Heterozygous Rabbits Research Articles

Demonstration of double allelic expression (allelic inclusion) of rabbit κ light chain allotypes on heterozygous lymphocytes by double allotype sequential stimulation

The hypothesis that individual lymphoid cells from rabbits heterozygous for genetically controlled immunoglobulin antigenic determinants (allotypes) are able to express only one of the allelic specificities (allelic exclusion) has been tested by double anti-allotypic sequential stimulation (DASS). DASS refers to stimulation by anti-allotypic serum directed to one specificity, followed, after blocking, by stimulation with an anti-allotypic serum directed to a different allotypic specificity. Previously published data reveal that most lymphocytes from the peripheral blood (PBLs) may be stimulated to undergo “blast transformation” by the addition of anti-immunoglobulin (anti-Ig) or anti-allotype antiserum and that this stimulation is mediated through surface immunoglobulin on the responding cells. In addition, the process of transformation may be reversed (blocked) by the addition of serum immunoglobulin which reacts with the stimulating antiserum. Such “blocked” cultures may be restimulated by removal of the blocking immunoglobulin and readdition of the stimulating antiserum. The present report demonstrates that lymphocyte cultures from heterozygous donors prestimulated by antiserum to one of the allelic specificities, after blocking and subsequent stimulation by antiserum directed towards the other allelic specificity, respond to a greater extent than do cultures stimulated by one antiserum only. This suggests that some individual PBLs of a heterozygous rabbit express both allelic Ig allotypes on their surface. The data suggest that it is possible that doubly expressing cells may produce both allelic markers sequentially or alternatively.

Evidence for active synthesis of two allelic b locus markers by single lymphocytes from heterozygous rabbits

The double expression on single cells of both b4 and b6 allotypic markers by the mixed antiglobulin technique was observed on single peripheral blood lymphocytes (PBL) from heterozygous rabbits which were treated with pronase and incubated overnight to allow for regrowth of surface immunoglobulin (Ig). The percent of double staining rosettes observed after lymphocyte processing prior to pronase stripping ranged from 10–30%. Following pronase treatment at concentrations allowing for maximum stripping but assuring high cellular viability, the total rosetting population was reduced to 1–3% in the majority of cases. Occasionally, pronase stripping was less effective. Overnight incubation in serum free medium resulted in the reappearance of surface Ig with double staining rosettes ranging from 8–30%. Exposure of b6 or b4 normal rabbit serum to b4 or b6 cells, respectively, resulted in uptake of b6 in which most of the passively acquired Ig eluted off during overnight incubation, while negligible uptake of b4 was seen. Coincubation of normal b4, 4 PBL which presumably are capable of donating Ig with pronase treated b6, 6 PBL and vice versa also resulted in the absence of mixed rosettes. That simultaneous active synthesis of both allelic markers results in double allotype expression was demonstrated by kinetics data. Supporting this was the observation that only after washing of cycloheximide inhibited cells did allotype reexpression resume.

Study of A100 allotypic specificity of rabbit immunoglobulins: the cross-reactivity between A100, a1 and a3.

Evidence for the genetic determination of A100 allotypic specificity suggests that it is the product of an allele at the alpha locus. The A100 allotypic specificity was detected in a wild rabbit lacking all the known specificities of the a series. None of the 8 rabbits possessing the A100 allotypic specificity possessed two allotypic specificities of the a series. The concentration of A100+ molecules is larger in the serum of a presumably homozygous rabbit than in that of a presumably heterozygous rabbit. In the sera of heterozygous A100+ rabbits, the A100 allotypic specificity and the known specificity of the a series are carried by different molecules. The determinants responsible for the A100 allotypic specificity are present both on IgG and IgM. They are located on the heavy chain and the Fab fragment of IgG. A family of variants of A100 specificity has been detected and cross-reactivity between A100, a1 and a3 specificities has been found. This was detected and studied with anti-a3 antisera. It seems that A100 allotypic specificity is closer to a3 specificity than a1 is to a3 specificity.

Effect of VHa Locus Allotype-Suppression on the Expression of Closely Linked VHx, VHy, and Cµn Genes in Heterozygous Rabbits

Abstract Injection of a1x-y-n81/a2x32y33n82 heterozygous rabbits at birth with anti-a2 allotype antiserum suppresses the production of the VHa2 gene product a2 Ig as well as the expression of the closely linked Cµn gene product n82 but not of the closely linked VHx and VHy gene products x32 and y33. This effect is related to the appearance of a2 and n82 on the same IgM molecules whereas a2, x32, and y33 appear on different Ig molecules.

In vitro studies on allotype suppression. III. compounds of antiallyotype serum active in release from allotype suppression.

Spleen cells of b4b6 rabbits, shown to be deficient in their ability to produce b4Ig due to prenatal exposure to anti-b4, formed anti-T2 antibodies marked with the b4 determinant in response to solubilized T2 phage (S-T2) only when cultured in the presence of antibodies specific for the nonsuppressed type (b6), thus confirming and extending the previously reported observation of release from b4 suppression in cultured cells of b4-suppressed b4b5 rabbits treated with anti-b5 serum. Only antiallotype sera made in b4 rabbits were active in reversing b4 suppression. Anti-b5 or anti-b6 sera from rabbits of allotypes b6 or b5, respectively, when used in concentrations which completely or partially inhibited the formation of anti-T2 antibodies marked with the corresponding nonsuppressed allotype of the spleen donor, proved to be almost completely ineffective in causing release of suppression. Exceptions were noted when spleen cells of rabbits advanced in spontaneous escape from suppression were tested with such sera. The addition of normal b4 serum to non-b4 antiallotypic sera rendered them as effective in releasing b4 suppression in vitro as were antisera from b4 rabbits. Furthermore, the capacity of a b4 antiallotype serum to cause reversal of b4 suppression could be potentiated by the addition of normal b4 serum, indicating that nonantibody b4 Ig is a limiting factor in such a serum. Thus, the release from allotype suppression observed in cultures of spleen cells from b4-suppressed heterozygous rabbits is dependent upon the presence of two components: antibodies directed against the nonsuppressed allotype of the donor and normal b4Ig. These findings are interpreted in terms of alternate hypotheses involving (a) a mechanism of b4 derepression and (b) inactivation of a suppressor cell with recognition for a b4-labeled target.

In Vitro Studies on Allotype Suppression

Abstract The regulatory effects of rabbit antibodies specific for light chain determinants (b locus) on the formation of rabbit serum immunoglobulins have been studied in an in vitro system which measures the response of unprimed rabbit spleen cells to solubilized T2 phage antigen. Treatment of spleen cells from b4b4 rabbits with anti-b4 serum, which was either incorporated into the culture medium or employed in appropriate pulse treatment of the cells before culture, prevented the formation of T2 neutralizing antibodies by such cells. Spleen cells of heterozygous (b4b5) rabbits formed anti-T2 antibodies which could be shown to be divided between the b4 and b5 specificities. Incorporation of anti-b4 or anti-b5 serum into the culture medium suppressed the specific anti-T2 response and, except in the instances noted in the text, did not significantly change the level of T2 neutralizing antibodies marked with the alternate allelic determinant. These findings are discussed in the light of the compensatory formation of an alternate immunoglobulin type which occurs during allotype suppression in vivo.

In Vitro Studies on Allotype Suppression

Abstract Spleen cells from heterozygous rabbits with b locus immunoglobulin allotypes b4 and b5 which had been exposed in utero to maternal antibodies against the paternal allotype (b4) were stimulated in vitro with solubilized T2 phage antigen. The cells of thoroughly suppressed rabbits formed T2 neutralizing antibodies which were identifiable only as having allotype b5. The presence of anti-b5 serum in the cultures inhibited b5 expression and resulted in the formation of antibodies having the previously suppressed allotype, b4. Normal rabbit serum of the same allotypic specificity as the anti-b5 serum (b4) had no effect on the allotype distribution of anti-T2 antibodies. Although partial inhibition of b5 production occurred when the anti-b5 serum was removed from cultures after 5 hr, no reversal of b4 suppression was observed. Possible mechanisms accounting for these results are discussed.

Studies of hyperimmune restricted and partially restricted anti-pneumococcal polysaccharide antibodies from allotype-defined pedigreed rabbits. I. Preparative liquid isoelectric focusing of the antibodies and characterization of the isolated fractions by electrophoretic techniques.

AbstractFour heterozygous rabbits (a2 a2, b4 b5; a1 a2, b6 b9; a2 a2, b5 b9; a3 a3, b4 b5) and one homozygous rabbit (a2 a2, b6 b6) were hyperimmunized with either type 3 or 8 pneumococcal vaccines. Four antisera were partially restricted and one antiserum was highly restricted. The immunoglobulins (Ig) were obtained by salt precipitation, ion‐exchange chromatography, and affinity chromatography. The total Ig, obtained by salt precipitation, were fractionated by preparative liquid isoelectric focusing (IEF) at various pH ranges. From each antiserum a number of fractions were obtained with significantly different isoelectric points. Many major fractions had reduced electrophoretic heterogeneity when compared with the Ig isolated before IEF. These fractions were tested for isoelectric purity by analytical IEF in polyacrylamide gels and by preparative refocusing over a narrow pH range. After complete reduction and alkylation, the light and heavy polypeptide chains of the focused antibody fractions were examined both by disc electrophoresis in alkaline urea gels and by IEF in polyacrylamide gels containing urea. From each antiserum, a number of fractions were obtained which yielded single light chains often with different electrophoretic mobilities. This method provided a successful means of preparatively obtaining different antibody fractions from a single antiserum.

Preferential allotypic expression of anti-hapten response in first and second generation heterozygous rabbits of al,3 allotype

Preferential allotypic expression of anti-hapten response in first and second generation heterozygous rabbits of al,3 allotype

Studies of hyperimmune restricted and partially restricted anti-pneumococcal polysaccharide antibodies from allotype-defined pedigreed rabbits. II. Allotypes and idiotypes in sera and electrofocused fractions.

AbstractThe expression of allelic forms of immunoglobulin during immunization of heterozygotes with pneumococcal vaccines was monitored using anti‐allotype antisera specific for the genetically different forms of rabbit kappa chains (b‐locus allotypes). We also used the a‐locus (VH) allotypic markers of rabbit heavy chains and idiotypic determinants associated with a restricted subpopulation of anti‐polysaccharide antibodies of an individual rabbit. Analysis of fractions obtained by preparative liquid isoelectric focusing (IEF) of antisera from heterozygous rabbits exhibiting restricted antibody responses to type 3 or type 8 pneumococcal polysaccharides, indicated an enrichment for one a or b locus allotype. Enrichment of a specific idiotype from ∼ 11 % in the starting serum up to as high as 65 % in a focused fraction was also obtained. The method of preparative liquid IEF can thus be used to isolate antibodies restricted in allotype and idiotype from antisera. The quantities obtained are sufficient for further chemical and immunological study.

Preferential allotypic expression of anti-hapten response in first and second generation heterozygous rabbits of al,3 allotype

Heterozygous rabbits of the al,2 or al,3 and b4 or b4,5 allotypes were injected with bovine serum albumin (BSA)‡ coupled with equivalent ammounts of p-azophenylarsonate (As) anions and p-azophenyl-N-trimethylammonium (TMA) cations. The isolated and purified anti-As, anti-TMA and anti-BAS antibodies were tested for their allotypes. All three types of antibodies from heterozygous rabbits of the allotypes al,2 and/or b4,5 gave strong precipitates with anti-a1, anti-a2, and/or anti-b4 and anti-b5 immune sera. However, in some of the al,3/b4 immune sera the anti-As antibodies gave strong preipitates only with anti-a1 and anti-b4, but very weak or no detectable precipitates with anti-a3, whereas the anti-TMA and anti-BSA of the same animals gave distinct precipitates with anti-a1, and anti-a3 and anti-b4 immune sera. The weak expression or the apparent lack of the a3 allotype in anti-As antibodies is transmitted through the least two generations according to simple Mendelian fashion. We attribute it to heterogeneity of the receptors of the a3 allotype; only some of them to be stimulated by the As-hapten of the injected As-TMA—BSA immunogen.

In vitro suppression of immunoglobulin allotype synthesis by Fab and F(ab′) 2 fragments of anti-allotype antibody

The F(ab′) 2 and Fab fragments were prepared from IgG containing antiallotype antibody (Ab) specific for the kappa light chains (i.e., anti-b4) and were tested for their ability to suppress b4 allotype production in vitro. Spleen cells from a b 4b 5 heterozygous rabbit were incubated for 24 hr with the F(ab′) 2 or Fab fragments and then washed and cultured for an additional 4 days. The tissue culture supernatant fluids were analyzed for the b4 and b5 allotypes by a radioimmunoassay. The results clearly indicated that both types of IgG fragments can suppress the production of the b4 allotype. Thus, neither the Fc fragment nor the bivalency of the intact Ab molecule is essential for suppressing activity. This makes it highly unlikely that the allotype suppression in vitro occurs as a result of cell death. When equivalent amounts of the IgG, F(ab′) 2, or Fab anti-b4 preparations, as determined by their antigen binding capacity, were compared for their relative efficiencies in the induction of b4 allotype suppression, the IgG and F(ab′) 2 were found to be equally efficient, while the Fab fragment was less efficient than the bivalent Ab preparations.

Allelic Selection of Light Chain Allotypes by Antibodies Raised in Heterozygous Rabbits

Abstract A marked selection for allotype b4 over b9 was observed for anti-streptococcal antibodies produced in rabbits heterozygous for allotypes of groups a and b. Less than 2% of the streptococcal antibodies was of allotype b9 while 20% of the IgG in preimmune sera from the same rabbits was of allotype b9. No such effect was observed with respect to the group a allotypes. In spite of this marked selectivity in b4,b9 heterozygotes for the production of antistreptococcal antibodies of allotype b4, no evidence for linkage between the gene controlling this allotype and immune responsiveness to streptococcal antigens could be detected. Equal amounts of antibody were produced by all immunized offspring (b4,b4; b4,b9; or b9,b9) of heterozygous rabbits.

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 b 4 b 5 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 b 4 b 5 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.

Altered quantitative expression of immunoglobulin allotypes in rabbits.

In 1962, Dray showed that the quantitative expression of allelic allotypes in heterozygous rabbits was affected by exposure of the neonatal rabbits to maternal isoantibodies directed against the antigenic type inherited from the father (Dray, 1962). This phenomenon has become known as allotype suppression. During the decade since the discovery of the phenomenon, a considerable body of information has accumulated about the genetics, structure and localization of the genetically controlled antigenic variants of rabbit immunoglobulins (immunoglobulin allotypes) (reviewed by Mage et al., 1973a). This review will summarize our knowledge of allotype suppression in the light of current concepts of immunoglobulin structure and cellular immunology.KeywordsLight ChainRabbit ImmunoglobulinImmunoglobulin AllotypeAllotypic DeterminantAllotypic SpecificityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Cell-free synthesis of peptides bearing rabbit immunoglobulin allotypic markers

A cell-free system derived from the spleens and lymph nodes of genetically characterized rabbits has been used to synthesize peptides bearing b4 and b5 allotypic specificities which are genetic markers for immunoglobulin light chains. Peptide synthesis requires polysomes, a pH 5 fraction, and an exgenous source of ATP. The system appears to complete peptides previously initiated on the polysomes in vivo. Newly synthesized peptides bearing b4 or b5 allotypic markers of immunoglobulin light chains were detected by their inhibition of the reaction of 125I-labeled b4 or b5 Fab fragments with the specific anti-allotype antiserum. Sucrose gradients were used to assay for this interaction. As expected, cell-free systems from b4 to b5 homozygous rabbits synthesized peptides bearing only b4 or br allotypic markers. Although both types of light were present in the serum of heterozygous rabbits, systems from b4b5 heterozygous rabbits tended to support the synthesis of either b4 or b5 peptides but not both.

ALLOTYPE SUPPRESSION IN THE RABBIT

The ability to distinguish immunoglobulin (Ig) of paternal allotype both on lymphocyte membranes and in the serum of neonatal b(4)b(5) heterozygous rabbits has allowed us to study the postnatal ontogeny of cells bearing endogenously synthesized Ig that could not have been derived from the mother. In normal b(4)b(5) rabbits, such endogenously synthesized Ig of paternal allotype is present on the membranes of bone marrow-derived (B) lymphocytes from birth, but does not appear as detectable circulating Ig until approximately 2 wk of age. In the neonate, cells bearing the paternal allotype are potential targets for the induction of chronic allotype suppression. Within 24 h of exposure to anti-allotype antisera in vivo, Ig of the suppressed paternal allotype is no longer detectable on the surface of neonatal lymphoid cells. Further, this lymphocyte membrane Ig is eliminated and not simply masked by the suppressing antibodies. Finally, cells bearing the suppressed allotype are deleted from all lymphoid organs for the duration of total allotype suppression, and reappear first in bone marrow and peripheral blood at the time of spontaneous escape from suppression.

Isolation and Characterization of Electrophoretically Homogeneous Rabbit Antihapten Antibody Populations: III. NH2-TERMINAL LIGHT CHAIN SEQUENCE ANALYSES OF FOCUSED ANTI-p-AZOPHENYLTRIMETHYLAMMONIUM AND ANTI-p-AZOBENZENEARSONATE ANTIBODY FRACTIONS

Abstract The structural heterogeneity of light polypeptide chains derived from rabbit antibodies directed against p-azophenyltrimethylammonium (Ap) and p-azobenzenearsonate (Rp) haptens from individual heterozygous rabbits was investigated. Antibodies were purified by adsorption on specific solid immunoadsorbents, followed by preparative liquid isoelectric focusing at either pH 5 to 8 or pH 7 to 10. The focused anti-Ap antibody fractions were refocused in pH gradients extending only 0.5 pH unit on either side of their respective isoelectric points. A number of focused antibody fractions demonstrated electrophoretic, structural, and functional features characteristic of homogeneous antibody populations. Amino-terminal sequences of the kappa light chains, obtained by mild reduction and alkylation followed by gel filtration, were determined for nine focused fractions of anti-Ap antibody from one rabbit, seven focused fractions of anti-Rp antibody from a second rabbit, and two focused fractions of anti-Rp antibody from a third rabbit. Amino acid sequence analyses extended for 6 to 39 positions (a few included the first hypervariable region) depending upon the quantity of light chain obtained from the focused fractions. Sequences obtained from light chains isolated from serum prior to immunization immunoglobulins or from specifically purified unfractionated antibodies were heterogeneous (2 to 4 residues at most positions), while those from the major focused antibody fractions were as homogeneous, for most of the first 25 to 30 positions, as human kappa Bence-Jones proteins sequenced by the same methods. The light chains from the focused antibodies demonstrated significant microheterogeneity and nonidentity within limited areas (positions 31 to 34). Focused antibody light chains had sequences representative of either the Vkii or Vkiii subgroups. This approach, therefore, permits structural studies to be performed on the homogeneous antibody constitutents of heterogeneous antibody preparations and demonstrates that the heterogeneity is relatively restricted to limited areas.

Comparative studies of Aal allotypic specificity in IgG and IgM of rabbits. II. Phenotypic expression of allotypic specificities in rabbits exposed in neonatal life to antibodies directed against Aal allotypic determinants specific for IgG.

Neonatal rabbits were treated with antibodies directed against Aal allotypic determinants specific for IgG and its effects on development of allotypically determined immunoglobulins were analysed in sera of- the treated animals. The treatment of Aa1Aa3 heterozygous rabbits resulted in a delayed appearance in serum of all or some Aa1+ immunoglobulins: In some animals neither Aal+ IgG nor Aal+ IgM were detected for 9 weeks after birth and in others no Aal+ IgG only Aal+ IgM was found up to 5 weeks after birth. In Aa1Aa1 homozygous rabbits, no similar effect was observed. Another effect of the treatment was manifested as supernormal levels of serum IgG and IgM with a maximum at 9 weeks. This effect was more remarkable in homozygotes than in heterozygotes in which phenotypic expression of some Aal allotypic patterns was suppressed. In other heterozygotes in which phenotypic expression of all Aal allotypic patterns was suppressed, IgG levels were lower than controls, suggesting that the suppressed phenotypic expression was not compensated. Quantitative analyses of IgG molecules carrying Aal allotypic specificity have revealed that approx. only 40% of IgG molecules carried the Aal allotypic determinants specific for IgG in an IgG preparation from an Aa1Aa1 homozygote in which more than 60% of the molecules possessed Aal allotypic specificity.

Contribution of the allelicMtz 3 andMtz 4 allotype genes to the formation of individual rabbit serum α2-macroglobulin molecules

The contribution of the allelicMtz 3 andMtz 4 genes to the formation of individual rabbit serum α2-macroglobulin (α2M) molecules was examined by precipitation of α2M from rabbits of known genotype with antiallotype antisera. The α2M was isolated fromz 3z3 andz 4z4 homozygous andz 3z4 heterozygous rabbits, iodinated with I125 and precipitated by sequential reactions with antiallotype antiserum and goat anti-rabbit IgG. Purified unlabeled α2M or α2M in serum was used to inhibit competitively the reaction of antiallotype antiserum and labeled α2M. Nearly all α2M molecules have z3 or z4 antigenic determinants; approximately 50% of α2M molecules in heterozygotes have both. Altogether, the z3, z3,4, and z4 molecules in heterozygotes have approximately 60% of the number of z3 and 40% of the number of z4 determinants as compared to the respective homozygotes. Unlike all other known allelic blood protein systems of rabbits, allelic exclusion does not occur in α2M molecules of heterozygotes; rather, hybrid molecules are formed.