Studies on the control of antibody synthesis. XVI. Effect of immunodepression on antibody affinity

The relationship between depression in the magnitude of the immune response and a decrease in affinity of the antibody produced was examined in three different models of immuno-depression (B-cell clonal deletion tolerance, specific suppressor T-cell activity, and anti-genie competition). In B-cell clonal deletion tolerance and in antigen-specific, suppressor T-cell-mediated immunodepression, a small decrease in magnitude (50% or less) is associated with a marked decrease in high-affinity, plaque-forming cells. In contrast, with nonspecific immunodepression, due to antigenic competition, a depression in affinity is only seen when there is a marked (85%) reduction in the magnitude of the response. The results are consistent with the view that when the mechanism of immunodepression involves interaction of antigen with antigen-specific B cells there is a disproportionate loss of high-affinity, antibody-producing cells relative to the decrease in magnitude. In contrast, with nonspecific immuno-depression, where the decrease in affinity is presumably due to inefficient expansion of high-affinity clones, an effect on affinity is only observed in association with a marked depression in the magnitude of the response.     

Studies on the control of antibody synthesis. IX. Effect of boosting on antibody affinity.

The effect of boosting on antibody affinity was studied in a haptenic system. Generally, boosting results in the prompt synthesis of high affinity anti-hapten antibody. However, repeated boosting frequently leads to a decrease in the amount and affinity of the serum antibody. Repeated boosting with hapten on a carrier different from that used for priming selectively stimulates synthesis of the highest affinity anti-hapten antibody and does not result in a decrease in affinity. Priming with soluble antigen without adjuvants results in the synthesis of low affinity antibody. After such priming, boosting stimulates low affinity antibody synthesis and repeated boosting leads to a moderate increase in antibody affinity.     

Studies on the control of antibody synthesis. XIV. Role of T cells in regulating antibody affinity.

The effect of limiting the number of helper T cells on the affinity of the primary antibody response to a T-dependent antigen (DNP-BGG) was evaluated in a cell transfer system. Lethally irradiated, thymectomized mice were reconstituted with either bone marrow or anti-brain θ antiserum plus complement-treated spleen as the source of B cells. In addition, they received various numbers of thymus cells as a source of helper T cells. The animals were immunized with DNP-BGG 1 day after cell transfer and their splenic anti-DNP PFC response was assayed for magnitude and affinity 3 weeks later. A marked restriction in helper T-cell activity resulted in a primary response which was of low magnitude, which lacked indirect PFC, and which had a very low affinity and restricted heterogeneity. When sufficient thymus cells were given to permit a switch to indirect plaque formation, a highly heterogeneous, high-affinity primary response was elicited. Further increase in the number of thymic cells resulted in a progressive increase in the magnitude of the primary response but had no effect on affinity. Thus, a reduction of 50% in the magnitude of the response as a consequence of limiting the number of T-helper cells had no effect on the affinity of the PFC. The results are consistent with the interpretation that the effect of restriction in T-cell help on antibody affinity is not due to a direct effect on precursors of high-affinity PFC but is secondary to inefficient selection for high-affinity cells when the degree of cell proliferation is markedly reduced.     

The genetic control of liver cAMP levels in mice

Steady-state level of liver 3,5-cyclic monophosphate, cAMP, has been shown to be under genetic control linked to the mouseH-2 complex. Liver cAMP levels are associated withH-2 haplotype in fully segregating crosses of strains C3H and C57BL/10. In crosses involving strain A, other loci have an effect that swamps that ofH-2. Results withH-2 recombinants indicate that liver cAMP levels are affected by more than oneH-2-linked locus.     

Studies of antibody affinity in plasmacytoma-bearing mice: Evidence for a maturational defect of B lymphocytes

The antibody response of plasmacytoma-bearing mice (PC-mice) is severely reduced. In order to understand the nature of the effect of the tumor on the cells making antibody, quantitative and qualitative studies of the humoral response of PC-mice were undertaken. In these studies, the affinity of the antibody produced by tumor-bearing and normal mice was compared to determine whether the small amount of antibody produced by PC-mice is the product of a normal or an altered population of B cells. Antibody to TNP-Ficoll made by PC-mice 3 days after immunization was less heterogeneous and of an affinity lower than that of antibody made by normal mice. However, at 7 days, the antibody made by PC- and normal mice did not differ significantly. These data suggest that, prior to antigenic stimulation, the B cells of PC-mice are relatively immature, reflecting a possible retardation in the generation and turnover of B lymphocytes. The process of antigen-driven selection of high-affinity antibody-producing cells, however, appears to function normally in PC-mice. These studies, then, reveal a qualitative as well as quantitative defect in the primary humoral response of PC-mice which may reflect an abnormality in the development and differentiation of B cells in these mice.     

Polymorphism ofTcrb andTcrg genes in Biozzi mice: Segregation analysis of a newTcrg haplotype with antibody responsiveness

Tcrb andTcrg gene polymorphism was investigated in high (H) and low (L) responder Biozzi mice from selection I, II, and GS by Southern blot analysis with appropriateV andC probes. No polymorphism of theTcrb haplotype was detected between H and L mice in all selections which were all found to be of the BALB/c type. The H-I and H-II g genotype was of BALB/c and DBA/2 type, respectively. In contrast, a newTcrg haplotype shared by L-I and L-II mice was identified and characterized by C1, 2, 3, C4, V1, 2, 3, V5, and V6 restriction fragment length polymorphisms (RFLPs).Tcrg genotypes were not fixed in the GS selection and two additional new haplotypes were identified in two L-GS mice. An attempt was made to correlate the L-Ig genotype with the low responder status by analyzingg haplotypes among highest and lowest responder (H-1 x L-I)F₂ hybrids immunized with sheep red blood cells (SRBC). No correlation was found in this segregation study, whereas a highly significant one was established with theH-2 haplotype, a locus already known to participate in the genetic control of H-I/L-I difference. The lack of correlation between SRBC response and theTcrg genotype was consistent with the heterogenousg haplotypes found in mice of the GS selection. Together, the present results suggest that H and L mice have the sameTcrab potential repertoire and that T-cell receptor (Tcr) genes cannot be considered as immune response genes in this model. Our results also indicate that the F₂ segregation analysis, given a polymorphic gene, is suitable for an investigation of its immune response functions.     

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.     

Studies on Tyzzer's disease: a long-term study of the humoral antibody response in mice, rats and rabbits.

Mice treated with an antigen prepared from livers infected with Bacillus piliformis developed antibodies to the microorganism which reached a peak on the 7th day and disappeared within 40 days: antibody titres in experimentally-infected mice remained at a high level throughout life. The antibody titres in naturally-infected mice, rats and rabbits ramined positive throughout life and followed the same pattern as that of the experimentally-infected mice.     

DonorIgh-linked genetic control of allotype-specific antibody response

Immunogenicity of allogeneic immunoglobulins in mice were studied, measuring the allotype-specific antibody activity by agglutination of allogeneic antibody-coated red blood cells. It was found that the serum from C.B-20 mice (Igh ^b , BALB/c-congenic) was uniquely immunogenic in BALB/c mice for allotype antibody response. Whereas the C57BL/6 (Igh ^b ) serum was immunogenic only when heat aggregated and/or combined with adjuvant, the ultracentrifugation-deaggregated C.B-20 serum was definitely immunogenic when administered in a moderate dose (100 μl/mouse). Even more surprising was the fast that very low doses (0.01–0.1 μl) of soluble C.B-20 serum, but not C57BL/6 serum, down regulated the allotype-specific response effectively. Genetic analysis on congenic mice suggested that the immunogenicity is controlled by donorIgh orIgh-V(Id-C.B) inasmuch as the serum from BALB/c-congenic C.B-20 (Igh-V ^b C ^b ), but not BALB/c-congenic BAB/14 (Igh-V ^a C ^b ), mice was active in BALB/c mice in soluble form. Further studies showed that the Id-C.B was dominantly expressed on the immunoglobulins of (BALB/c×C.B-20)F₁ and (C56BL/6×C.B-20)F₁ strains, and was originally derived from the C57BL/Ka strain. The major determinant for the antibody production was encoded inIgh-C, but not inIgh-V. It is suggested thatId-C.B controls the allotype-specific antibody response in an unusual manner, possibly acting as a unique determinant activating helper T cells.     

Studies on the genetic control of tryptophan pyrrolase in Drosophila melanogaster

Summary A reexamination of the role of the vermilion cistron (v: 1–33.0) in the control of tryptophan pyrrolase in Drosophila melanogaster uncovered several, previously unknown features about this enzyme: 1. Crude extracts of Drosophila possess one or more inhibitors which may be removed by Norit treatment during homogenization. 2. Contrary to previous assertion, crude wild type extracts are stimulated by an exogenous source of hematin, and upon purification, become heavily dependent upon added methemoglobin. 3. The wild type enzyme exhibits a lag period in time course of product accumulation which is eliminated by preincubation with methemoglobin and -methyltryptophan. 4. The molecular weight of the wild type enzyme is estimated at approximately 150000 Daltons. 5. A linear increase of tryptophan pyrrolase activity as a function of dosage of v ⁺ alleles supports the contention that the vermilion cistron is the structural gene for this enzyme. 6. A sex difference in response to gene dosage (dosage compensation) is seen.A suppressor locus (su(s): 1-0.0) is known, whose mutants operate as recessive suppressors of certain vermilion alleles and not others. Experiments are described which bear upon the mode of suppressor action, and a model is offered which is based upon the present data as well as other work reported recently.     

The ontogeny of thymic independent antibody responses in vitro in normal mice and mice with an X-linked B cell defect.

The primary in vitro antibody response of neonatal spleen cells to three thymic independent antigens has been examined. The time of onset of responsiveness to TNP-Brucella abortus and TNP-lipopolysaccharide was significantly earlier than the onset of responsiveness to TNP-Ficoll. This ontologic sequence was not affected by T cell depletion or antigen presentation on adult macrophages. In neonatal mice bearing the X-linked CBA/N defect, the response to TNP-Brucella abortus and TNP-lipopolysaccharide was much delayed and no response to TNP-Ficoll developed. We conclude that different thymic independent antigens address different subpopulations of B cells, one of which appears earlier in ontogeny than the other.     

Memory in the B-cell compartment: antibody affinity maturation

In the humoral arm of the immune system, the memory response is not only more quickly elicited and of greater magnitude than the primary response, but it is also different in quality. In the recall response to antigen, the antibodies produced are of higher affinity and of different isotype (typically immunoglobulin G rather than immunoglobulin M). This maturation rests on the antigen dependence of B-cell maturation and is effected by programmed genetic modifications of the immunoglobulin gene loci. Here we consider how the B-cell response to antigen depends on the affinity of the antigen receptor interaction. We also compare and draw parallels between the two processes, which underpin the generation of secondary-response antibodies: V gene somatic hypermutation and immunoglobulin heavy-chain class switching.     

Structural and thermodynamic basis of affinity in anti-dinitrophenyl antibody.

The thermodynamic quantities of the anti-dinitrophenyl antibody-hapten interaction are reported for rabbit, goat, and guinea pig antibodies. Rabbit and goat antibodies had similar exothermic enthalpy changes for their reaction with 2,4-dinitrophenyl-L-lysine (-13.9 and -14.8 kcal/mol, respectively). The enthalpy change with guinea pig antibody was much less exothermic (-8.7 kcal/mol), and this value was the same for two guinea pig antibody preparations that differed in affinity by almost two orders of magnitude. A heterogeneous goat anti-dinitrophenyl antibody preparation was fractionated on a molecular sieve column in the presence of a bivalent ligand, a procedure that has been reported to separate antibodies according to differences in the depth of interaction with the ligand. The relationship of these differences in apparent site depth to changes in interactions with the hapten tail was examined by comparing the affinities of various fractions for two haptens. The results show that the presumed deeper sites have stronger interactions with the hapten tail. These studies suggest that the heterogeneity of anti-dinitrophenyl antibodies with respect to affinity results from differences in entropy driven lysyl side-chain interactions which arise from a heterogeneity in antigen binding site depth.