Evidence for distinct polygenic regulation of antibody responses to some unrelated antigens in lines of mice selected for high or low antibody responses to somatic antigen of Salmonella

The effect of the selective breeding of mice for high or low antibody production to complex immunogens is largely nonspecific, since it modifies the responsiveness of high (H) and low (L) lines to many antigens unrelated to the selection antigen. However, the nonspecific effect of the polygenic control operating in these lines is not a general feature. For example, the group of genes in selection IV, carried out for responsiveness to somatic antigen of Salmonella, does not modify the responses to sheep erythrocytes (SE). Despite equivalent responses in H and L mice of selection IV, a large variability was found in individual responses of F₂ interline hybrids, which demonstrates the presence of alleles with high or low effect on responses to SE. A selective breeding (Selection IV-A) was therefore initiated from this F₂ population for responsiveness to SE. A progressive interline divergence occurred during the first seven generations of selection; the interline separation was due to polygenic regulation (about four independent loci from a preliminary estimate).Equivalent responses to the s antigen of Salmonella are observed in the two lines. This constitutes additional evidence for distinct polygenic regulation of responses to SE and to somatic antigen. Moreover, the pattern of responses to several unrelated antigens (nonspecific effect) also differs between Selections IV and IV-A.Abbreviations H high responder lines - L low responder lines - s somatic antigen of Salmonella - f flagellar antigen of Salmonella - R response to selection - S selection differential - F₀ foundation population - h² heritability (realized) - RGG rabbit gamma globulin - CE chicken erythrocyte - HE human erythrocyte - PE pigeon erythrocyte - SE sheep erythrocyte     

Inverse modification of antibody responsiveness to RGG in lines of mice selected for high or low responses to somatic antigen of Salmonella

High (H/s) and low (L/s) antibody responder lines of mice selected according to their response to the somatic (s) antigen of Salmonella (Selection IV) have unexpected inverse capacity for antibody production to rabbit gamma globulin (RGG): H/s mice are low or even nonresponders to this antigen, whereas L/s mice are high responders. It was shown that the phenotypic variability within each line is due to environmental factors. RGG was a selection antigen in Selection V; the high (H/p) and low (L/p) responder mice are therefore considered as homozygous for the RGG genes. Responsiveness to RGG was investigated in F₁ and F₂ hybrids obtained by crossing the phenotypically similar RGG responder or nonresponder mice of Selections IV and V. The results support the hypothesis that the same genes control the response to RGG in L/s and H/p lines as well as in H/s and L/p lines. This means that the genes specific for RGG responsiveness were independent from those regulating responses to the s antigen. Unaffected by the selective breeding in Selection IV, they have been fixed by chance in an inverse way in H/s and L/s lines.     

Variable Region Identical Immunoglobulins Differing in Isotype Express Different Paratopes

The finding that the antibody (Ab) constant (C) region can influence fine specificity suggests that isotype switching contributes to the generation of Ab diversity and idiotype restriction. Despite the centrality of this observation for diverse immunological effects such as vaccine responses, isotype-restricted antibody responses, and the origin of primary and secondary responses, the molecular mechanism(s) responsible for this phenomenon are not understood. In this study, we have taken a novel approach to the problem by probing the paratope with ¹⁵N label peptide mimetics followed by NMR spectroscopy and fluorescence emission spectroscopy. Specifically, we have explored the hypothesis that the C region imposes conformational constraints on the variable (V) region to affect paratope structure in a V region identical IgG₁, IgG_2a, IgG_2b, and IgG₃ mAbs. The results reveal isotype-related differences in fluorescence emission spectroscopy and temperature-related differences in binding and cleavage of a peptide mimetic. We conclude that the C region can modify the V region structure to alter the Ab paratope, thus providing an explanation for how isotype can affect Ab specificity.     

Somatic cell hybridization of mouse myeloma cells

Somatic cell hybrids between different mouse myeloma cell lines have been readily isolated using modifications of existing techniques. The hybrid nature of these cells was established by HAT or HAT-ouabain selective procedures, their chromosome number, and, in one case, H-2 surface antigen expression. Three hybrid cell lines are described here in detail: an IgG_2b, ? X IgG_2a, ?; an IgG₁, ? X IgG_2b, ?; and an IgG₁, ? X IgM, λ. In all cases, both parental types of H and L chains are expressed in the hybrid cells and no new chains are observed. However, molecules possessing disulfide-bonded mixtures of parental H and/or L chains are seen. Analysis of subclones of these hybrids indicates considerable stability in the expression of the immunoglobulins for up to 13 months. However, segregant clones no longer synthesizing one or more of the parental H or L chains arise frequently.     

Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae: I. Analysis of antibody and T-lymphocyte responses in mouse strains developing differing levels of immunity

The kinetics of cellular and humoral responses directed against schistosomula were examined in mice of three inbred strains which demonstrate differences in the degree of resistance induced by immunization with irradiated cercariae. T-Cell reactivity was observed during the first 4 weeks after vaccination but declined to control levels thereafter. Anti-schistosomulum antibody was first detected 2 weeks after vaccination, peaked by 6 weeks, and persisted as late as 15 weeks. In sera obtained at 6 weeks, antibody activity was detected in affinity chromatography-purified fractions containing IgM, IgA, IgG₁, IgG_2a, and IgG₃ immunoglobulins. In general, the cellular and humoral responses observed in C57B1/6J mice, which consistently developed a high level of immunity after vaccination, were not significantly different from those observed in C3H/HeJ or CBA/J mice, which achieved only low to moderate levels of immunity. Thus, although antibody production appears to correlate more closely than T lymphocyte responsiveness with the typical long-term resistance pattern observed in this model, the absence of striking differences in parasite-specific antibody levels between mice of these different strains suggests that additional mechanisms may be involved in the development of immunity after vaccination.     

Amplification of the high production and low production nature of antibodies produced by the genetic selection of mice. Correlation between immunodeficiency and the incidence of lymphoma

Five bidirectional selective breedings have been carried out in mice for quantitative antibody responses to various natural immunogens. Appropriate crosses between the five "high responder" lines and between the five "low responder" lines resulting from these selections, were performed to obtain balanced hybrid populations. These populations were further selected for antibody production on the basis of multiple responses i.e., responses to all the antigens used in the previous separate selections. The result was a marked amplification of the interline divergence. The low line in particular has a profound deficiency for both primary and secondary responses. Moreover a high incidence of lymphomas was observed among these low responder mice (up to 42%).     

Selective breeding of high and Low antibody-responder lines of guinea pigs

High (H) and Low (L) antibody-responder lines of guinea pigs were produced by selective breeding for the character quantitative agglutinin responsiveness to optimal doses of sheep erythrocytes (SE) and chicken erythrocytes (CE). The non cross-reacting SE and CE were alternated with each generation in order to avoid the specific interference of maternally transmitted antibodies. The continuous distribution of the phenotypes in the foundation population (F₀) and the progressive interline divergence during eight consecutive generations of selective breeding demonstrates that the character investigated is submitted to polygenic regulation. The difference in the peak agglutinin titers between the H and L lines of the eighth generation (HF₈/LF₈) is eight fold. The mean heritability (h²) of the character investigated, measured from the interline divergence of the eight generations of selection is 0.18±0.04. The h² value was established by the linear regression of the cumulated response to selection (R) on the cumulated selection differential (S). The selective breeding has a nonspecific effect because it also modifies the antibody responsiveness to other immunogens, such as f and s antigens ofSalmonella typhimurium or human erythrocytes, that are immunologically unrelated to those used in the selection.     

Immunoglobulin isotype distribution of malaria-specific antibodies produced during infection with Plasmodium chabaudi adami and Plasmodium yoelii

The antibody response of mice to Plasmodium chabaudi adami and Plasmodium yoelii has been compared using a solid phase isotype-specific radioimmunoassay and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Serological cross-reactivity between these parasites was substantial. Studies using a radioimmunoassay detecting all classes of malaria-specific antibody demonstrated that during the early part of infection it was not possible to distinguish between homologous and heterologous reactions. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 50% or more of the protein antigens detected were apparently shared by both parasites although the intensity of bands was always greater with homologous reactions. However, the distribution of isotypes in the antibody (Ab) response differed in the two infections. P. chabaudi infections were characterized by a predominant and persistent IgM response, moderate IgG₂ and IgG₃ and little significant IgG₁ response during a primary infection. By contrast, IgM antibodies were transient in P. yoelii infection, IgG₂ was the predominant isotype, and both IgG₁ and IgG₃ antibodies were present during a primary infection. These differences in isotypes were also detected when sera were tested on the heterologous antigen extracts suggesting that antigens shared by P. chabaudi and P. yoelii do not necessarily induce similar antibody responses in the two infections.     

Polygenic control of quantitative antibody responsiveness: restrictions of the multispecific effect related to the selection antigen

Among the differences observed between the various high (H) and low (L) antibody responder lines of mice resulting from distinct bidirectional selective breedings, one of the most puzzling is the variation in the multispecific effect, i. e., in the modification of antibody responses to antigens unrelated to those used during the selection. The best examples are the H and L lines of selection IV, selected on the basis of responses to somatic antigen of Salmonella which do not differ in their antibody responses to sheep erythrocytes (SE). However, a wide range of variability is observed in the responses of (H_IV x L_IV)F₂ hybrids to this antigen, and it was therefore hypothesized that distinct groups of genes might regulate antibody responses to SE and the somatic antigen. Indeed, a new selection (IV-A) for anti-SE responsiveness started from these (H_IV x L_IV)F₂ successfully produced a high and a low anti-SE responder line. The results of selection IV-A and the variance analysis of (H_IV-A × L_IV-A)F₂ hybrids are reported. They are roughly similar to those in selection I, also carried out for anti-SE responsiveness. In vivo attempts to identify the major regulatory mechanism which contributes to the interline difference indicate that the efficiency of macrophage accessory function has been modified in selection IV-A, as was observed in selection I, whereas this function did not differ in Hév and Lév lines. Probably in relation to the involvement of macrophage function there is a notable increase of the multispecific effect in selection IV-A when compared with selection IV. The results of selection IV-A demonstrate that responsiveness to heterologous erythrocytes and to somatic antigen of Salmonella are under separate polygenic control operating through distinct regulatory mechanisms. The choice of the selection antigen and immunization procedure is of major importance for defining the gene interaction operating in each selective breeding experiment and the extent of its multispecific effect.Abbreviations used in this paper f. ag. S. flagellar antigen of Salmonella - H high responder line (roman numeral is the selection number) - h₂ heritability - HE human erythrocytes - HoGG horse gamma globulin - L low responder line (roman numeral is the selection number) - PE pigeon erythrocytes - R response to selection - RGG rabbit gamma globulin - S selection differential - s. ag. S. somatic antigen of Salmonella - SE sheep erythrocytes     

Immunoglobulin heavy H chain isotypes in a teleost fish

Three mouse mAb were derived which identified different populations of the approximately 700,000-Da tetrameric Ig of channel catfish (Ictalurus punctatus). Immunoprecipitation analyses using the three mAb in various combinations showed that the catfish Ig population identified by each mAb was antigenically distinct. Each Ig population contained both classes of catfish L chains (F and G). Solid-phase binding assays indicated that the mAbs preferentially identified H chains rather than L chains. Comparative peptide mapping analyses of the H chains defined by each mAb indicated that H chains were different, although each had the same apparent mass (approximately 70,000 Da). This structural distinction was not based upon allotypic variation because the analysis of the serum from individual catfish showed the presence of each H chain in fish examined. Therefore, the different H chains represent different isotypes; each representing approximately 20% of the total serum Ig. There is at least one undefined additional H chain isotype found in the serum Ig population. Partial sequence information was obtained on the first variable framework region (FR1) of two of the three H chain isotypes. These results indicated that the variable region associated with each isotype was heterogeneous; two and sometimes three different residues were represented in the majority of positions. The sequence information also showed that the FR1 of one H chain was distinct from the other; there was less than a 50% match of the primary residues. The analyses suggest that the V region genes which code for the FR1 are differently associated with each H chain isotype. Finally, the relative levels of the three H chain isotypes were monitored during the temporal immune response of individual catfish to the dinitrophenyl hapten. These results indicated that one H chain isotype was preferentially expressed early in the immune response (approximately 3 wk) and remained the predominantly expressed isotype during the humoral immune response.     

Detection of immunoglobulin heavy chain IgG3 polymorphism in wild mice with xenogeneic monoclonal antibodies

We have generated four xenogeneic rat antimouse IgG₃ monoclonal antibodies recognizing at least three different antigenic determinants (epitopes) on BALB/c IgG₃ molecules. These antibodies were used in solid-phase blocking radioimmunoassays for detection of the epitopes in sera of 40 inbred strains and 134 wild mice. These antibodies detect genetic polymorphism of IgG₃ isotype among wild mice even though there is no polymorphism found among 40 inbred strains tested (except X-chromosome-linked immunodeficient CBA/N strain which lacks IgG₃ molecules). An IgG₃ variant was also isolated from hybridomas derived from Mus spretus.Abbreviations Igh-C immunoglobulin heavy chain constant region - PVC polyvinyl chloride - RIA radioimmune assay - ELISA enzyme linked immunosorbent assay     

Capillaria hepatica: Granuloma formation to eggs: III. Anti-immunoglobulin augmentation and reagin activity in mice

Sera from mice with primary and secondary Capillaria hepatica egg granulomas were examined for 48-hr homologous PCA activity and for the presence of IgM, IgG₁, and IgG₂ using a microtiter anti-globulin augmentation assay following indirect hemagglutination testing. All three antibody types assayed were present. Secondary granuloma formation produced an antibody response characterized by the initial production of IgM followed by IgG₁ and IgG₂ during the latter phase of the test period, however primary granulomatous sera demonstrated a more varied antibody response with the presence of all three during the entire test period. Forty-eight-hour PCA tests demonstrated the presence of reagin activity in sera from granulomatous and infected mice. Reagin activity occurred more frequently in primary than secondary granulomatous mice. These studies thus confirm the presence of antibody during granuloma formation to C. hepatica eggs and conclusively demonstrate the presence of at least two classes of antibody.     

Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells

Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG₁, IgG₂ and IgG₄ thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG_2a with humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development.     

Salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection

Susceptibility to Salmonella typhimurium infection was compared in H (high Ab responder) and L (low Ab responder) mice obtained by several selective breeding experiments (Selections I, II, III, IV and IV A). H mice were always much more susceptible to infection than their L mice counterparts within a continuous LD 50 variation range. In three of the selections (I, II and IV A) the low responsiveness character is known to result mainly from rapid Ag degradation in L mice macrophages. It was hypothesized that resistance to multiplication of intracellular pathogens could be related to an increased catabolic activity towards Ag. This was actually demonstrated, in F2 segregant hybrids of selection IV A, by the significant inverse correlation between capacity for Ab production and resistance to infection.     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

Salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection

Abstract Susceptibility to Salmonella typhimurium infection was compared in H (high Ab responder) and L (low Ab responder) mice obtained by several selective breeding experiments (Selections I, II, III, IV and IV A) [10,19,22]. H mice were always much more susceptible to infection than their L mice counterparts within a continuous LD 50 variation range. In three of the selections (I, II and IV A) the low responsiveness character is known to result mainly from rapid Ag degradation in L mice macrophages. It was hypothesized that resistance to multiplication of intracellular pathogens could be related to an increased catabolic activity towards Ag. This was actually demonstrated, in F₂ segregant hybrids of selection IV A, by the significant inverse correlation between capacity for Ab production and resistance to infection.     

Ig H and L chain contributions to autoimmune specificities

An Ig H chain expression vector has been constructed by using the V region of 3H9, an antibody that binds ssDNA, dsDNA, and cardiolipin. The H chain construct was transfected into six hybridoma cell lines expressing Ig L chains. All resulting H and L chain combinations had at least some affinity for ssDNA, whereas five also bound dsDNA to a similar degree as 3H9. The loss of dsDNA binding was correlated with a single amino acid difference between two V kappa 8 L chains. A further characteristic of 3H9, its immunofluorescent staining pattern, was shared by four of the recombinant antibodies, whereas its specificity for cardiolipin was shared with five. The transfections reported here show that a V kappa 3 L chain confers specificity for an RNA-associated epitope and that a V kappa 21E L chain prevents cardiolipin binding. These experiments suggest that the 3H9 H chain contributes essential determinants required for binding to DNA as well as cardiolipin but that L chains can modulate or prevent this binding. L chains may also expand the specificity of a recombinant antibody.     

Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus colubriformis infected sheep

Sheep infected with the nematode parasite Trichostrongylus colubriformis showed anti-T. colubriformis acetylcholinesterase. (AChE) antibodies in the IgG₁ but not the IgG₂ or IgM fractions prepared from their serum. Using the fluorescent antibody technique with representative sera, antibodies in the IgG₁ fraction exhibited specificity for antigens in the subventral glands of the worm excretory system. IgA antibody specificity for antigens in the excretory glands and intestine of the worm was also demonstrated.     

Adaptive evolution of variable region genes encoding an unusual type of immunoglobulin in camelids

A typical immunoglobulin (Ig) molecule is composed of four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains. This tetrameric structure is conserved in almost all jawed vertebrate species. However, it has been discovered that camels and llamas (family: Camelidae) possess a type of dimeric Ig that consists of two H chains only. These H chains do not associate with L chains, and they do not have the first constant region (CH1), which is present in the conventional Ig. In spite of these changes, the dimeric Ig maintains the normal immune function. To understand the evolution of the dimeric Ig, we studied the phylogenetic relationships of the variable region (V(H)H) genes of the dimeric Ig from Camelidae and those (V(H)) of the conventional Ig from mammals. The results showed that the V(H)H genes form a monophyletic cluster within one of the mammalian V(H) groups, group C. We examined the type of selective force in complementarity-determining regions (CDRs) and framework regions (FRs) by comparing the rate of synonymous (dS) and nonsynonymous (dN) substitutions. We found that the results obtained from V(H)H genes were similar to those from V(H) genes in that CDRs showed an excess of dN over dS (indicating positive selection), whereas the reverse was true for FRs (purifying selection). However, when the extent of positive selection or purifying selection was investigated at each codon site, three major differences between V(H)H and V(H) genes were found. That is, very different types of selective force were observed between V(H)H and V(H) genes (1) at the sites that contact the L chain in the conventional Ig, (2) at the sites that interact with the CH1 region in the conventional Ig, and (3) in the H1 loop. Our findings suggest that adaptive evolution has occurred in the functionally important sites of the V(H)H genes to maintain the normal immune function in the dimeric Ig.