Structural and antigenic studies of an idiotype-bearing murine antibody to the arsonate hapten.

Mice of strain A/J responded to repeated intraperitoneal injection of Limulus hemocyanin derivatized with arsanilic acid by producing large quantities (approximately 5 mg/mL of ascites fluid) of IgG antibodies specific for this hapten. The antibodies possessed a characteristic idiotypic determinant and exhibited restricted heterogeneity as demonstrated by isoelectric focusing and primary N-terminal amino acid sequence analysis of isolated light and heavy polypeptide chains. Both light- and heavy-chain sequences were comparable to those of myeloma proteins in lack of heterogeneity. The N terminus of the light chain exhibited V KI sequence and only one position in the first 30 residues showed more than one amino acid. No variability was observed in the first 10 N-terminal residues of the heavy chain. Rabbit antiserum to the idiotype blocked binding of hapten by the purified antibody. The presence of both light- and heavy-chain antigenic determinants was required for optimal formation of the idiotypic determinant.     

Specific suppression of the antibody response in vitro by serum from paralyzed mice.

BALB/c mice immunized with either the whole vaccine or the C-polysaccharide obtained from the R36A strain of pneumococcus produce antibody to phosphorylcholine. Mice injected i.v. with a single high dose of the C-polysaccharide are specifically unresponsive to immunization to phosphorylcholine for many months and are considered paralyzed. The induction of paralysis does not eliminate cells reactive to phosphorylcholine; however, serum from paralyzed mice specifically suppresses the response of cultures of normal spleen cells to phosphorylcholine. Paralyzed mice have an early low antibody response to phosphorylcholine and to the receptor for phosphorylcholine as indicated by plaque-forming cell assays. The factor or factors present in serum which may suppress cultures, and, by presumption, be responsible for paralysis are complexes of antigen, antibody, and antibody to the receptor for phosphorylcholine.     

The expression of Ia antigenic determinants on macrophages required for the in vitro antibody response.

A subpopulation of antigen-presenting macrophages required for an in vitro antibody response to burro erythrocytes was deleted by pretreating the splenic macrophages with anti-Ia serum and complement (C). The in vitro response of the macrophage depleted T-B cell population could not be restored by the addition of macrophages resistant to anti-Ia antibodies and C (Ia-). The response of Ia- macrophages and the macrophage-depleted T-B cells was only reconstituted by the addition of Ia+ macrophages. Macrophages pretreated with anti-Ia antibodies restricted to react with determinants of one I subregion could not support the in vitro antibody response when added to cultures whose macrophages were pretreated with anti-Ia serum and C specific for the I-J subregion. These results confirmed that Ia determinants of the I-A, the I-E, and the I-C subregions were all expressed on the I-J+ macrophage required for an in vitro antibody response.     

Augmentation of the antibody response by hapten help: I. Dominant genetic control

The objective of the present work was to characterize those genetic factors that determine susceptibility to “hapten help,” i.e., the augmentation of B-cell responses by hapten-reactive T cells. After mice had been sensitized to the hapten azobenzenearsonate (ABA), one of two approaches was used to assay hapten help. In the first, circumvention of tolerance to low doses of bovine γ-globulin (BGG) was augmented in CBA but not in C57BL/6 mice, as measured by serum anti-BGG antibody after challenge with ABA-BGG. Second, similar strain differences but on a larger scale were demonstrated in the anti-BGG plaque-forming cell (PFC) response of spleen cells from hapten-primed nontolerized mice after challenge with ABA-BGG. Results with the F1-hybrid of a cross between a high responder and a low responder for hapten help demonstrated that high responsiveness is dominant. Experiments with recombinant inbred mice from high- and low-responder progenitor strains suggested that hapten help is associated not with the major histocompatibility complex (H-2) so much as with minor histocompatibility antigens such as H-22 and/or H-24, both of which are on chromosome 7.     

Induction of primary and inhibition of secondary antibody response to hapten by hapten conjugates of type III pneumococcal polysaccharide.

Tri- or dinitrophenylated pneumococcal polysaccharide type III (TNP- or DNP SIII)) induced a primary 19S anti-TNP response without generating immunological memory to the hapten in LAF₁ mice. Hapten-hemocyanin (TNP-KLH) or hapten conjugates of B. abortus organisms (DNP-BA) induced both 19S and 7S primary responses and memory to the hapten. Spleen cells from mice immunized with TNP-KLH or DNP-BA did not give adoptive memory responses upon challenge with hapten-SIII and, in fact, were inhibited from responding to their homologous hapten conjugates by simultaneous injection of hapten-SIII. Incubation of TNP-KLH-primed spleen cells for as short as 5 min at 0 °C with 10 μg of TNP-SIII per milliliter virtually abolished their ability to give 19S and 7S memory responses to TNP-KLH upon transfer into irradiated recipients. It is suggested that a difference in avidity and/or number of anti-TNP receptors per cell between virgin and primed B cells may be an important factor in determining whether the cells will be stimulated or inhibited by exposure to hapten-SIII. Another factor may be a difference between virgin and memory cells in their requirement for T-cell help.     

Inhibition of anti-DNP antibody formation by high doses of DNP-polyacrylamide molecules; effects of hapten density and hapten valence

High-dose inhibition of anti-DNP antibody formation by a series of DNP-polyacrylamide molecules of varying hapten density and hapten valence was measured. It was found that a molecule's inhibitory ability correlated directly with its hapten density, but not with its hapten valence nor with its own ability at optimal dose to stimulate an anti-DNP response.     

FcgammaRIIB in IgG-mediated suppression of antibody responses: different impact in vivo and in vitro.

The suppressive effect of IgG on Ab responses to particulate Ags such as erythrocytes is well documented. IgG-mediated suppression is used clinically in rhesus prophylaxis to prevent RhD-negative mothers from becoming immunized against their Rh D-positive fetuses. We have recently shown that IgG anti-SRBC, passively administered together with SRBC, can induce efficient suppression of primary Ab responses to SRBC in mice lacking the known FcRs for IgG (FcgammaRI, FcgammaIII, and FcgammaRIIB or the neonatal FcR). The lack of a demonstrable effect of the inhibitory FcgammaRIIB was particularly surprising, and, in this study, the involvement of this receptor is further investigated during broader experimental conditions. The data show that SRBC-specific IgG administered up to 5 days after SRBC can induce suppression both in wild-type and FcgammaRIIB-deficient mice. Suppression of secondary Ab responses to SRBC in vivo was similar in the two strains. In contrast, IgG-mediated suppression of Ab responses in vitro was impaired in cultures with primed FcgammaRIIB-deficient spleen cells. In conclusion, inhibition of in vivo Ab responses to SRBC by passively administered IgG can take place via an FcgammaRIIB-independent pathway. This pathway causes >99% suppression and operates during all experimental conditions studied so far. The nature of the mechanism can at present only be hypothesized. Masking of epitopes and/or rapid elimination of IgG-Ag complexes would both be compatible with the observations.     

Long-lasting in vitro immune response to a distinct antigenic determinant of a bacterial protein. Cyclic changes of antibody titer and affinity.

Long-lasting (60 days or more) antibody responses in vitro by rabbit lymph node fragments to a distinct determinant of Escherichia coli beta-D-galactosidase were obtained by supplementing culture medium with fetal calf and horse serum. Antibodies released in the supernatant were removed every 3rd to 5th day together with the spent medium, without pooling to minimize intermixing of molecules synthesized far apart in time. Antibody titer, association constant, and heterogeneity index were measured in medium samples collected throughout the response in order to draw profiles of their changes under conditions whereby a limited number of clones synthesize antibodies in a closed system without connection to antigen depots, central lymphoid organs, and circulating cell and antibody pools. It was found that antibody affinity changes cyclically and that such cycles may be repeated. Cycles are composed of an ascendant limb with a gradual increase in affinity and a parallel diminution of heterogeneity. A descendant limb follows with the opposite modifications. High affinity antibodies predominate at the peak of the cycles, whereas low affinity molecules take over at the end of the cycles until the next ascendant limb begins; these persist after the last cycle has waned.