Immunoelectron microscopic localization of idiotypes and allotypes on immunoglobulin molecules

We have developed a novel approach to the analysis of antigenic (allotypic and idiotypic) determinants on intact immunoglobulin molecules. Immune complexes composed of IgG in combination with anti-idiotype or anti-allotype antibody were "visualized" by transmission electron microscopy. Individual Fab fragments of anti-idiotype or anti-allotype antibody, when bound to the IgG, altered the "Y" configuration in a reproducible and interpretable manner. Anti-idiotype antibody (either as Fab or IgG) bound to the terminus of the presumed V region of the IgG molecule, thus extending the apparent length of the Fab arms. Analysis of a rabbit VH framework allotype (a1) revealed that the determinant(s) is (are) located on the lateral portion of the V region of IgG. Binding of the anti-a1 Fab fragments was always at approximately right angles to the axis of the Fab arms of IgG. Fab antibody to the rabbit kappa light chain (b4) allotype bound to the lateral portion of the terminal half of the IgG Fab arms. This technique should be of value in localizing less well defined immunoglobulin determinants.     

IgA allotype suppression in mice: A cellular implication for the IgA preponderance in the gut

Paternal IgA allotype suppressions were induced in (A/HeJ x SJL/J)F₁, (BALB/cJ x CB-20) F₁, and (BALB/cJ x SJL/J)F₁ mice by perinatal exposure of the neonates to antibody against paternal IgA allotype (Ig-2b). All the Ig-2b allotype suppressions were temporary and were prominently seen at the age of 4–10 weeks. These transient Ig-2b allotype suppressions were manifested by a suppression of Ig-2b allotype in the serum and a suppression of Ig-2b allotype-producing cells in the gut lamina propria. During the suppression, no replacement of the Ig-2b allotype by IgM and IgG plasma cells was seen in the gut lamina propria. The transient IgA allotype suppression was primarily due to a depletion of IgA allotype precursors in Peyer's patches; suppressor T cells were not actively involved. These results suggest that IgA preponderance in the gut is due to the migration of the patch IgA precursors and their accumulation and maturation in the gut lamina propria.     

Auto-antibody to an Ig VH region allotype: induction of anti-a1 antibody in an a1-suppressed a1a2 heterozygous rabbit.

Two a1a2 heterozygous sibling rabbits were first suppressed for the paternally inherited a1 VH region allotype and then immunized with a1 IgG. Anti-a1 antibody was detected in the serum of one of the rabbits. The anti-a1 auto-antibody reacted with the same amount of a1 IgG as did a conventional anti-a1 allo-antibody. Most of the IgG and IgM of this rabbit was of the a2 allotype and no significant amount of the a1 allotype was detected as would be expected for an a1 suppressed a1a2 heterozygous rabbit. However, allotype suppression in this rabbit is maintained by endogenous anti-allotype antibody. Rabbits with anti-allotype auto-antibody may be exploited to produce litters of heterozygous and homozygous rabbits efficiently suppressed for selected allotypes.     

Sequential double immunocytochemical staining for in situ identification of an auto-anti-allotype immune response in allotype-suppressed rabbits

Immunocytochemical staining has been used to detect putative autoimmune B-cells in rabbits undergoing chronic allotype suppression. This condition is seen in heterozygous rabbits exposed perinatally to antibody against the paternal immunoglobulin allotype. Such animals develop lifelong suppression for this allotype and have been used as models for study of antibody-induced disturbance of immune regulation. Normal rabbits deliberately immunized against a heterologous allotype were used to establish the feasibility of identifying cells forming anti-allotypic antibodies in cryostat sections of rabbit lymphoid tissues. Incubation and staining of tissue sections from suppressed rabbits then revealed the presence of autoimmune B-cells, with antibody specificity for the suppressed allotype, in all chronically suppressed adult rabbits tested. Sequential incubation and staining with allotype- and anti-allotype-enzyme conjugates established that such cells were of non-suppressed origin. Auto-anti-allotype antibody-forming cells were not found in normal heterozygotes or in chimeric rabbits. The immunocytochemical techniques described here permitted simultaneous detection of specificity (i.e., anti-allotype) and origin (allotype) of antibody-forming cells involved in an autoimmune response, as well as their anatomical correlation with other B-cells of suppressed or non-suppressed origin. Since the method described can be adapted to detection of alternate cell markers, we believe it to have potential application to the study of other autoimmune phenomena.     

Generation in vivo of non-T suppressor cells with the use of anti-allotype antibody

Anti-Igh-1b antiserum induced allotype-specific suppression of adult mouse spleen cells in an adoptive transfer system. Suppression of Igh-1b anti-sheep red blood cell plaque-forming cells was measured as late as 4 wk after the injection of allotype heterozygous (Igha/b) spleen cells, antiserum, and sheep red blood cells. Suppression was maintained on retransfer of the allotype-suppressed spleen cells to further irradiated recipients in the absence of additional exogenous anti-allotype antibody. Mixing experiments were performed to test the putative inhibitory effects of allotype-suppressed spleen cells from the first adoptive transfer (stage I) on the antibody response of normal spleen cells in a second adoptive transfer (stage II). No suppression was observed by using unfractionated stage I spleen cells. In contrast, when these allotype-suppressed spleen cells were depleted of T cells, they strongly inhibited the antibody production of admixed normal spleen cells in stage II. This inhibitory activity of antibody-induced stage I spleen cells was directed primarily toward the target allotype, but some suppression of the Igh-1a plaque-forming cell response and total IgG production also occurred. Although removal of adherent cells did not affect the inhibitory activity of allotype-suppressed spleen cells from stage I, removal of Ig+ cells completely abrogated the inhibitory activity. These results suggest that antibody-induced regulatory B cells may play a role in maintaining long term allotype suppression.     

From cell biology to immunology--a short trip

Immunologic memory and immunoglobulin allotype suppression are discussed as problems in Cell Biology. Memory, the ability of an animal after a first antigenic exposure to give a heightened and faster immune response upon a second exposure to the same antigen, is shown to be a property of bone marrow-derived cell lines. Expression of this memory depends on interaction with thymus-derived cells from either non-immunized or immunized mice. Chronic allotype suppression is described for the first time. It is initiated by in utero or neonatal exposure of (SJL × BALB/c)F₁ mice, allotype a/b, to anti b antibody. Suppression lasts for long periods and continues in irradiated recipients which have received lymphoid cells from suppressed donors. Selection against b allotype producing cell precursors can explain suppression.     

A Waldenstr?m's macroglobulin with anti-G3m(b1) antibody activity.

A human monoclonal macroglobulin (IgM, K) from a patient (KI) with Waldenstr?m's macroglobulinemia was shown to have antibody activity against a human IgG (Gm) allotype. In hemagglutination tests, only one anti-D serum with G3m(b0b1) reacted with macroglobulin KI. Antiglobulin specificity of macroglobulin KI was determined to be an anti-G3m(b1) antibody by hemagglutination inhibition tests. Fab fragments from macroglobulin KI could react with human IgG3 protein possessing G3m(b1), but Fc fragments could not react. Gm phenotype in IgG isolated from serum KI was determined to be Gm(a,z,g,b0,s,t,u). This is the first report of a Waldenstr?m's macroglobulin with antiglobulin specificity against a Gm allotype.     

Recurrent idiotopes and internal images.

A rabbit was immunized with rabbit immunoglobulins of a different allotype. The anti-allotypic antibodies produced by this rabbit were used to immunize a second rabbit which produced anti-idiotypic antibodies. To explain the occurrence, among these anti-idiotypic antibodies, of "internal images" of the original immunizing allotype, a restricted and a more general hypothesis are developed. The first assumes that B-cells can be triggered when idiotopes on their receptor molecules are recognized by the paratopes of the immunizing antibody. The second denies the existence of a specially constructed combining site on the variable domain of an antibody molecule.     

In vivo activation of quiescent B cells by anti-immunoglobulin. I. Induction of latent VH allotype production in adult rabbits by treatment with heterologous antibodies or antibody fragments

We demonstrated previously that injection of adult rabbits with homologous anti-VHa1 allotype antibody induces the appearance in serum of genetically unexpected (latent) a1 immunoglobulin (Ig) and a1-like internal images. We have now investigated the mechanism for this induction effect and have found that treatment of animals with polyclonal goat or monoclonal mouse anti-a1 antibody similarly results in production of unexpected a1 determinants. The N-terminal amino acid sequences of deblocked heavy chains showed that latent a1 Ig induced by monoclonal antibody treatment was identical to nominal a1 Ig at 18 of 19 residues, with the one amino acid difference at position 13 probably due to a single nucleotide change. Like nominal a1, these molecules expressed multiple VHa1 framework epitopes, as demonstrated by direct immunoelectron microscopic visualization of immune complexes. Whereas F(ab)'2 fragments of rabbit anti-a1 antibody retained inducing activity, F(ab) fragments were effective only when administered in an aggregated form; this result suggests that cross-linking of surface Ig receptors plays a critical role in the induction process. We conclude that all rabbits contain dormant B cells expressing germ-line-encoded latent allotypes, and that in vivo administration of anti-Ig antibody causes activation of these cells directly rather than through perturbation of an allotype regulatory network.     

Effect of allotype on activation of neutrophils by FcgammaRIIIB cross-linking

Human neutrophils constitutively synthesize two receptors for the constant region of IgG, FcgammaRII, and FcgammaRIIIB. Fluo-3-loaded neutrophils were treated with biotinylated Fab fragments of anti-FcgammaR antibodies and cross-linked with streptavidin, and intracellular calcium ([Ca2+](i)) was monitored by flow cytometry. Polymerization of filamentous actin was quantitated by NBD-phallacidin using flow cytometry. Cross-linking of FcgammaRII by monoclonal antibody (mAb) IV.3 induces an increase in [Ca2+](i), superoxide generation, and the polymerization of actin. [Ca2+](i) responses from cross-linking of FcgammaRIIIB by mAb 3G8 varied from minimal to no release. To determine whether discrepancies in 3G8-induced [Ca2+](i) release were due to allotype variation, we selected five donors who were homozygous for the NA1 allotype of FcgammaRIIIB and five who were either heterozygous or homozygous for the NA2 allotype and compared their [Ca2+](i) response and actin polymerization induced by FcgammaRIIIB cross-linking. Cross-linking of FcgammaRIIIB by 3G8 produced minimal [Ca2+](i) release and polymerization of actin irrespective of donor allotype.     

Normal and altered phenotypic expression of immunoglobulin genes.

Genetically controlled intraspecific differences between immunoglobulins (allotypes) provide valuable markers for the study of the quantitative expression of allelic and nonallelic alternative forms of immunoglobulins (Igs) during the normal development of rabbits. Heterozygous rabbits are mosaics of cells expressing different Ig-genes since fully differentiated productive cells generally secrete only one of alternative forms of Ig. The proportions of cells that differentiate to produce allelic forms of immunoglobulins during normal development depend on the particular heterozygous genotype. The normal proportions of some markers can be drastically altered if the differentiation of lymphoid cells in the young rabbit occurs in the milieu of antibody specific for one form (allotype suppression). An initiating step in the establishment of persistent allotype suppression is probably the interaction of antiallotype antibody with allotype-bearing receptors on lymphoid cell surfaces, but the mechanism for the maintenance of a state of chronic suppression may well be more complex. Allotype suppression can be viewed as one example of numerous immunological phenomena that reflect specific and finely tuned regulatory mechanisms governing the differentiation and clonal expansion of lymphoid cells destined to secrete immunoglobulins.     

In vitro immunoglobulin allotype synthesis by spleen cells of heterozygous rabbits. Specific suppression by anti-allotype antibody

The production of immunoglobulin (Ig) bearing the b4 and b5 allotypic markers by b⁴b⁵ heterozygous spleen cells cultured in vitro was assessed by means of a sensitive and reproducible radioimmunoassay. Ig synthesis was demonstrated by the increasing amounts of the b4 and b5 allotypes appearing with time in the supernatant fluids. To determine the effect of anti-b4 or anti-b5 antibody on the synthesis of the b4 and b5 allotypes, spleen cells from b⁴b⁵ heterozygous rabbits were incubated for 24 hr in the presence of anti-b4 or anti-b5 and then washed and cultured for an additional 4 days. Anti-b4 suppressed the production of the b4 allotype with no effect on b5 production, whereas anti-b5 suppressed the production of b5 allotype with no effect on b4 production. This suppression of allotype synthesis in vitro presumably results from an antigen-antibody reaction occurring on the surface of lymphoid cells by a mechanism which may be similar to that which brings about allotype suppression in vivo for fetal and newborn rabbits.     

Allotype suppression in the chicken. V. Abnormal isotype ratios of chronically suppressed IgM and IgG allotypes

Chronic allotype suppression was generated in M-1 (C mu), G-1 (C gamma) heterozygous B14 line chickens either by embryonal injection or by maternal transfer of anti-M-1b allotype antibodies. Using a sensitive radioimmunoassay to detect the suppressed IgM-1b, a striking disparity was observed between the degree of suppression of the IgM-1b allotype and that of the genetically linked IgG-1i allotype. The amount of suppressed IgM-1b ranged between 0.2 and 3% of total serum IgM in chronically suppressed birds. The levels of the genetically linked IgG-1i, however, comprised 5 to 25% of total serum IgG in such birds. The allotypes measured in suppressed chickens were qualitatively identical to those in normal, nonsuppressed birds by serological criteria. These results are discussed within the context of isotype regulation in the chicken.     

Unidirectional IgG allotype- and isotype-specific suppressor cells in congeneic mice

By the use of allotypic markers on immunoglobulin molecules of isotypes IgG1 and IgG2a, in transfers of spleen cells between Igh haplotype congeneic partner strains BALB/c (Igha) and CB20 (=BALB/c-Ighb), the expression of donor and recipient lymphocytes could be followed differentially. BALB/c donor's allotype a was produced in nonirradiated CB20 recipients for months. By contrast, CB20 donor's allotype b disappeared in nonirradiated BALB/c recipients shortly after transfer. These BALB/c recipients of CB20 spleen cells ("CB20-primed") developed lymphocytes which were able to suppress the autochthoneous allotype b production of CB20 irradiated or CB20 nu/nu or neonatal F1 (BALB/c female X CB20 male) recipients immediately after transfer. Titers decreased with a half life of about 4 days, resembling that of immunoglobulin molecules. The suppression was restricted to the IgG2a isotype of allotype b. Neither the other isotype IgG1 of allotype b, nor, in the reciprocal transfer experiment, IgG1 or IgG2a of allotype a was affected. Analogous transfers between Igh congeneic partners on a C57B1/6 genomic background revealed the same susceptibility of allotype b-producing cells from C57B1/6 donors toward suppression by C57B1/6-Igha mice as recipients. Allotype suppression, induced by cell transfer, is thus unidirectional in that Igha haplotype mice react against allotype b but not vice versa, and it is isotype-specific, only directed against IgG2a, and not IgG1.     

Tritrichomonas foetus extracellular cysteine proteinase cleavage of bovine IgG2 allotypes

Bovine trichomoniasis is a sexually transmitted disease associated with reproductive failure. Systemic immunization results in protective IgG antibodies in uterine and vaginal secretions. Because bovine IgG2 is a better opsonin than IgG1, it is potentially important in defense. Yet, Tritrichomonas foetus extracellular cysteine proteinase (TFECP) cleaves bovine IgG2, evading protective IgG2 responses. Variations in resistance of the 2 IgG2 allotypes to digestion may explain inherited differences in protection. To address this hypothesis, TFECP was incubated with both IgG2 allotypes at different concentrations and times. The digestion products were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained, and quantitated by image analysis. IgG2a was digested faster by TFECP than IgG2b. Differences in the sizes and numbers of digestion products were observed, but the presence of bands the size of Fc and Fd fragments indicated that both allotypes were cleaved at the hinge. Cysteine in the digestion mixture reduced the antibody molecules and increased the rate of digestion, but IgG2a was still more susceptible to cleavage than IgG2b in the absence of cysteine. Thus, not only reduced H chains can be cleaved by cysteine proteinase secreted by T. foetus but also intact functional antibody molecules. Because parasites may evade protective antibody responses by cleaving IgG2, animals with the more resistant IgG2b allotype may be better protected by immunization than animals with the more readily digested IgG2a allotype.     

Antibody-induced suppression and postsuppression stimulation of complement in vitro. III. Long-term C4 suppression is actively maintained by a soluble suppressor factor (FsC4)

Previous work in our laboratory established that individual complement components can be regulated in vivo by administration of specific antibody or immunocompetent cells to newborns and in vitro by administration of specific antibody to cultured peritoneal macrophages or splenic fragments. Antibody-induced suppression of C4 was much longer lasting in cultured guinea pig splenic fragments than in cultured guinea pig peritoneal macrophages, suggesting that splenic fragments contained elements necessary for long-term suppression that were not present in the macrophage monolayers. This publication presents data in support of this concept. Antibody-treated splenic fragments from normal guinea pigs--but not from C4-deficient guinea pigs--elaborated a soluble factor (FsC4) that suppressed C4 production in previously untreated splenic fragments. FsC4 activity was most potent in splenic fragment culture supernatants at those times when intracellular and secreted C4 hemolytic activity and C4 antigen were at their lowest. C4 itself or a fragment of C4 was therefore unlikely to mediate suppression in this system. Residual anti-C4 antibody was ruled out as a mediator of FsC4 activity since it was shown by two independent methods that the amount of anti-C4 antibody carried over with the supernatant was orders of magnitude less than the amount necessary to cause suppression or to neutralize fluid phase C4 in fresh splenic fragment cultures. Preliminary data revealed that FsC4 activity may be mediated by two or more distinct molecular species or may be mediated by a single molecule that exhibits secondary size and charge heterogeneity. The identification of factors that are capable of regulating C4 suggests that, as with immunoglobulins, complement components may be regulated by complex networks of immunocompetent cells and their soluble products.     

Variation in the N-terminal sequence of heavy chains of immunoglobulin G from rabbits of different allotype

The sequences of the N-terminal peptides prepared by Pronase digestion of the heavy chain of rabbit immunoglobulin G of allotype Aa1, Aa2 and Aa3 were determined and were shown to be related to the allotype. An N-terminal fragment of about 34 residues was also prepared from the allotype heavy chains, by cleavage with cyanogen bromide; the yield varied with the allotype. The sequences of the cyanogen bromide fragments from the Aa1 and Aa3 heavy chains contain allotype-related variations similar to those found in the N-terminal Pronase peptides, and these sequences are thought to be representative of the whole heavy-chain populations. There is about 60% homology between the two sequences, and superimposed on the differences between them there are a number of positions within each sequence at which at least two amino acids are present.     

Video enhanced imaging of the fluorescent Na+ probe SBFI indicates that colonic crypts absorb fluid by generating a hypertonic interstitial fluid

We report a rare ‘hypomorphic’ C4 allotype detected during routine screening in controls for the Rogers:1 epitope. C4B^*15 was distinguished by having only faint staining when using polyclonal anti-C4 antibody on agarose inimunoelectrophoresis (e.g. hypomorphic), having relatively weak hemolytic activity but being strongly reactive with monoclonal antibody to Rodgers 1. TaqI restriction fragment length polymorphism (RFLP) demonstrated that C4B^* 15 segregated with 7 kb and 5.4 kb C4 gene fragments and with the haplotype HLA-A2,C-, B50,BW6,DR7,DQ2,DR52,S07C2(1,15). The 5.4-kb fragment was more intense than the 7.0-kb fragment, suggesting duplication of the 5.4-kb fragment. This hypomorphic C4 allotype (genotype FREQUENCY = 0.0088) has diminished expression of C4 epitopes commonly recognized by polyclonal anti-C4 and may be missed by standard phenotyping methods.     

Antibody-induced suppression and postsuppression stimulation of complement in vitro. II. Intracellular and extracellular changes in C4 during long-term C4 suppression in guinea pig splenic fragments

Suppression of the synthesis of the fourth component of complement in vitro was originally accomplished by exposing cultured guinea pig peritoneal cells to anti-C4 alloantisera. When guinea pig splenic fragments were used instead of peritoneal cells, equivalent antibody treatment produced C4 suppression of significantly longer duration, lasting weeks instead of days after removal of antibody. As with peritoneal cell monolayers, antibody treatment induced specific suppression of C4 followed by nonspecific stimulation of C4 and other proteins such as C2. Although IgG2 is more readily sequestered by splenic tissue, both IgG1 and IgG2 antibodies were effective in inducing and maintaining suppression. Experiments with radiolabeled antibody demonstrated that a small amount (less than 5%) of the original dose of antibody was retained by the splenic fragments. Because there was no continuous slow release of that antibody, long-term suppression of C4 cannot be explained as a fluid-phase neutralization reaction. Because antibody treatment might induce production of aberrant C4 molecules with no functional activity, C4 antigens was also studied. Tissue culture supernatants were assayed by using an ELISA for C4. In none of these experiments was extracellular C4 antigen detectable immediately after antibody treatment. Extracellular and intracellular C4 were immunoprecipitated from biosynthetically labeled tissue cultures and analyzed by SDS-PAGE. Antibody treatment suppressed intracellular C4 as well as extracellular C4. Although extracellular C4 levels of antibody-treated cultures eventually returned to levels comparable to untreated cultures, intracellular C4 levels of treated fragments remained lower than controls for the full period of observation (22 days). Therefore, a short (4-day) exposure to anti-C4 antibody induced long-term effects that profoundly altered regulation of C4 synthesis and secretion by cultured splenic macrophages.     

Factors affecting natural antiallotype antibody production in rabbits

Since not all rabbits that have receivedb locus-incompatible antigens from the mother respond with natural antiallotype antibody, various factors were studied to determine their role in the expression or nonexpression of antibody production in the individual rabbit. It was found that the mother-offspring allotype relationship is important in determining the probability that a given individual will respond to maternally derived allotype differences, and that the ability to respond is under genetic control. The genetic basis appears to be polygenie with the response status of a given animal determined by a threshold of response-positive genes needed for response. A greater number of response-positive genes seems to be required if there is an antigenic relationship between the incompatibleb-locus antigen from the mother and the individual's ownb-locus antigens.Abbreviations used in this paper are as follows BDB bis-diazotized benzidine - _c ² chi-square corrected for continuity - HPA high probability antibody - LPA low probability antibody - HPA(+) or LPA(+) animal produced the natural antiallotype antibody designated - HPA(–) or LPA(–) animal did not produce the natural antiallotype antibody designated, even though it had the potential to do so