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.     

Monoclonal anti-alpha (1----3) dextran antibodies of Igha BALB/c and Ighb C.B20 mice display striking similarities

Allotype Ighb congenic C.B20 mice when immunized with dextran B1355S are unable to produce anti-alpha (1----3) dextran antibodies that express the VH-associated cross-reactive IdX idiotype. This intrastrain-specific idiotype is normally associated only with the anti-dextran response of Igha mice of which BALB/c is a prototype strain. In this study we have obtained monoclonal hybridoma antibodies specific for the alpha (1----3) glucosidic linkage of dextran from C.B20 mice that were presensitized with rabbit anti-IdX antibodies. These antibodies display the light chain isotype distribution, the H chain amino terminal sequence, share VH-associated IdX idiotypic determinants, and finally the similar fine specificity for dextrans observed for anti-alpha (1----3) dextran antibodies of BALB/c mice.     

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.     

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 clones responding to dextran B1355S. II. Response of T-dependent and T-independent precursors

The existence of precursors for TI and TD alpha 1 leads to 3 dextran antigens in BALB/c mice was demonstrated. A T-dependent dextran antigen was prepared by coupling dextran B1355S to hemocyanin and subsequent digestion with dextranase. The PFC response of BALB/c mice primed with hemocyanin to dextran-hemocyanin was found to be 8 times higher than in unprimed animals. The splenic focus assay was adapted for the analysis of precursors responding to T-dependent and T-independent dextran antigens. Pretreatment of recipients with anti-thymocyte serum abolished the response in fragment cultures to dextran-hemocyanin but did not affect the response to dextran B1355S. The frequencies of precursors in the adult BALB/c mouse responding to dextran and dextran-hemocyanin were determined by limiting dilution analysis. The frequency of T-dependent precursors was found to be almost 3 times greater than the frequency of T-independent precursors.     

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.     

Estimation of antibodies specific for dextran.

Methods are described for the isolation and characterization of picogram quantities of anti-dextran antibodies. 14C-dextrans produced by using the dextransucrases of Leuconostoc mesenteroides strains B1355 and B512 were used in a radioimmunoassay. The specificity of this assay was verified by using cell cytoplasmic lysates from mouse plasmacytomas, J558 (anti-alpha 1 leads to 3 dextran) and W3129 (anti-alpha 1 leads to 6 dextran). Dextran produced by strain B1355 and insolubilized with epichlorohydrin was used as an immunoabsorbent.     

Genetics of the alpha 1,6-dextran response: expression of the QUPC52 idiotype in different inbred and congenic strains of mice

Antibodies to dextran B512 were raised in various strains of mice and were assayed by a radioimmunoassay procedure. Idiotypic antibodies to the IgA(k) dextran B512 binding myeloma proteins QUPC52 and W3129 of BALB/c origin were prepared in rabbits. After adsorption each antiserum was specific for the immunizing myeloma protein and did not react with hundreds of other myeloma proteins; nonetheless, antibodies to dextran B512 from various strains of mice cross-reacted in these test systems. Of the 2 idiotypes tested, the W3129 idiotype was more universally expressed in different strains of mice. The QUPC52 idiotype was the predominant idiotype in BALB/c anti-dextran B512 antibodies and was found in only a few other inbred strains. Using a battery of congenic and inbred strains, it was shown that the QUPC52 idiotype was controlled by genes linked to the Igh complex locus (chromosome 12) and to the Ig kappa complex locus (chromosome 6). The W3129 idiotype was found in a number of stocks of mice in the genus Mus recently isolated from the wild. The QUPC52 idiotype thus far was found only in inbred mice.     

A characterization of the BALB/c lambda and kappa class antibodies to alpha (1,3) glycosidic linkages

The avidity relationships of kappa and lambda antibodies with specificity for the glucosyl-alpha (1,3)-glucose disaccharide epitope were compared using a solid-phase hapten inhibition assay. The kappa class antibodies, induced by the T-cell-dependent antigen, nigerosyl-keyhole limpet hemocyanin (N-KLH), and the lambda class antibodies, induced by the T-cell-independent antigen, Dextran B1355, were only marginally distinguishable (approximately equal to 10-fold) on the basis of hapten inhibition using the free disaccharide, nigerose, as inhibitor. Analysis of the binding site sizes of antibodies induced by N-KLH through inhibition with oligosaccharides of different sizes showed that the disaccharide protein antigen did not select for antibodies with smaller binding site sizes. The cellular basis for these findings is discussed.     

Comparison of the homogeneous, primary anti-dextran B1355 antibody raised in BALB/c mice with protein 104E

The primary antibody response to dextran B1355 in BALB/c mice is largely a homogenous IgM antibody. This antibody appears to be similar if not identical to the myeloma protein, protein 104E, for the following reasons: a) isoelectric focusing of the 7S monomers, separately and together in co-isoelectric focusing, give the same pattern, both in the presence and absence of 4M urea; b) the inhibition of lysis of the dextran-coated SRBC by specifically purified anti-dextran antibody and by protein 104E required essentially the same concentration of dextran B1355. This similarity was further demonstrated by plaque assays with dextran-coated SRBC, in which the formation of plaques was inhibited by free dextran. Inhibition of plaques produced by both types of cells required essentially the same concentration of dextran B1355. On these bases, there appears to be no difference in properties (or in structure) between the myeloma protein and the induced antibody.     

A comparison of the IgG isotype expression in the so-called thymus-independent immune responses to alpha(1----3) and alpha(1----6) dextran

This paper presents data on the IgG antibody response against two "thymus-independent" dextran (Dex) antigens from Leuconostoc mesenteroides, alpha(1----3) Dex B 1355S and alpha(1----6) Dex B 512F in BALB/c and C57BL/6 mice, which are considered to be responders or low responders to the respective antigen. The data point toward three common rules governing the two anti-Dex responses despite immunogenetic and antigenic disparities: (1) age dependency of the IgG isotype regulation of the response; (2) down-regulation of IgG isotype expression by T cells; and (3) individually determined preposition for IgG isotype formation in a given animal.     

Secondary IgG responses to type III pneumococcal polysaccharide. II. Different cellular requirements for induction and elicitation.

Mice primed with a thymus- (T) dependent form of Type III pneumococcal polysaccharide (S3), i.e., S3 coupled to erythrocytes (S3-RBC) produce S3-specific IgG antibody after secondary challenge with either S3 or S3-RBC. The production of IgG antibody by mice challenged with S3 was shown to be T independent since secondary responses were enhanced when mice were treated with anti-lymphocyte serum (ALS) at the time of secondary challenge with S3 and T-depleted spleen cells responded as well as unfractionated spleen cells to S3 in an adoptive transfer system. Secondary S3-specific IgG responses in mice challenged with S3-RBC were shown to be T dependent by the same criteria. The results obtained by using S3 as the antigen indicate that IgG-producing B cells (B lambda cells) can recognize and respond to antigen in the absence of helper T cells. On the other hand, T cells were required for the induction of S3-specific memory B lambda cells since mice depleted of T cells by treatment with ALS at the time of priming with S3-RBC failed to produce S3-specific IgG antibody after secondary challenge with either S3-specific IgG antibody after secondary chall-nge with either S3 or S3rbc. Since RBC-specific memory cells were induced in T-deprived mice the results suggest that T cell regulation of IgG antibody production may vary for different antigens.     

Genes on different chromosomes influence the antibody response to bacterial antigens

B6.C congenic strains of mice, possessing histocompatibility (H) alleles from high responding BALB/cBy (C) mice on the genetic background of low responding C57BL/6By (B6) mice, were assayed for their ability to make an antibody response to Type III pneumococcal polysaccharide (SSS-III) and the (13) epitope of bacterial (Leuconostoc) dextran B-1355. The results affirmed that the antibody response to SSS-III is multigenic and that genes making a positive contribution to responsiveness are located on different chromosomes, i. e., chromosomes 1, 3, 4, 5, and 9. At least one other gene also influences responsiveness to SSS-III; it is linked to the H-17 locus, which has not yet been assigned to a specific chromosome. Genes on chromosomes 1, 4, and 5 influence the magnitude of the antibody response to dextran B-1355. Some of these genes may be antigen-specific in their mode of action; however, others may not since they appear to exert a positive influence on the antibody response to both SSS-III and dextran B-1355.     

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.     

Serum antibody and cellular immune response in mice to dextran B512.

Serum antibodies to dextran started to appear 3 days after immunization of C57BL/6 mice. Synthesis of IgM antibodies was followed by IgG3 and IgGA. Other immunoglobulin classes (IgG1, IgG2b, and IgG2a) were very low or absent. The immune response to dextran was also thymus independent with regard to IgG3 and IgA synthesis as demonstrated by the use of nu/nu mice. CBA and C57BL/6 mice were high responders to dextran with regard to IgM synthesis. C57BL/6 mice produced high levels of IgG3 and IgA antibodies, whereas CBA, A/J, and A.TL only synthesized IgM antibodies. A/J and A.TL strains were most frequently low responders with regard to IgM synthesis and CBA/N mice were completely nonresponders with regard to all immunoglobulin classes. The ability to produce anti-dextran antibodies increased with age in high responder strains. This was most pronounced for IgG3 and IgA antibodies, which reached adult levels 3 months after birth. The affinity of anti-dextran antibodies was high and homogeneous in antisera from C57BL/6 mice. Preimmune matural antibodies and antibodies from immunized low responder strains had a low and variable affinity for dextran.     

Igh restriction of the anti-alpha (1-3) dextran response: polyclonal B cell activators induce the synthesis of anti-alpha (1-3) dextran antibodies in lymphocytes from Igha mice only

The only strains of mice which are able to synthesize lambda 1-bearing antibodies in response to alpha (1-3) Dextran are those expressing the Igha allotypic haplotype or those having an Igh V region identical to Igha mice. The experiments reported here were designed to investigate whether the nonresponsiveness of mice which do not express the Igha haplotype is a consequence of an absence of a polyclonal B cell receptor for the alpha (1-3) Dextran TI-antigen. B cells of several mouse strains were stimulated with polyclonal B cell activators (PBA) known to either stimulate non-overlapping B cell subsets or to stimulate B cells at different stages of maturation, i.e., lipopolysaccharide, Nocardia delipidated cell mitogens and alloreactive T helper cells. Whereas all three PBA induced B cells from Igha mice to secrete lambda 1-bearing anti-alpha (1-3) antibodies, the PBA were incapable of inducing B cells from non-Igha mice to mount an anti-alpha (1-3) Dextran response. The data suggest that non-Igha mice lack a functional VH Dex gene for the lambda 1-bearing anti-alpha (1-3) Dextran response.     

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.     

Ir gene-controlled response to haptenated hen ovomucoid: isotypic specificity and dominant nonresponsiveness

We have examined the isotypic pattern of the response of mice to the Ir gene-controlled antigen, dinitrophenyl-ovomucoid (DNP-OM). H-2 kappa mice are high responders (HR); (H-2b,d mice are low responders (LR). The isotype patterns of HR and LR strains differ both quantitatively and qualitatively. In the primary response to doses of 20-100 micrograms DNP-OM, HR strains produce IgM and IgG antibodies, whereas LR strains produce only IgM. Background genes modify the kinetics of the IgGl primary response in HR strains, but no background was found which allowed an IgG response in a LR strain. In secondary responses, priming with 0.2 microgram DNP-OM increases secondary responses in HR strains, and decreases them in LR strains. Control of this response maps to I-A, and is not altered by the bm 12 I-Ab mutation. The LR phenotype is dominant in (HR X LR)F1 mice.     

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.