A reevaluation of rabbit anti-allotype antibody for the presence of cross-reactive idiotypes. II. Expression of rabbit a1-like images on goat antibody after immunization with anti-a1 antibody

In an effort to generate heterologous anti-idiotype (Ab2) molecules to a suspected IdX on rabbit anti-a1 antibody (Ab1), goats were immunized with either rabbit or guinea pig Ab1. The goat Ab2 preparations reacted with each of 13 rabbit Ab1, as well as two goat Ab1 samples in serologic assays. From 8 to 50% of the molecules in purified rabbit Ab1 preparations reacted with each goat Ab2. Electron microscopy of immune complexes composed of rabbit Fab anti-a1 and goat Ab2 reveals that the Fab anti-a1 binds to the side of the variable region of most goat Ab2 molecules, rather than at the tip (i.e. in the CDR) as expected. This configuration indicates that the goat Ab2 actually represents a population of induced or enhanced Ig molecules expressing a1-like allotypic or isotypic determinants, rather than an anti-IdX Ab or a paratope-associated internal image of a1.     

Cross-reactive idiotypes on heterologous anti-allotype antibody

Specific anti-a1 Ab was isolated from rabbits, guinea pigs, mice, chickens, and a goat. Each of these preparations was able to inhibit the reaction between a1 IgG and rabbit anti-a1 Ab as well as the reaction between rabbit IdX (anti-a1 Ab) and rabbit anti-IdX (anti-anti-a1 Ab). Maximal levels of inhibition ranged from 75 to 100% in the latter assay. Although the relative binding efficiencies of each preparation varied widely, there was generally a positive correlation between the ability of an anti-allotype reagent to bind to a1 IgG and to anti-IdX Ab. Each of the heterologous anti-a1 Ab samples was able to form precipitin bands with rabbit anti-Id Ab. These bands fused with each other and with rabbit anti-a1 Ab. These results weaken the interpretation that the ubiquitous expression of IdX previously observed in the rabbit reflects shared conserved genes. We suggest that either 1) the anti-Id Ab represents high fidelity internal images of the a1 epitopes, or 2) latent a1 allotype Ig is present in the anti-IdX preparation. In either case, any anti-a1 Ab would be potentially reactive with the anti-IdX preparation.     

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.     

Partial amino acid sequence and genetic control of latent a2 allotype induced in rabbits by immunization with anti-a2 antibody

We have shown that after immunization of homozygous a1 rabbits of the B immunoglobulin (Ig) heavy chain haplotype with anti-a2 antibody (Ab) a population of molecules appears that has all of the serologic characteristics of the a2 allotype. We have now isolated these putative latent a2 molecules, have separated the heavy chains, and after enzymatic deblocking, have determined the first 19 N-terminal amino acids. For all eight allotype-associated residues, these putative latent a2 molecules have the amino acid residues typical of a2 allotype. As expected, the preimmune IgG from this a1a1 rabbit has the amino acids typical of the a1 allotype. Thus by partial amino acid sequence analysis, we provide additional evidence that the latent a2 allotype can be induced in a1a1 rabbits of the B heavy chain haplotype by immunization with anti-a2 Ab. Rabbits of other heavy chain haplotypes were also immunized with anti-a2 Ab and were tested for their ability to synthesize latent a2 allotype. Thus far, a1a1 rabbits of the A, B, C, and I heavy chain haplotypes all synthesize latent a2 allotype. In contrast, a3a3 rabbits of the G and H heavy chain haplotypes did not synthesize latent a2 allotype.     

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.     

Preferential assignment of allotype a1 globulin for the production of early IgM anti-para-azobenzenearsonate antibodies in heterozygous a1a3 rabbits.

Rabbits of allotype a1a3 were injected on days 0, 2, and 4 with mixtures containing equal amounts of pigeon erythrocytes (Prbc) coupled to para-azobenzenearsonate (AA) and to para-azobenzene-N-trimethylammonium (TMA). On day 6, the allotypes of antibody from plaque-forming cells (PFC) of the blood were determined by observing the inhibition of plaque formation by anti-allotype sera. Anti-AA PFC appeared to consist for the most part of cells making antibody of allotype a1 since 65% of them were inhibited by anti-a1 serum and only 8% by anti-a3. Anti-TMA PFC, on the other hand, appeared to consist mostly of cells making antibody of allotype a3, since less than 1% of them were inhibited by anti-a1 but 47% by anti-a3. Antibody allotype for spleen PFC was also determined on day 6 and was similar to that found for blood PFC. Anti-AA PFC were inhibited 74% by anti-a1 serum and 15% by anti-a3 whereas anti-TMA PFC were inhibited 19% by anti-a1 and 43% by anti-a3. Serum hemolysin specific for AA hapten from a1a3 animals was also strongly inhibited by anti-a1 serum but not by anti-a3 whereas the converse was true for hemolysin against TMA hapten. The a1a3 rabbits, in whcih the anti-AA was restricted to allotype a1, were mated to produced homozygous a3a3 animals. When the PFC and serum antibodies of these a3a3 offspring were examined by specific inhibition, the anti-AA activity was found to be of allotype a3 rather than being a-negative. The number of anti-AA PFC in the blood of a3a3 rabbits was lower than that in blood of a1a3 or a1a1 animals. In addition, the TMA hapten appeared to inhibit the response to the AA hapten. Thus a1a3 rabbits immunized with AA-Prbc alone had 14-fold more anti-AA PFC or 18-fold higher anti-AA hemolysin titer than a3a3 animals immunized with both AA-Prbc and TMA-Prbc. Our results are discussed in relation to various explanations which have been offered for an imbalance of allotypes in a given antibody.     

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.     

Heavy chain variable region allotypic subspecificities of rabbit immunoglobulins. II. Selective escape of the a1-AB Ig subpopulation after the induction of auto anti-a1 antibody.

An a1a2 rabbit (P286-3), neonatally suppressed for the expression of the a1 allotype, was immunized with autologous a1 IgG at 2 months of age. Both auto anti-a1 Ab and a1 IgG molecules were found in the serum of this rabbit after the auto-immunization. The auto anti-a1 Ab and the IgG from the auto anti-a1 Ab-depleted serum were isolated. Of the previously defined a1-AB, a1-AC, and a1-AD Ig subpopulations, the a1 IgG in the IgG preparation from the rabbit P286-3 were all of the a1-AB Ig subpopulation. The auto anti-a1 Ab from rabbit P286-3 did not react with the a1-A, a1-B, and a1-C allotypic subspecificities; thus, it was presumably specific for the a1-AC and a1-D allotypic subspecificity. Thus, the a1-AB Ig subpopulation escaped from allotype suppression in rabbit P286-3, whereas the a1-AD Ig subpopulation remained suppressed. The a1-AD Ig subpopulation will probably remain suppressed for a long time and perhaps permanently since rabbit P286-3 has produced circulating auto-Ab specific for the a1-D allotypic subspecificity. These results indicate that the a1 Ig subpopulations are synthesized by distinct clones of lymphocytes under separate control.     

Characterization of a monoclonal antibody to guinea pig T cells that inhibits rosette formation of the cells with rabbit erythrocytes: similarity of the antigen to E-receptor on human T cells

A monoclonal antibody, GPT-1, was prepared by fusion of the splenic cells of mice immunized with guinea pig thymocytes with a mouse myeloma cell line. GPT-1 completely inhibited spontaneous rosette formation of T cells with papain-treated rabbit erythrocytes. GPT-1 reacted with 90% of thymocytes, 70% of peripheral blood lymphocytes, and 45% of splenic lymphocytes, but not with B cells. These results indicate that GPT-1 has pan-T reactivity. The antibody specifically bound to a single polypeptide chain with a molecular size of 50-65 kD. The surface density of the antigen was higher on thymocytes than on peripheral T cells, suggesting that the antigen is a certain differentiation antigen on T cells. Phytohemagglutinin-activated T cells expressed more antigen molecules than resting T cells. In addition, GPT-1 suppressed the proliferation of T cells induced by the mitogen, indicating that GPT-1 recognizes a T cell-specific surface antigen which is associated with T cell activation. Based on these results, it was concluded that GPT-1 reacts with a guinea pig T cell surface antigen which is similar to the E-receptor protein on human T cells (CD2 molecule).     

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.     

Lysis of tumor cells by antibody and complement. II. Lack of correlation between amount of C4 and C3 fixed and cell lysis.

Two antigenically distinct diethylnitrosamine-induced guinea pig hepatoma cell lines, line-1 and line-10, sensitized with rabbit anti-Forssman or with tumor-specific antibody, were more susceptible to killing by human complement (HuC) than by guinea pig complement (GPC). This difference could not be ascribed to differences in the amount of C1, C4, and C3 fixed: millions of C4 and hundreds of thousands of C3 were detected on cells whether they were killed or not killed by the C sources. Tumor cells sensitized with anti-Forssman IgM antibody generally had more GP C4 and C3 than Hu C4 and C3 bound to their surfaces. Cells sensitized with anti-tumor antibody generally had more Hu C4 and C3 than GP C4 and C3 bound to their surfaces. The resistance to killing of nucleated cells by antibody and C may be due in part to intrinisic properties of the cell.     

Effects of heterologous sera on fertilized rabbit ova

Undiluted blood serum of various species was used to culture two-celled rabbit ova for 24 hours. It was found that there is an ovocidal factor present in the serum of man, sheep, cattle, goat, and fowl. The factor is lethal rather than inhibitory; exposure to it for 10 minutes will cause the death of the ova. This factor is unstable, thermolabile (destroyed at 55 degrees C. in 30 minutes), and of large molecular size. The strength or concentration of this factor varies according to the origin of the serum, increasing in the order man, sheep, cattle, goat, fowl. The blood serum of rabbit, horse, dog, guinea pig, rat, and pig contains no ovocidal factor against rabbit ova. The ovocidal factor differs from the spermicidal factor, which is present in all the sera of the different species studied with rabbit spermatozoa. Immunization of the guinea pig against rabbit ova is possible. Normal development of young rabbits was obtained by transplantation of ova cultured in the heated or normal serum of other species after 24 hours.     

Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.     

The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies

The rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for the generation of therapeutic human antibodies. The humanization of rabbit antibodies, however, has not been reported. Here we use phage display technology to select and humanize antibodies from rabbits that were immunized with human A33 antigen which is a target antigen for the immunotherapy of colon cancer. We first selected rabbit antibodies that bind to a cell surface epitope of human A33 antigen with an affinity in the 1 nm range. For rabbit antibody humanization, we then used a selection strategy that combines grafting of the complementarity determining regions with framework fine tuning. The resulting humanized antibodies were found to retain both high specificity and affinity for human A33 antigen.     

Phagocytosis of Candida albicans by rabbit alveolar macrophages and guinea pig neutrophils.

Studies of host-parasite relationships at the cellular level, using Candida albicans and rabbit alveolar macrophages or guinea pig neutrophils are presented. Guinea pig neutrophils killed the intracellular candida cells presumed by myeloperoxidase-halide-hydrogen peroxide system. In contrast, rabbit alveolar macrophages did not kill the intracellular candida cells although their phagocytic rate was almost comparable to that of neutrophils. Phagocytizing macrophages were eventually destroyed by the intracellular proliferation of candida cells and formation of germ tubes and pseudomycelia. No significant improvement of candidacidal activity was observed with macrophages from normal and immunized rabbits in immune serum. The mode of phagocytosis by macrophages and neutrophils were also studied under the scanning electron microscope.     

Lysis of tumor cells by antibody and complement. III. Lack of correlation between antigen movement and cell lysis.

The increase in susceptibility to killing by rabbit antibody and guinea pig complement of guinea pig hepatoma cells (line-10), after treatment with certain metabolic inhibitors, did not correlate with the mobility of antigen on the cell surface as measured by indirect immunofluorescence.     

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.     

Isolation and characterization of a specific antibody population against human fibrinogen

A specific antibody population against human fibrinogen was isolated from a rabbit antiserum by affinity chromatography on fibrinogen-bound Sepharose gel. Using a sensitive competitive radioimmunoassay, the antibody population was found to recognize epitopes on native fibrinogen but crossreacted minimally with fibrinogen fragment D and an early plasmin-degraded fibrinogen A alpha-chain product, but not at all with fragment E or fibrinopeptides A and B. Fibrin monomers shared part of these epitopes. The antibody population crossreacted to a small extent with bovine, horse and baboon fibrinogens and not at all with fibrinogens from sheep, rat, pig, goat, guinea pig, dog and rabbit.     

Immunochemistry of prostaglandin endoperoxide-forming cyclooxygenases: the detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence.

Rabbit antiserum has been prepared against the prostaglandin endoperoxide-forming cyclooxygenase (EC 1.14.99.1) purified from sheep vesicular glands. Ouchterlony double diffusion and immunoelectrophoretic analyses indicate that the anti-cyclooxygenase serum is monospecific for the enzyme. The anti-cyclooxygenase serum reacts with both active and inactivated forms of the sheep vesicular gland (SVG) cyclooxygenase. Furthermore, the immune serum precipitates solubilized microsomal cyclooxygenases from each of six other tissues examined, including bovine seminal vesicle, rabbit kidney medulla, guinea pig lung, dog spleen, sheep uterus, and human platelets. Anti-SVG cyclooxygenase serum was used in combination with fluorescein isothiocyanate )FITC)-labeled goat anti-rabbit IgG to detect cyclooxygenases in cryostat sections from rat, rabbit and guinea pig kidneys by immunofluorescence. Highly prominent fluorescence was associated only with the epithelial cells lining the collecting ducts in rabbit and guinea pig kidneys, and except for the nucleus, was uniformly distributed within the interior of these cells. In rat kidney, fluorescence was detected not only in collecting tubules but also in the interstitial cells of the renal papilla. Our results are consistent with the emerging hypothesis that PGE2 produced intrarenally plays a physiological role in natriuresis.     

Differentiation autoantigen of testicular cells and spermatozoa in the guinea pig.

Guinea pigs, immunized with autologous testes, produce antibody that reacts with surface antigen(s) of epididymal sperm and testicular cells. Both IgG and F(ab')₂ fragments of the antibody molecules bind to testicular cells. Only 50% of the testicular cells had detectable surface antigens and they are identified by rosette formation with Staphylococcus aureus, which bind only to testicular cells and sperm coated with guinea pig IgG. Rosette-positive testicular cells (percentage of cells positive) are: early round spermatids (8%), oval late spermatids (98%), cytoplasmic droplets (93%), and heads (and midpieces) of testicular and epididymal sperm (100%). As determined by a quantitative cellular radioimmunobinding assay, antibody to testicular cells is absorbed by testis or sperm but not by other male or female guinea pig tissues. The antigen, which could be multiple, is therefore sperm and late spermatid specific, and is designated testicular cell-sperm differentiation autoantigen. The surface distribution of this antigen is modulated by antibody to exhibit temperature-dependent translational movement along the plasma membrane.