the spleen and bone marrow of these mice. Random rearrangement of endogenous V kappa genes, in the absence of a subsequent receptor-driven selection, should give rise to equal numbers of T15- and M167-id+ B cells. The observed 100-500-fold amplification of M167-id+ B cells expressing an endogenous encoded V kappa 24]kappa 5 light chain in association with the M167 VH1-id transgene product appears to be an antigen driven, receptor-mediated process, since no amplification of non-PC-binding M167 VH1/V kappa 22, T15-id+ B cells occurs in these mu-only transgenic mice. The selection and amplification of antigen-specific, M167-id+ B cells requires surface expression of the mu transgene product; thus, no enhancement of M167-id+ B cells occurs in the M167 mu delta mem-transgenic mice, which cannot insert the mu transgene product into the B cell membrane. Surprisingly, no selection of PC-specific B cells occurs in M167-kappa-transgenic mice although large numbers of B cells expressing a crossreactive M167-id are present in the spleen and bone marrow of these mice. The failure to develop detectable numbers of M167-id+, PC-specific B cells in M167-kappa-transgenic mice may be due to a very low frequency of M167-VH-region formation during endogenous rearrangement of VH1 to D-JH segments. The somatic generation of the M167 version of a rearranged VH1 gene may occur in less than one of every 10(5) bone marrow B cells, and a 500-fold amplification of this M167-Id+ B cell would not be detectable by flow cytometry even though the anti-PC antibody produced by these B cells is detectable in the serum of M167-kappa-transgenic mice after immunization with PC.