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Germinal center formation is the result of antigenic stimulation of B cells in a T cell-rich area. B cells cycle through the germinal centers, and a small percentage survive to become plasma cells or memory B cells. The transformation from a mature B cell into a germinal center B cell and finally into a terminally differentiated B cell is not well understood. Human CD38 is highly expressed on both germinal center B cells and plasma cells, and is useful in delineating these B cell subsets and in understanding the signaling events involved in the development of these B cells. To determine whether CD38 expression on activated germinal center B cells and postgerminal center B cells influences germinal center differentiation, we studied the expression of CD38 in the mouse. CD38 is expressed on follicular B cells in the Peyer's patches but is down-regulated on germinal center B cells located within the Peyer's patches. CD38dim/-B220+ germinal center B cells are also found in the spleens of immunized but not control mice, suggesting that Ag-stimulated germinal center formation is involved in the production of CD38dim/-B220+ B cells. Furthermore, mature plasma cells isolated from in vitro LPS cultures do not express CD38, but do contain high levels of cytoplasmic Ig. These results are in contrast to studies in humans in which CD38 is not found on follicular B cells but is highly expressed on germinal center B cells and plasma cells.

BALB/c mice were inoculated i.p. with a cross-reactive anti-Idiotypic mAb (designated FD5-1) in the absence of Ag or adjuvants. Injection with unmodified FD5-1 resulted in the induction of serum antibodies reactive with both FD5-1 (Ab3) and the hapten DNP (Ab1'). Endpoint titers of the Ab3 response showed an increase in serum IgM, which was dose-responsive to both the number of injections and the amount of FD5-1 antibody injected. The serum IgM Ab3 response was found to be thymus dependent and idiotypically specific for FD5-1. Athymic mice injected with FD5-1 were unable to produce a serum IgM Ab3 response, whereas their euthymic littermates produced strong Ab3 responses. Serum Ab3 responses and Ab1' were detectable only in the IgM isotype; no specific IgG responses were observed. Indeed, IgG recognized by FD5-1 appeared to be suppressed by FD5-1. Injection of mice with FD5-1 modulated serum IgM responses to DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one (OX), phosphorylcholine (PC), and alpha 1,3-dextran (DEX) in a thymus-dependent manner. FD5-1 injection induced IgM responses against DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one, and DEX but decreased IgM binding to PC. No detectable Ab1' responses to any of the aforementioned molecules were found when the same sera were probed for IgG. The specificity of serum Ab1' from FD5-1-injected mice was evaluated by antigenic inhibition. Binding of serum Ab1' to DNP-BSA was inhibitable by DNP-lysine, whereas equivalent concentrations of lysine alone had no inhibitory effect. The antigenic specificity of IgM from normal serum binding to PC-BSA was demonstrated by inhibition with free PC, and the binding of Ab1' from FD5-1-injected mice to DEX-coated plates was shown to be inhibitable by DEX. We have described in vivo network perturbation in adult BALB/c mice injected with anti-Id antibody in the absence of Ag or adjuvants. Our findings show that injection of the cross-reactive anti-Id FD5-1 can induce thymus-dependent Ag-specific responses. In two systems where FD5-1 functions as an anti-anti-anti-Id antibody (PC and DEX), thymus-dependent responses were also observed. FD5-1 injection suppressed antibodies binding to PC, whereas DEX-specific responses were induced.

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.

The most D-proximal functional VH gene, VH81X, is preferentially expressed in the mouse fetal B cell repertoire; however, it is expressed in few B cells in the adult. To determine when VH81X gene expression affects size and phenotype of particular stages in B cell differentiation, transgenic mice have been developed expressing a germline fetal liver-derived VH81X-mu rearrangement. Comparative analysis of B lymphopoiesis reveals similarities and differences between fetal liver and adult bone marrow which pinpoint developmental stages in mice during which VH81X-expressing B cell progenitors expand or deplete compartment sizes. These include a similar reduction in c-kitR+ and establishment of a predominant CD43low/HSAhigh phenotype within the B220+ CD43+ compartment which is dependent on the association of the transgene with lambda 5. In contrast, the CD43- pre-B and immature B cell compartments are expanded in the fetus but not in the adult. In addition, there are other factors that later disfavor the survival of VH81X-expressing B1 and B2 cells. Thus the failure to detect VH81X-bearing B cells in the adult is the result of a multistep selection process occurring at all stages during B repertoire expansion.

Human CD7 is a 40 kDa protein expressed on thymocytes, early T, B, NK and myeloid lineage cells in bone marrow, and on mature T and NK cells. Previous studies suggested human CD7 may be involved in T and NK cell activation and/or adhesion, and that CD7-mediated cell activation may be transduced via the lipid kinase phosphatidylinositol 3-kinase (Pi3-kinase), a heterodimeric cytosolic protein consisting of an 85 kDa adaptor subunit that is coupled to a 110 kDa catalytic subunit. It has recently been shown that a sequence motif present in the cytoplasmic tall of both human and mouse CD7 bound with high affinity to recombinant SH2 domains present in the p85 subunit of Pi3-kinase. In this work, we used co-precipitation with anti-CD7 mAb 3A1 and recombinant p85 SH2-GST fusion proteins and peptide competition analysis to demonstrate that the cytoplasmic tail of CD7 interacts with a functional Pi3-kinase via the pTyr-X-X-Met motif. Furthermore, we show that cross-linking of CD7 markedly increased the amount of Pi3-kinase activity associated with CD7. The interaction of CD7 with the Pi3-kinase signal transduction pathway provides a mechanism for the previously observed functional responses attributed to CD7-mediated T and NK cell activation.

Monoclonal hybridoma antibodies to the human IgA subclasses were produced by immunizing mice with purified myeloma proteins. These antibodies were shown to be specific for the appropriate IgA subclass by enzyme-linked immunoabsorbant assay (ELISA) and by immunofluorescent staining of myeloma plasma cells and B cells from normal individuals. These antibodies were used to demonstrate age-related shifts in the proportions of IgA1- and IgA2-bearing B cells that could be correlated with 3 distinct staining patterns. In the newborn equal numbers of IgA1 and IgA2, B cells were found. These cells had only small amounts of surface IgA in a patchy distribution. They also expressed surface IgM. In the infant, large lymphoblastoid cells were observed that bore more IgA in a homogeneous pattern but did not express IgM. Of these cells, 98% were positive for IgA1. In the adult, 80% of the IgA B cells were positive for surface IgA1, and 20% were positive for IgA2. These were small lymphocytes brightly stained for IgA and negative for IgM. In culture, the adult B cells responding to pokeweed gave rise to roughly equal numbers of IgA1 and IgA2 plasma cells. These results suggest that there are equal numbers of precursor cells for IgA1 and IgA2 whose expansion, further differentiation, and migration are selectively affected by immunoregulatory controls.

We used four mouse monoclonal antibodies (Mab) as probes of functional sites of human complement protein B. Two Mab, HA4-1B (gamma 2a kappa) and HA4-15 (gamma 2a kappa), reacted with the same or adjacent epitopes on the Bb fragment of B, while the other two, HA4-1A (gamma 1 kappa) and FD3-20 (gamma 1 kappa), reacted with distinct epitopes on Ba. All reactive epitopes were expressed on native B and only one, recognized by the anti-Ba Mab HA4-1A was more reactive on isolated Ba than on B. These binding specificities were determined by direct binding radioassays and confirmed by inhibition studies. Immunoelectron microscopy of B and Bb in complex with anti-Ba and anti-Bb revealed that the recognized epitopes are on opposite sides of the molecule and are on discrete domains. All four Mab inhibited the hemolytic activity of B, although with different efficiencies and through different mechanisms. The main effect of the two anti-Bb Mab was an increased rate of loss of hemolytic sites from preformed EC3bBb C3 convertase presumably through accelerated dissociation of Bb. On the other hand, the main effect of the two anti-Ba Mab was inhibition of binding of B to C3b. HA4-1A was more efficient, inhibiting by 50% the binding of [125I]B to EC3b at 10 micrograms/ml as IgG and at 13 micrograms/ml as Fab. The data suggest that a binding site for C3b on intact B is located on the Ba portion of the molecule.

We have used two rat monoclonal antibodies (Mab) (Bet-1 and 331.12), a mouse Mab (AF6-78.25), and an alloantiserum (SJA anti-BAB/14) in two-color immunofluorescence analysis and enzyme-linked immunosorbent assays to examine the expression of IgM allotypes at various stages of mouse B cell development, especially at the pre-B cell stage. In agreement with findings previously reported by others, these antibodies displayed the patterns of allotypic reactivity with IgM+ B cells and plasma cells from the appropriate strains of mice: Bet-1 antibody is specific for a allotype, AF6-78.25 and SJA anti-BAB/14 for b allotype, and 331.12 for both a and b allotypes but not e allotype. These antibodies, however, did not react with isolated mu-heavy chains prepared from secreted molecules or synthesized in situ by normal pre-B cells or pre-B cell-derived hybridomas. The requirement of light chain participation in the expression of IgM allotypic determinant(s) was additionally suggested by analysis of a fetal liver-derived hybridoma (F1-26-11) that has been shown to secrete IgM heteromolecules composed of C57BL/6-derived mu-chain and BALB/c-derived kappa-chains. In contrast to the nonreactivity with C57BL/6 mu-only pre-B cell-derived hybridomas, all three antibodies with specificity for the b allotype (AF6-78.25, SJA anti-BAB/14, and 331.12) reacted with this particular hybridoma. After treating surface IgM molecules on B lymphocytes with papain to cleave Fab fragments, the b allotype reactivity of AF6-78.25 mouse Mab, but not 331.12 rat Mab, disappeared, whereas the a allotype reactivities of two rat Mab (Bet-1, 331.12) were not altered. These results suggest that the IgM allotypic epitopes defined by these antibodies appear to be sterically dependent on the assembly of the whole IgM molecules, and rat Mab 331.12 and mouse Mab AF6-78.25 define two separate b allotype specificities, one on the Fc portion and the other on the Fab portion of the IgM molecules, respectively.

By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immunoglobulin nonproducing plasmacytoma cell line, a B cell hybridoma was isolated (MM-60) that has been shown by multiple criteria to produce a bona fide auto-anti-(anti-T15 idiotype) antibody. In vivo administration of MM-60 antibody suppressed T15+ anti-PC antibody production in an idiotope-specific manner by activation of an intervening set of anti-T15 B cells. These T15-specific B cells i) appeared to express germline-encoded variable region gene products, ii) developed in parallel to, but independent of, T15+ B cells, and iii) suppressed the anti-PC response in a T cell-independent fashion. Variants of T15+ anti-PC B cells possessing aberrant immunoglobulin heavy chain D region structure escaped from the suppression imposed by this anti-T15 B cell set, suggesting that a function of the heavy chain D region may be to contribute to the formation of molecular target sites for idiotype-directed regulatory cells and/or antibodies. The indigenous nature of these particular populations of anti-idiotypic and anti-(anti-idiotypic) B cells and the ability of their immunoglobulin products to regulate antigen-specific B cells in vivo provides strong supportive evidence for the significant role idiotype-directed network interactions play in regulating specific antibody production during a normal immune response.

BALB/c mice were injected with IgM mAb specific for Moloney murine leukemia virus (M-MuLV)-determined cell surface Ag in an attempt to inhibit Moloney sarcoma growth. The monoclonal IgM significantly inhibited sarcoma growth when given to the mice after inoculation with Moloney murine sarcoma/leukemia virus, and also potentiated the in vivo antibody response specific for M-MuLV Ag. These responses were significantly greater than the primary response to the virus alone in age- and sex-matched control mice, and were also seen in mice which were injected with the IgM antibody only and not with virus, suggesting that an Ag-independent mechanism may be involved. The M-MuLV-specific serum antibody responses induced by the monoclonal IgM, with or without prior virus inoculation, were predominantly of the IgG1 isotype, with some IgG2a; no other isotypes were found to have titers significantly higher than in the normal response to virus alone. M-MuLV-specific IgG1 was detected only in mice injected with monoclonal IgM, and not in the response to virus alone. The same sera also had high titers of anti-idiotypic antibodies, (Ab2), as well as anti-anti-idiotypic antibodies (Ab3). It appears, therefore, that passive immunization with M-MuLV-specific IgM mAb activates an idiotypic network, which results in both Ab2 and Ab3 responses; the M-MuLV-specific response may be considered a subset of Ab3.

The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.

Monoclonal antibodies to idiotypic determinants are being used with increasing frequency for analysis and treatment of B cell malignancies. In the present study we have compared the idiotypic specificities of a panel of 39 mouse monoclonal anti-idiotype (anti-Id) antibodies developed against 16 monoclonal human immunoglobulins (Ig). The Id cross-reactivities of these antibodies with Ig products of normal and abnormal B cells were examined by immunofluorescence and immunochemical methods. The reactivity patterns of these anti-Id antibodies with a normal population of plasma cells were highly variable in the immunofluorescence assay. Six were reactive with 2 to 10% of normal plasma cells, 30 with 0.1 to 2% of plasma cells, and three with less than 0.1% of plasma cells from blood, bone marrow, spleen, or tonsils. These reactivity patterns were relatively consistent among samples from 23 Caucasian, black, and Oriental adults. Although the reactivities of most anti-Id antibodies in the panel were not restricted to a particular Ig isotype, several were preferentially reactive with a particular heavy or light chain isotype: one IgM-, two IgA-, two kappa-, and three lambda-restricted antibodies. The immunofluorescence data was confirmed by biosynthetic analysis of Id+ molecules produced by a normal plasma cell population. When the reactivity of this panel of anti-Id antibodies with nonhomologous B cell neoplasms was examined, seven of 30 myelomas or leukemia-derived products and one of nine B cell leukemias or lymphomas without paraproteins were found to be cross-reactive with one or two of the anti-Id antibodies. Although clearly significant, the cross-reactivity between the Id of these paraproteins appeared to be of lower affinity than the reactivity of the homologous Id with their respective anti-Id antibodies. The results reveal a remarkable diversity in the specificities of monoclonal antibodies classified by conventional criteria as anti-Id antibodies, and indicate the potential usefulness of a panel of antibodies for analyzing clonal diversity in normal and abnormal B cell development.

The clonal origin of an IgA1 kappa B cell leukemia in a 71-year-old man (WF) was examined using a monoclonal anti-Id antibody and a panel of monoclonal anti-VH antibodies. Immunofluorescent studies revealed that all surface IgA1 kappa + leukemic cells in WF's blood and 10% of the IgM+ B cells in his bone marrow expressed the WF Id. Three percent of the IgA1 kappa + leukemic cells in blood also expressed gamma-chains in their cytoplasm. Approximately 0.1%, 1%, and 10% of bone marrow mononuclear cells, respectively, expressed mu-chains, gamma-chains, and alpha-chains in their cytoplasm, but no detectable light chains or surface immunoglobulins. These mu, gamma, and alpha-positive cells had the convoluted nucleus and narrow cytoplasm characteristic of normal mu+ pre-B cells. Sequential isotype switching among this unusual pre-B population was indicated by co-expression of mu-chains and alpha-chains by 11% and 63%, respectively, of the gamma pre-B cells. These pre-B cells and the surface alpha-chains and cytoplasmic gamma-chains of the leukemic B cells were reactive with one of four monoclonal anti-VH antibodies. The data suggest malignant transformation of the clone before isotype switching, and also imply light chain precommitment at the pre-B cell level of differentiation.

Molecular relationships between β2m and other cell surface antigens (H-2, Tla, Ia, and Thy-1) were studied with the double immunofluorescence method. Cells were incubated with an antiserum against one antigen, capped, and then tested with an antiserum against a second antigen. Capping of β2m on thymocytes led to simultaneous capping of H-2 and Tla but not Thy-1 antigens; capping of H-2 and Tla (but not Thy-1) antigens resulted in capping of all β2m detectable by the immunofluorescence method. Similarly, capping of β2m on B or T lymphocytes resulted in capping of H-2 and vice versa. Ia antigens on B lymphocytes were not capped after the redistribution of β2m. We conclude from these data that, in the cell membrane of thymocytes, virtually all the β2m molecules are associated with H-2 and Tla, but not with Thy-1, and that on the cell surface of T or B lymphocytes, virtually all β2m is associated with H-2 but not with Ia. We found no evidence of any significant free β2m on either thymocytes or splenocytes.

A large panel of enteric organisms was screened for agglutination with a panel of lambda monoclonal antibodies of different heavy chain isotypes specific for alpha 1-3 dextran (DEX). Two strains were initially isolated that were bound by most of the anti-DEX antibodies. One organism, Enterobacter cloacae strain MK7, which was characterized in detail, induced a typical lambda anti-DEX response in Igh-Ca mice that had a fine idiotope profile comparable with that induced by purified B1355S dextran containing alpha 1-3 glucosidic linkages (alpha 1-3-DEX). The determinant on the bacterial surface was shown by binding inhibition with nigerotriose to contain alpha 1-3 linkages. Hyperimmunization with these organisms of normal, athymic (nu/nu), or germ-free mice induced large amounts of IgM antibodies but very little IgG. This is the first description of an organism isolated from the normal gut flora of mice that can be shown directly to be bound by alpha 1-3-DEX antibodies and to induce the typical germ-line response of the DEX family of antibodies.

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.

Monoclonal anti-DNA antibodies from 13 cloned hybridoma cell lines have been used to analyze the clonal heterogeneity of autoimmune anti-DNA antibodies. The hybridomas were obtained by fusing spleen cells from a single, autoimmune (NZB X NZW)F1 mouse. With the use of the criteria of isotype, isoelectric focusing patterns, fine specificity for various nucleic acids, and idiotype, the monoclonal anti-DNA antibodies were found to be heterogeneous in that 12 different clonotypes were identified among the 13 monoclonal antibodies studied. However, eight out of the 13 monoclonal antibodies have similar antigen specificity and share a common idiotype. This suggests that although autoimmune anti-DNA antibodies within a single (NZB X NZW)F1 mouse are the products of a large number of different antibody-producing clones, many of the clones produce antibodies with similar antigen-binding regions. Furthermore, in the majority of (NZB X NZW)F1 mouse sera the appearance of detectable levels of the common idiotype coincides with the appearance of anti-DNA autoantibody.

We describe a surface determinant shared by human monocytes and cells of the megakaryocytic axis that has been identified using a mouse monoclonal antibody. This monocyte-platelet antigen (MPA) is expressed on all (greater than 99%) of peripheral blood monocytes, platelets, and megakaryocytes. It is also expressed weakly on the monocytic cell line U937 and the promyelocytic line HL60 and is present on cells from 3 of 4 AML patients examined. It is absent from polymorphonuclear leukocytes, T and B lymphocytes, erythrocytes, and a panel of hematopoietic cell lines. MPA is stripped from monocyte membranes with pronase and is reexpressed overnight. The determinant is carried on a noncovalently linked biomolecular complex with molecular weights of 93,000 and 135,000.

The potential light chain repertoires expressed by IgM antibodies of fetal liver and adult spleen cells were compared. B cells from these sources were stimulated by the polyclonal mitogen bacterial lipopolysaccharide and after 3 days were fused to construct hybridomas. The light chains synthesized by these hybridomas were isolated by electrophoresis and their heterogeneity was examined by isoelectric focusing. A total of 109 BALB/c and 77 CBA/J hybridoma-derived light chains obtained from fetal liver and adult spleen cells were analyzed in this way. Fifty-three and 45 unique light chains were obtained from BALB/c and C57BL/6 hybridomas, respectively. Some of these were repeated more frequently than others, but no significant differences were seen in the heterogeneity of the light chain spectrotypes of fetal and adult CBA/J and BALB/cJ mice. Furthermore, the kappa:lambda ratio of light chains were the same in fetal liver as adult-derived hybridomas. These results show that the heterogeneity of light chains as determined by IEF and the kappa:lambda ratio of IgM immunoglobulins is established very early in ontogeny.

Our findings indicate that a subset of VHIII antibodies, which we refer to as J606-GAC, contains a determinant(s) that is present on 5 to 15% of normal splenic B cells and plasma cells as detected by immunofluorescence. This subpopulation is detected by purified antibody, 0-1, which was prepared against a murine anti-group A carbohydrate (anti-GAC) hybridoma antibody. The J606-GAC subset includes the beta 2, 1 fructosan myelomas J606, EPC109, W3082, ABPC4, and UPC61, as well as 13 anti-GAC hybridomas. The 0-1 antiserum failed to react with hybridoma and myeloma immunoglobulins from murine VH groups I and II or other VHIII antibodies. By Western blot analysis, it was observed to react with isolated heavy, but not light, chains of J606-GAC-bearing antibodies. 0-1 failed to react with myelomas XRPC44 and J539, which have the same J region as J606 but a very different VH region. These observations indicate that 0-1 is detecting a VH region determinant. The J606-GAC marker recognized by 0-1 was expressed as early as 4 days after birth and was expressed at similar frequencies in germfree and conventional mice. Immunoprecipitation of both surface and biosynthetically labeled proteins from spleen cells or J606-GAC-positive hybridoma cell lines, respectively, confirmed that 0-1 was recognizing an immunoglobulin determinant.

The antibody response to alpha 1 leads to 3 dextran (DEX) in BALB/c mice consists of a family of closely related yet highly heterogeneous molecules. Although these antibodies have been previously characterized both idiotypically and structurally, detailed analysis of responding clones has not been possible using conventional anti-idiotype antibodies. Monoclonal syngeneic and allogeneic anti-idiotype antibodies (MAIDs) specific for anti-DEX antibodies were used in this study to dissect the serum antibody response to DEX in BALB/c mice. The constructed MAIDs showed considerable heterogeneity by isoelectric focusing and by their binding characteristics to a series of DEX specific myeloma and hybridoma proteins. The predominant heavy chain isotype of these MAIDs was gamma 1. These antibodies were used to identify individual idiotypic structures (IdI) on J558, or M104E as well as cross-reactive determinants common to both (IdX). Although both IdX and IdI MAIDs were obtained, IdI specific antibodies were obtained more frequently. BALB/c mice immunized with DEX produced antibodies expressing both IdI but in highly variable amounts. A large percentage of, but not all DEX specific antibody, could be accounted for by IdX bearing antibodies. Suppression of adult and neonatal mice by IdI specific MAIDs was effective with precise elimination of only those clones expressing IdI determinants leaving the total lambda bearing anti-DEX response intact. Suppression of adults and neonates by an IdX specific MAID resulted in a temporary and partial suppression of the total lambda bearing anti-DEX response along with total suppression of the IdX portion of the response. Unlike other systems these monoclonal antibodies produce only suppression, and under a variety of conditions enhancement of anti-DEX responses has not been observed.