IgA anti-dextran B1355 responses.

Injection of mice bearing the Ig-1a allotype with dextran B1355 results in an IgM antibody response that is generally regarded as thymus independent. Moreover, the antibody is directed to alpha[1,3] determinants on dextran B1355 and shares cross-reacting idiotypic determinants with a lambda 1 IgA (J558) myeloma protein as well as a lambda 1 IgM (MOPC 104E) myeloma protein. In this study, we show that BALB/c (Ig-1a) mice injected with dextran B1355 produced highly significant IgA anti-dextran responses with specificity directed to the alpha[1,3] epitope. Kinetics of the IgA anti-dextran response in BALB/c mice paralleled kinetics of the IgM response. However, the magnitude of the IgA response was markedly T cell dependent and age dependent.     

Analysis of the diversity of murine antibodies to dextran B1355. I. Generation of a larger, pauci-clonal response by a bacterial vaccine.

Mice immunized with a combination of dextran B1355 in adjuvant followed by three injections of 2 x 10(9) Escherichia coli B organisms produced an average of 14.5 mg/ml of anti-dextran antibodies. It was demonstrated that the stimulating effect of E. coli B was due to antigenic determinants cross-reactive with B1355 and not solely because of adjuvant properties of the organism. The anti-dextran antibodies were distributed among both 7S and 19S components. Isoelectric focusing of the 7S antibodies showed several spectrotypes of antibody, most of which were shared by the majority of the individual sera. The limited spectrotypic heterogeneity of the 7S antibodies was supported by idiotypic studies. Thus, a heterologous, anti-idiotypic serum, rabbit anti-M104, was prepared which distinguished between two closely related myeloma proteins, M104 and J558,with specificity for alpha-(1 leads to 3) dextran. This antiserum demonstrated that some, but not all, of the 7S and 19S anti-dextran antibodies possessed variable region determinants cross-reactive with M104.     

Detection and characterization of idiotype-specific enhancing cells generated in mice immunized with idiotype

Cellular interaction between MOPC-104E (M104E) cross-reactive idiotypic (CRI) antibody-producing B lymphocytes and lymphocytes generated by immunization with the relevant idiotype, M104E, was investigated. Adoptive transfer of M104E idiotype-primed and normal spleen cells into 600R x-irradiated syngeneic recipient mice resulted in striking enhancement of the M104E-CRI positive antibody response upon simultaneous immunization of recipients with dextran B1355S. The enhancement was not attributable to a simple additive effect but was due to synergistic cooperation between the two lymphocyte populations. This synergistic enhancement of the anti-idiotype immune cells producing CRI antibody was specific for MOPC-104E CRI, and was reproducible in an in vitro culture system. Because of the cellular characteristics of the enhancing cells, they were assumed to be B lymphocytes specific for the corresponding idiotype, since the activity was not abrogated by treatment with anti-Thy-1, anti-Lyt-1, anti-Lyt-2, or anti-brain-associated theta antisera plus complement, but was eliminated by means of a planning method using a rabbit-anti-mouse immunoglobulin-coated or idiotype-coated dish. The mechanisms of interaction between the CRI-positive B cells and anti-idiotypic B cells in response to the thymus-independent antigen dextran B1355S are discussed.     

Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: Specificities and reactivities of the antibodies formed in rabbits to stearyl-isomaltosyl oligosaccharides

The specificities and the sizes and shapes of the antibody combining sites of the 15 antisera raised against various stearyl-isomaltosyl oligosaccharides were studied by quantitative precipitin and precipitin inhibition. The antibodies precipitated well with dextrans B512 and B1424 but less well with B1299S and B1355S. Only 3 of the 15 antisera reacted with linear dextrans; however, with about 50% of the added antibodies being precipitated, showing that most of the antibodies cannot bind to internal determinants along the dextran molecules and are similar to myeloma protein W3129 in having cavity-type sites which bind only to terminal nonreducing ends of α1 → 6 dextran. Antibodies differing in the sizes of their antibody combining sites were elicited in different rabbits by the same antigen. Of the 15 antisera studied, four have antibody combining sites as large as IM3, five as large as IM4, three as large as IM5 and three as large as IM6. The association constants for various isomaltose oligosaccharides of an antiserum (R-862) showing fewest bands in isoelectric focusing gel were determined by affinity electrophoresis and were comparable to W3129.     

Idiotype-specific T lymphocytes. I. Regulation of antibody production by idiotype-specific H-2-restricted T lymphocytes

Cytotoxic T lymphocytes (CTL) specific for MOPC-104E myeloma cells of BALB/c origin could be induced in BALB/c, (BALB/c X BALB.B)F1, and (BALB/c X BALB.K)F1 mice. (BALB/c X BALB.B)F1 CTL activity specific for MOPC-104E was effectively inhibited by anti-H-2d but not by anti-H-2b alloantiserum. However, the activity was hardly blocked by specific anti-idiotypic antibodies to MOPC-104E. For further analysis of the recognition of idiotype on target cells by CTL, the effect of those lymphocytes on anti-dextran B1355S antibody-producing B lymphocytes, which have a cross-reactive idiotype to MOPC-104E, was investigated. Lymphocytes from the CTL population did inhibit antibody production by dextran-immune spleen cells, but those from the CTL population specific for irrelevant myeloma cells (MOPC-167) did not. The (BALB/c X BALB.K)F1 CTL population suppressed the antibody production of BALB/c but not of BALB.K. This indicates that F1 cells can preferentially see H-2 antigens of immunizing myeloma cells on target B lymphocytes. The inhibition of antibody production was antigen specific and was only restricted to the PFC that were inhibitable by anti-idiotypic antibodies. The surface phenotypes of the cells that inhibited the antibody production were Thy-1+, Lyt-1-, Lyt-2+, and I-J-. These results strongly suggest that CTL specific for MOPC-104E recognize self H-2 antigens simultaneously with idiotypic determinants on B lymphocytes. Possible immunoregulatory roles of idiotype-specific CTL on antibody production systems are also suggested.     

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

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

Regulation of the IgM and IgA anti-dextran B1355S response: synergy between IFN-gamma, BCGF II, and IL 2

T cell help is required for the induction of the humoral antibody response to dextran B1355S, a type II thymus-independent bacterial polysaccharide antigen. In the present study we have identified three B cell growth and differentiation factors that can substitute for T cells in the induction of IgM and IgA antibody responses to alpha(1,3) glucan determinants on dextran B1355S. Dextran B1355S stimulated murine B cell cultures supplemented with a combination of murine recombinant interferon-gamma (IFN-gamma) and a late-acting B cell growth and differentiation factor, BCGF II, produced both IgA and IgM anti-alpha(1,3) dextran plaque-forming cells (PFC). Interleukin 2 (IL 2) was not required for those responses. In contrast, recombinant IFN-gamma and recombinant IL 2 in combination supported the induction of IgA but not IgM anti-alpha(1,3) dextran PFC. In all cases, depletion of surface IgA-bearing B cells significantly decreased IgA but not IgM anti-dextran responses, indicating that the B cells responding to those lymphokines already were committed to IgA expression. These studies indicate that B cell growth and differentiation factors can exhibit differential effects on the induction of IgA compared with IgM responses.     

Generation of helper T cells that recognize a cross-reactive idiotype through a network mechanism.

T cells that recognize the cross-reactive idiotype expressed on the heavy (H) chain of M104E (IgM, lambda 1) were induced in BALB/c mice by immunization with Dextran B-1355. T cells derived from mice immunized with 1 mg of Dextran B-1355 showed a marked proliferative response against M104E, whereas T cells from mice immunized with Ficoll or lesser amounts of Dextran B-1355 did not. BCL1Id, which had an immunoglobulin isotype identical to M104E, did not induce proliferation of the T cells. These T cells also proliferated against J558 (IgA, lambda 1) which shared the cross-reactive idiotype of the anti-alpha (1----3) glucosidic linkage antibody with M104E on the H chain. The T cells proliferated more efficiently against F(ab')2-104E, Fab-104E and H104E, the H chain of M104E, than against intact M104E. The T cell proliferative response against the idiotype on M104E or even H104E required macrophages as antigen-presenting cells (APC) and the response was inhibited when APC were treated with NH4Cl or chloroquine, inhibitors of antigen processing. Moreover, anti-CD4 antibody or anti-Ia antibody inhibited the proliferative response. These results indicated that anti-idiotypic T cells of the helper type, which recognized a cross-reactive idiotype associated with Ia molecules in processed form, could be induced physiologically through a network mechanism.     

Failure of PFC inhibition assays to distinguish idiotypically between clonotypes that are readily distinguishable by RIA analysis

Two different hemolytic plaque assay protocols that are commonly used to quantitate idiotype-positive antibody-secreting cells have been compared to a standard radioimmunoassay (RIA) to test for their ability to discriminate between related, but idiotypically distinct, clonotypes. The idiotype proband used in this analysis is the individual specific idiotype associated with the dextran-binding myeloma protein M104E (M104E IdI). Antibodies specific for this private idiotype (anti-M104E IdI) were purified by a combination of adsorption and affinity chromatography of the immunoglobulin fraction isolated from the sera of rabbits repeatedly immunized with M104E. The same affinity-purified anti-M104E IdI antibodies were used in the hemolytic plaque assays and in the RIA. In one of the plaque assays, the putative idiotype-positive antibody-forming cells were scored by lysis of target erythrocytes to which the anti-idiotype had been covalently coupled. In the other plaque assay, the idiotype-positive cells were determined indirectly by anti-idiotype inhibition of PFC produced on dextran-coupled target erythrocytes. The fidelity of these two assays to quantitate the M104E private idiotype expression in individual BALB/c mice after a single immunization with dextran B-1355S was determined by comparing the plaque assay data to the data generated by a double-antibody, post-precipitation RIA of either the antibodies in the serum or of monoclonal antibodies produced by hybridomas. Our data indicate that both plaque assay protocols reflect an overestimate of the actual expression of M104E private idiotype. By using a library of dextran-specific hybridomas (that have been characterized in an RIA with respect to their M104E IdI cross-reactivity), we have shown that the PFC overestimate of the M104E expression observed in dextran-immune mice is due to the inability of both plaque assay protocols to distinguish between dextran-specific clonotypes that express idiotypes cross-reactive with, but not identical to, the 104E IdI. We conclude that the plaque assay should be used only in conjunction with an RIA to estimate the idiotype expression. This is especially true in situations where closely related cross-reactive idiotype families exist.     

Characterization of the primary IgM response to GAT and GT: conditions required for the detection of IgM antibodies.

Primary IgM antibody responses to synthetic linear copolymers of L-glutamic acid, L-tyrosine, and L-alanine were investigated. The appearance of primary IgM anti-GAT antibodies was detected in BALB/c mice by using a solid phase radioimmunoassay (SPRIA) procedure. The finding was verified for GAT in responder mice and GAT-MBSA and GT-MBSA in nonresponder mice in an indirect plaque forming cell (PFC) assay by using a rabbit antiserum directed against the mulambda myeloma protein, MOPC 104E. Facilitated IgM PFC could be inhibited by a purified muK myeloma protein, TEPC 183. Maximal facilitated IgM plaque response was found to precede the IgG response by several days. A direct plaque assay was developed for the detection of IgM anti-GAT plaques using poly-L-lysine (PLL) to couple GAT to sheep erythrocytes (SRBC). GAT-SRBC coupled by the PLL method optimally couple 4 to 5 times less antigen to the indicator cell surface than does the CrCl3-coupling method routinely employed in our laboratory. These findings were extended to a conventional antigen, chicken gamma globulin (CgammaG). We found that a less dense epitope coat on the indicator cell surface favors detection of direct IgM PFC, whereas a more densely coated indicator cell favors the detection of facilitated IgM and IgG PFC responses.     

Regulation of clones responding to dextran B1355S. III. The thymic-independent and thymic-dependent antibody responses

The primary antibody response of different mouse strains challenged with two antigenic forms of alpha (1-3) dextran, dextran B1355S and dextran-hemocyanin, was examined. Only BALB/c mice responded with both kappa and lambda antibodies. The kappa to lambda ratio was affected by factors such as the antigenic form of dextran, the time at which the serum was analyzed, and the priming regimen. Surprisingly, priming with hemocyanin in adjuvant increased the kappa portion of the response not only to dextran-hemocyanin but also to dextran B1355S. Other strains of mice responded with only lambda antibodies. These results extend our previous results on the analysis of dextran-specific B cell precursors.     

Role of dextran-specific suppressor T cells in the regulation of the immune response by a reactive form of dextran

Reactive forms of antigens or haptens have been shown to induce a state of hyporesponsiveness mediated in part by suppressor T cells. Injection of Balb/c x C57B16 F1 (CB6F1) mice with a reactive form of dextran B1355S (periodate oxidized dextran, dex-P) specifically reduced responses to dextran immunization within 1 day after dex-P treatment. This unresponsiveness lasted at least 23 days and required a reactive form of dextran for its induction since native dextran and oxidized/reduced dextran failed to induce tolerance. Furthermore, hyporesponsiveness could be induced by iv injection of dextran-coupled cells, especially peripheral blood lymphocytes, a result which suggests that in vivo coupling to cellular antigens is involved in dex-P-induced hyporesponsiveness. Suppression of the anti-dextran response could be transferred to normal mice with T-cell-enriched spleen cell populations from dex-P-injected mice. Interestingly, the presence of B cells in the transferred cell preparations interfered with detection of suppression. Both Lyt 1+2- and Lyt 1-2+ cells were involved in the dex-P-induced suppression; indeed, mixtures of these types of T cells led to the most profound degree of suppression. The suppressive activity of spleen cells from dex-P-injected mice could be removed by passage over dextran-coated plates. Moreover, cells eluted from the plates specifically suppressed anti-dextran responses of normal mice, indicating that dex-P injection induces a population of antigen-binding suppressor cells. This system will allow the study of the suppressor-T-cell receptors in a well-defined idiotypic system.     

Mechanisms of B cell tolerance. I. Tolerance to dextran B1355 induced with the oxidized dextran.

The bacterial dextran B1355, which is normally a potent thymus-independent immunogen, was made tolerogenic by oxidation. The injection of the oxidized dextran into BALB/c mice before, at the same time, or up to 4 days after the injection of the immunogenic form of the dextran resulted in a marked immunologically specific suppression of the number of anti-dextran antibody-forming cells found in the spleen. This suppression resulted from a direct inactivation of antibody-forming cell precursors rather than from either inhibition of antibody secretion or the exhaustive utilization of precursor B cells that have been observed in other tolerance systems. A substantial degree of tolerance was achieved after only a 1-hr in vivo exposure of the spleen cells to the tolerogen. At a dose of 1 mg of oxidized dextran per mouse, tolerance persised for at least 3 weeks. A complete recovery was apparent by 10 weeks. The stability of the tolerance was demonstrated by transferring tolerant spleen cells to irradiated recipients. The response in the recipient animals to an immunogenic dextran challenge remained suppressed. It appears that the tolerogenicity of the oxidized dextran is due to its ability to couple covalently with free amino groups in or near the receptor site of the cell membrane via the reactive dialdehyde groups of the dextran.     

Homologous monoclonal antibodies specific for alpha 1-3 dextran stimulate polyclonal proliferation of Lyb-5+ B cells

In this paper we describe the polyclonal blast response induced in nonimmune murine splenocytes by two homologous alpha 1-3 dextran-binding myeloma protein, J558 and MOPC104E. This stimulation appears to be independent of MHC or IgCh gene complex control, and proceeds entirely in a T-dependent fashion. The responding B cell population appears to belong to the more mature Lyb-5+ subset. This response was elicited by several independently prepared batches of J558 and MOPC104E, each of which was conclusively shown to be free of endotoxin contamination. Experiments are presented that suggest that this stimulation is being mediated via the Fab portion of these two myeloma proteins, in particular, a shared IdX, rather than through their Fc.     

Regulation of the anti-alpha (1,3) dextran response: two populations of dextran-reactive B cells that differ in their T cell requirements for induction to antibody synthesis

The in vitro antibody response to dextran B1355S, a thymus-independent Type 2 antigen, requires T cell-derived lymphokines but is not thought to require an activation signal from an antigen-specific T helper cell. The present study demonstrates that there are two dextran-reactive B cell populations in BALB/c mice with respect to the T cell requirements for the generation of antibody-forming cells. One population found among dextran-reactive spleen B cells from 12- to 14-mo-old BALB/c mice generated anti-dextran PFC in the presence of B cell growth factor (BCGF II) and IL 2 or the combination of BCGF II, IL 2, and IFN-gamma. A second population of dextran-reactive B cells found in spleen and Peyer's patches of 2-mo-old unprimed mice did not respond to these same lymphokines, but did generate anti-dextran plaque-forming cells in the presence of Thy-1.2+, L3T4+ T cells from Peyer's patches. However, splenic B cells obtained from 2-mo-old mice that had been primed with dextran 2 to 3 days after birth were shown to be responsive to the same lymphokines as dextran-reactive B cells from 12- to 14-mo-old mice. These results suggest that previous priming with dextran B1355S induces a dextran-specific B cell population that can be activated to antibody-forming cells in the presence of antigen and T cell-derived lymphokines, whereas a second, unprimed population requires an additional activation signal from L3T4+ T cells.     

Anti-immunoglobulin autoantibodies are not preferentially induced in polyclonal activation of human and mouse lymphocytes, and more anti-DNA and anti-erythrocyte autoantibodies are induced in polyclonal activation of mouse than human lymphocytes

Using a plaque assay with immunoglobulin (Ig)-coated SRBC, we and others have previously reported that the majority of polyclonally activated mouse lymphocytes secreted antibodies that appeared to be IgM anti-IgG autoantibodies. Careful reexamination of this assay, with application of several highly purified mouse serum and myeloma IgG and IgM preparations, revealed that IgM, which was a minor contaminant of Ig preparations, rather than IgG, was responsible for the formation of these plaques. High numbers of plaques could also be detected in assays with polyclonally activated human lymphocytes, Ig-coated SRBC, and anti-Ig developing sera. Of all IgG-, IgM- or IgA-secreting cells, 40 to 100% were detected with SRBC coated with gamma-globulin or Ig of the same isotype as the isotype to which the developing serum was specific; in general, low proportions of all PFC were detected with SRBC coated with Ig of a different isotype. Studies on the sequence of events leading to the formation of plaques with Ig-sensitized SRBC (both in humans and mice) revealed that antibodies detected in these assays were not able to bind to the Ig-coated SRBC (without the presence of developing serum), and therefore were not anti-Ig autoantibodies. It is our conclusion that the plaque assays with Ig-coated SRBC represent another type of a reverse hemolytic PFC assay that detects cells secreting antibodies regardless of their specificity, and these plaques are formed due to the cross-linking by the anti-Ig developing serum of the Ig coated on SRBC and the Ig secreted by lymphocytes. Our results confirmed preferential induction of anti-DNA antibody secreting cells in mice by showing that these antibodies indeed bind to DNA coated on SRBC. In cultures of polyclonally activated human lymphocytes, anti-DNA and anti-erythrocyte autoantibody-secreting cells were over 10 to 100 times less frequent than in mice. These results, therefore, disprove the concept of preferential induction of anti-Ig autoantibodies in the polyclonal activation of mouse and human lymphocytes, and show that anti-DNA and anti-erythrocyte autoantibodies are easily induced in the polyclonal activation of mouse, but not human, lymphocytes.     

Studies of mouse immunoglobulin allotypes by reverse plaque assay: Detection of spleen cells secreting immunoglobulins with specific allotype in C57BL/6J mice

A reverse hemolytic plaque assay for the detection and enumeration of mouse spleen cells secreting immunoglobulins bearing a particular allotypic specificity is described. Sheep red blood cells (SRBC) coated with protein A or anti-mouse gamma globulin antibody were employed as indicator cells and an anti-allotype antibody was used as developer. A comparison of the efficiency of protein A, goat anti-mouse or rabbit anti-mouse gamma globulin antibody-coated SRBC as indicator cells in the plaque assay indicated that the rabbit anti-mouse gamma globulin-coated SRBC gave the best results in terms of number and morphology of the plaques. The number of indicator cells in the assay mixture also significantly affected the quality of the plaques formed. When the mouse spleen cells were assayed with the indicator cells and an anti-allotypic antibody as developer in presence of complement in a liquid medium, only those cells secreting the immunoglobulin of the given allotypic specificity formed hemolytic plaques.     

Polysaccharides as labels for antibodies in electron microscopy.

Two polysaccharides, dextran 250 and dextran 70, were covalently linked to antibody molecules, antihuman immunoglobulin G and antihuman type O red blood cells. In electron microscope preparations exposed to lead citrate, polysaccharides, because they chelate lead, were quite dense. Polysaccharides served as a tag for the antibody molecules. Also, bacterial dextran 1355 was used to demonstrate antibody molecules on the surface of ascites tumor cells which are known to be producing a specific antibody to bacterial dextran 1355. The varying sized polysaccharide molecules that are readily available commercially, the high electron density of the polysaccharides after lead staining and a mild procedure for covalently linking polysaccharide to antibody make polysaccharides attractive as particulate labels for antibody in electronmicroscopy.     

Antibody response of BALB/c mice to dextran B1355S: alterations in the expression of an idiotype associated with the depletion of idiotype-binding cells

In this study, we established that BALB/c mice recognize and respond to the idiotype (M104E IdI) of a major dextran-specific clonotype within the BALB/c mouse repertoire. This idiotype recognition is established by demonstrating the presence of idiotype-binding cells and by the production of antibodies specific for the private M104E idiotype. To determine whether or not the idiotype-recognizing cells play a regulatory role during an immune response to dextran, the idiotype-binding cells were selectively removed either by panning or by radiation-induced killing. Two significant effects are observed when the depleted spleen cells are immunized with dextran. First, there is a substantial increase in the proportion of anti-dextran antibodies that are M104E IdI+. The second effect of the idiotype-specific cell depletion is the production of significant amounts of M104E IdI+ immunoglobulin molecules which do not bind dextran. The depletion experiments produced no alteration in the concentration of anti-dextran antibodies found in the serum or in the number of dextran-specific PFC in the spleen. The data indicate that idiotype-reactive cells can play a role in regulating the level of individual clonotype expression (i.e., the M104E clonotype), but that an alternative mechanism must exist for regulating the absolute amount of anti-dextran antibody produced after immunization.     

IgM complex receptors on subpopulations of murine lymphocytes.

Murine lymphocytes from spleen, lymph node, and thymus were examined for IgM complex receptors. Lymphocytes from all three organs were found to bind SRBC sensitized with IgM from various sources including: primary anti-SRBC serum, murine and rabbit anti-Escherichia coli LPS sera, and a murine IgM myeloma (MOPC 104E). Rosette formation by lymphocytes with IgM-sensitized SRBC was inhibited by soluble antigen-IgM complexes but not by IgM or antigen alone. Rosette formation was also inhibited by human IgM (Fc)5mu but not by Fab mu. Antiserum and complement treatment of the cells and subsequent recovery of the viable cells by trypsinization, filtration, and washing revealed the IgM rosette-forming cell (RFC) in the thymus to be a T cell. Spleen on the other hand was found to contain both B and T cells capable of binding IgM sensitized SRBC. Removal of both B and T cells from spleen cell suspensions eliminated all IgM RFC. The IgM complex receptor was found to be trypsin insensitive. Anti-Ig column fractionation enriched IgM RFC in spleen and lymph node suspensions passed through the columns, whereas cells bearing surface Ig, IgG complex receptors, and C3 receptors were retained in the columns.