Is there really a “lack of natural tolerance to allotypic γ-globulins in rabbits”?

We present data of extended studies on the possibility of maternally derived allotype Ig inducing a state of natural immunological tolerance to a non-inherited allotype in the offspring. Rabbits homozygous at the a locus, encoding allotypes in the variable region of immunoglobulin heavy chains, and rabbits homozygous at the unliked b locus, encoding allotypes of the constant region of kappa 1 chains, were immunized at the age of 2 months against the non-inherited allotype of their heterozygous mothers to which they had been exposed in utero and in early life. As control, we immunized rabbits of the same Ig phenotype but born to homozygous mothers, and therefore not exposed to that allotype. Immunization was done in a3/a3 offspring of either a1/a3 or a3/a3 mothers, by injecting a 1 IgG, and in b6/b6 offspring of b4/b6 or b6/b6 mothers, by injecting b4 IgG. The IgG was injected either in a soluble form or emulsified in adjuvant. Injection of soluble IgG elicited only a low response, if any, revealing no differences between the various groups. All rabbits responded upon immunization with IgG in adjuvant. We have not found any good evidence for natural tolerance to a non-inherited allotype, although progeny of a1/a3 mothers had slightly decreased responses to a1. On the contrary, progeny of b4/b6 mothers responded even better than offspring of b6/b6 mothers, upon such immunization with b4. To induce tolerance experimentally, we injected newborn rabbits, either from heterozygous a1/a3 or from homozygous a3/a3 mothers, with a1 serum or IgG. Newborn of heterozygous b4/b6 or of homozygous b6/b6 mothers were injected with b4 serum or IgG in the same way. Such treatment resulted in partial tolerance to each allotype. In an attempt to amplify the tolerizing effect of the maternal a1 Ig, we injected newborn rabbits of a1/a3 mothers with the serum of their mother. The response upon subsequent immunization with a1 allotype of another individual did not differ significantly from the response of control rabbits. The response was much poorer when rabbits were injected with nonmaternal tolerogen at birth, and when the same Ig preparation was used as immunogen. In a control experiment, neonatal injection of xenogeneic proteins, human IgG or bovine serum albumin, clearly resulted in tolerance. We speculate that tolerance to allotypes is established in the T cell repertoire only but bypassed by recognition of idiotypic determinants on antigen molecules by helper T cells, which trigger anti-allotype antibody formation by allotype-specific B cells. The end result of it is a lack of natural tolerance.     

Kinetics of Ig VH region gene expression: Allotypic and idiotypic specificities of anti-p-azophenylarsonate antibodies produced by al-suppressed a1a3 rabbits

The amount of anti-p-azophenylarsonate (anti-Ars3) antibodies produced by al-suppressed a¹a³ heterozygous rabbits was similar to the amount produced by nonsuppressed a¹a³ rabbits. However, when the al allotype emerged from suppression, the previously observed preferential expression of the al allotype for these antibodies was not restored: the al anti-Ars Ab produced in these rabbits remained very low. These observations suggest that the establishment of clones of the nonsuppressed-a3 allotype early in the immune response prevented the preferential expression of al anti-Ars Ab later on. By radioimmunoprecipitation, the kinetics of the idiotypic specificities of the anti-Ars antibodies were studied, along with the VHa locus allotypic specificities, from two rabbits. The idiotype of the al anti-Ars Ab produced, after the emergence of the al allotype from suppression, appeared to be different from the a3 anti-Ars Ab produced in the same rabbit, rabbit R2-3. In a second rabbit, R3, producing exclusively anti-Ars Ab of the a3 allotype, however, two transitions in idiotype expression were observed; the first idiotype disappeared for a period of about 30 weeks, and then reappeared. This transition in idiotypes may reflect a mechanism of autoregulation, that requires further investigation.     

The activity of homologous and heterologous anti-allotypic sera in transformation and allotypic suppression in rabbits

Rabbit anti-allotype sera produce transformation of rabbit peripheral blood lymphocytes and when injected neonatally into genetically heterozygous rabbits can cause suppression of the paternally contributed allotypic immunoglobulin. In contrast sheep anti-rabbit allotype sera, although causing similar transformation do not cause allotypic suppression when injected neonatally.     

In utero suppression of kappa 2 isotype in homozygous bas/bas rabbits through embryo transfer.

Allotypes of rabbit Ig provide a useful tool for the study of quantitative expression of alternative allelic forms. In the rabbit, maternal Ig is transmitted to the foetus and protects the immunologically immature newborn during several of the first weeks of life. Induced maternal antibodies directed towards a paternally inherited allotype of the offspring can influence the expression of that allotype drastically. Normal Ig level in heterozygous allotype suppressed animals is provided by increased expression of the alternative allele. On the other hand, allotype suppression in homozygous animals leads to increased expression of non-allelic alternative gene products. We quantitatively analysed the expression of bas, a marker on the kappa 2 light chain isotype of the mutant strain Basilea, in homozygous bas/bas rabbits which had been fostered in utero of b4/b6 mothers producing anti-bas. All of the offspring studied expressed the kappa 2 isotype at a very low level at two months of age, and, in some individuals, bas was hardly expressed after 6 to 12 months. A suppressed rabbit, which was immunized to produce auto-antibodies against bas, continued to do so for at least 2.5 years. The long duration of homozygous bas suppression contrasts with homozygous suppression of other allotypes (CL kappa 1 and VHa) which generally lasts for a much shorter period.     

Allotype suppression in the chicken. I. Generation of chronic suppression in heterozygous but not in homozygous chickens.

The injection of antibody directed against either the IgM-1 a or IgM-1 b allotype into heterozygous (M-l^a/M-l^b, G-l^g/G-lⁱ) B 14-line chickens produced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes in their serum, as quantitated by single radial immunodiffusion. Suppression of the IgM-1 and IgG-1 allotypes was associated with a compensatory increase in the alternative IgM-1 and IgG-1 serum allotype levels. This suppression was induced (a) by passive injection of anti-allotype antiserum into 13-day-old embryonal or neonatal recipients, or (b) by egg yolktransmitted antibody in chickens hatched from homozygous B 14 A (M-l^a, G-l^g) hens immunized against the IgM-l b of the homozygous B14C (M-l^b, G-lⁱ) rooster. Heterozygous chickens injected embryonally with anti-allotype antiserum were profoundly suppressed for at least 16 weeks after hatching, while neonatally injected chickens showed a gradual recovery of both IgM-1 and IgG-1 allotypes over the same period. Suppression of the IgM-1 b allotype could be induced in heterozygotes which had inherited the M-l^b allele either maternally or paternally. However, no suppression of either IgM-1 or IgG-1 levels could be detected in homozygous chickens injected with the relevant anti-allotype antiserum. Hence, allotype suppression only occurred in M-1 heterozygous chickens which had an alternative source of B cells available. The involvement of a B cell surveillance mechanism in allotype suppression is postulated and the possible role of suppressor cells is discussed.     

Allotypic determinants on the surface of rosette-forming cells in the rabbit

Antigen-binding cells to sheep erythrocytes from non-injected rabbits and from rabbits after a single injection of sheep red blood cells (SRBC) have been detected by the immunocyto-adherence technique (rosette formation). The expression of immunoglobulin allotypic determinants on these antigen-binding cells has been investigated by inhibition of rosette formation with anti-allotype antisera. The results indicate that a and b locus allotypes are expressed by the great majority of rosette-forming cells (RFC) in the lymph nodes of injected and non-injected rabbits. Ms3 allotype is expressed by practically all RFC from non-injected rabbits, while, after injection of SRBC, a population of RFC appears, which does not express this allotype. An antiserum to d 11 consistently failed to inhibit RFC. Pretreatment of lymphocytes from rabbits heterozygous at the b locus with antiserum to only one allelic product produced an inhibition of rosette formation which was approximately half that obtained when antiglobulin reagents to both allelic products were added. The results suggest that in heterozygous animals, the two allelic markers are expressed on two different populations of antigen-binding cells. Electron microscopy studies on the morphology of RFC showed that the majority of the RFC were formed around small lymphocytes.     

Clonal heterogeneity and other properties of auto anti-allotype antibodies produced in allotype-suppressed rabbits

Auto anti-allotype antibodies to the b6 determinants of rabbit immunoglobulin K light chain have been produced in heterozygote rabbits suppressed perinatally for the synthesis of molecules carrying this allelic product. These auto antibodies show equivalent degrees of clonal heterogeneity as conventional rabbit antibodies to both a locus and b locus allotypes, as determined by flat bed isoelectric focussing and overlayering the plates with target allotype-coated sheep red blood cells and complement, to produce bands of lysis. Furthermore the auto anti-allotype antibodies are capable of inducing blast transformation of peripheral blood lymphocytes from normal b6 phenotypic rabbits and of inducing suppression of synthesis of b6 immunoglobulin molecules in heterozygote rabbits injected neonatally with the sera.     

Escape from b locus allotype suppression in the rabbit is mediated by IgM molecules bearing an allotope-restricted variant of kappa light chain

Rabbits homozygous for b6 at the kappa light chain b locus were suppressed for the expression of the b6 allotype and then induced to produce auto anti-b6 antibody. Rabbits which subsequently escaped suppression produced auto antibody with restricted allotype specificity. Escape from allotype suppression was mediated by IgM bearing a kappa chain variant with a restricted number of b6 allotopes and having a diminished interaction with auto anti-b6 antibodies from the same and other rabbits escaping b6 suppression. This suggested that there were allelic variants or subpopulations of the b6 light chain which were under independent regulation of expression, clearly influenced by the specificity of auto anti-allotype antibody. Since escape from suppression was mediated by IgM it is proposed that a normal pathway of B cell differentiation occurs during recovery from suppression.     

Regulation of allotype expression in heterozygous rabbits. II. Concomitant suppression of b4 and b6 allotypes in the same cell.

Cells from heterozygous b4b6 rabbits were treated at 4 degrees with anti-b4 or anti-b6 antibodies and then warmed at 37 degrees. A disappearance of both b4 and b6 allotypes (concomitant modulation) ensued. When cells which had undergone extensive comodulation were cultured overnight we noted that those cells were unable to re-express either allotype at pre-modulation levels. This suppression was likely linked to the initial events which culminated in comodulation. Those cells were not further suppressible when suppressive antibodies were added to the cultures whereas cell cultures which had undergone little or no previous modulation or comodulation were readily suppressed for both allotypes after anti-allotype antibodies had been added to the cultures overnight (concomitant suppression). This indicated that in vitro suppression of allotype may depend on cell surface allotype being present at a sufficiently high density. We present data which show that events at the cell surface may play a role in the regulation of cell surface allotype expression and propose that concomitant suppression may have bearing on cellular mechanisms which control allotype expression and also allotype suppression.     

Relative expression of light-chain allotypic specificities on the surface of rabbit lymphocytes as a function of age

Lymphoid cells of heterozygous Ab4/Ab9 rabbits of various ages were stained with fluorescent anti-allotype antibodies. In foetal and newborn rabbits, the percentage of Ab-positive cells in low; it increases with age, reaching the adult value when the animals are 100 days old. The rate of increase in the percentage of Ab4-positive cells is higher than in that of Ab9-positive cells. Thus, the Ab4/Ab9 ratio, which is initially 1, increases with age and reaches 2.2. in spleen and 4 in appendix of young adult animals. We conclude that the change in Ab4/Ab9 ratio is due to differential clonal expansion which we attribute to charge-related differences in the ability of Ab4 and Ab9 receptor-bearing cells to capture antigen when environmental antigens are encountered in postnatal life.     

Serologic and biochemical analysis of latent a1 IgG

Immunization of rabbits with anti-allotype antibody (Ab) induces at least two populations of highly cross-reactive molecules. One of these populations bears the nominal VHa allotype of the producing rabbit and is designated the VHa-positive anti-IdX Ab. The other population lacks the expected nominal allotype and thus could represent an induced latent allotype-bearing Ig. To define better the putative latent allotypes, they were subjected to serologic, electron microscopic and biochemical analyses. The induced latent-like population was compared to nominal a1 and found to be indistinguishable in inhibition assays incorporating both rabbit anti-a1 Ab and mouse monoclonal anti-a1 Ab. In contrast, the latent-like Ig was less inhibitory than normal a1 Ig in assays with goat and guinea-pig anti-a1 Ab. The isolated anti-IdX population was less inhibitory than either nominal or latent a1 Ig in all assays. Immunoelectron microscopic analysis indicates that complexes composed of latent-like a1 molecules and Fab anti-a1 ab resemble allotype/anti-allotype (i.e. a1/anti-a1) complexes. Tryptic digests of the putative latent a1 H-chains reveals that these molecules share an a1-specific peptide with digests of nominal a1 H-chain. The peptides from both nominal and latent a1 IgG appear to have blocked N-terminal residues and have a similar though not identical amino acid composition. The composition of these peptides is correlated with the first nine amino acids of the nominal a1 H-chain. The data suggest that the induced latent a1-like molecules share the same major a1 epitope with nominal a1 but may differ in some subtle respects. The possible genetic bases for these observations are discussed.     

Autospecific and allospecific antibodies raised in allotype suppressed rabbits

Allotype-suppressed rabbits were injected with immunizing material containing the ‘suppressed’ allotypic specificity (Ab9). This invariably resulted in formation of antibodies against Ab9, even if the immunized animals already started to recover from suppression and had circulating Ab9 in concentrations as high as 1% of the normal homozygous value. By various techniques it was possible to detect Ab9 and anti-Ab9 present simultaneously in sera of a large proportion of animals. Antisera raised in suppressed-immunized rabbits were found to contain antibodies directed only to some determinants of the Ab9 allotype. Circulating antibodies did not react with autologous antigen, but reacted with antigens from sera of other suppressed-immunized rabbits.

At least some of the antisera contained also autoantibodies which reacted with autologous antigens. These ‘hidden’ autoantibodies were isolated by Sephadex G-200 chromatography; they reacted with Ab9 antigen isolated from the same serum sample.     

Allotype suppression induced in the adoptive transfer system: the variables of the system and an apparent absence of a role for T cells.

A study has been made of the variables concerned in allotype suppression of adult spleen cells in the adoptive transfer system. These are; SRBC (antigen) dose; the dose and timing of injection of anti-allotype serum IgG; the number of spleen cells transferred and whether these cells were taken from primed or unprimed donors. Adoptively transferred primed cells are considerably less susceptible to suppression by concomitantly injected anti-allotype serum IgG than are unprimed spleen cells. Injection of anti-allotype serum during the period after adoptive transfer, has shown that primed cells loose their susceptibility sooner (2 days) than the unprimed cells (4 days). Allotype heterozygous CBA spleen cells are less susceptible heterozygous CBA spleen cells are less susceptible to allotype suppression than either allotypically homozygous or heterozygous non-H-2k cells (H-2b,d, or s). Allotype suppression of the TI IgG response to DNP-Ficoll was measured 7 days after adoptive transfer of allotype-homozygous cells from both normal and nude CBA mice (unprimed). The results indicate that T cells do not play a role in the initiation of short-term allotype suppression in the adoptive transfer system.     

Quantitative and spectrotypic analysis of paternal IgG2a expression in normal and allotype-suppressed mice.

The synthesis and clonal diversity of IgG2a molecules bearing the paternally inherited immunoglobulin allotype have been examined in the offspring of matings between BALB/c mothers (Igh-1a) and SJL or C57BL/10 males (both Igh-1b) using a sensitive quantitative single radial immunodiffusion in gel assay and isoelectric focusing with autoradiography. In normal litters, the first detectable paternally-marked IgG2a is extensively polyclonal in both F1 crosses (i.e. diversity precedes expression); however, there is a delay of 2-3 weeks in the first appearance of the clonally diverse set of molecules when these are coded by the SJL genome, compared with the C57BL/10. Delayed maturation of allelically-excluded Igh-1b-expressing B cells in the (BALB/c X SJL)F1 may explain the unique susceptibility of these offspring to chronic allotype suppression when exposed to maternal anti-Igh-1b antibodies in early life. We find that, although such suppressed mice may begin life with a (delayed) synthesis of polyclonal IgG2a of paternal allele (Igh-1b), the condition of chronic suppression later imposed in the majority of mice is associated with spectrotype (clonal) simplicity.     

Induced latent allotypes within rabbit anti-cross-reactive idiotype reagents. Direct immunoelectron microscopic evidence

Immunization of rabbits with anti-VHa allotype antibody leads to the production of an "anti-cross-reactive idiotype (IdX)" reagent which appears to recognize a highly conserved IdX on anti-allotype antibodies. In many rabbits, a large percentage of this "anti-IdX" reagent does not express the nominal VHa framework allotype of the rabbit from which it was derived and is otherwise serologically indistinguishable from the original antigen, i.e. a1 or a2 allotype. To determine whether these anti-IdX molecules were in fact anti-IdX (or internal images) or were induced immunoglobulins bearing latent VHa allotypes, immunoelectron microscopic analysis of the molecules in complex with Fab anti-allotype antibody was conducted. The results show that the complexes do not resemble idiotype-anti-idiotype interactions but are essentially indistinguishable from allotype-anti-allotype complexes. We conclude that a major component of the "anti-idiotype" response of these anti-VHa-immunized rabbits is the production of Ig molecules bearing latent VHa allotypes.     

Demonstration of the clonality and specificity of an auto-antibody response to a suppressed immunoglobulin allotype of the rabbit kappa chain b locus

A regulatory idiotypic network is proposed to control allotype expression in normal rabbits. We have used suppression of the kappa chain b6 allotype in an attempt to restrict the number of regulatory idiotypes involved in an induced auto anti-allotype response. These auto anti-b6 antibodies were examined by an agarose imprint immuno-fixation IEF technique using iodinated allotype-bearing IgG. All totally b6-suppressed rabbits produced a clonally complex response which was generally spectrotypically unique - thus contradicting previous claims of a dominant common idiotypic pattern. The compensating light chains thus have available as many regulatory V genes as does the kappa 1 light chain. However, the b6/b6 homozygotes breaking b6 suppression produce an auto anti-b6 antibody which does not interact with their "escaping" molecules and which is clonally restricted. We propose a regulatory mechanism limiting the V genes utilised to produce the autoantibody, the latter then allowing only molecules bearing non-interactive allotopes to "escape" suppression.     

Idiotype suppression by maternal influence.

In BALB/c mice, antibodies to the alpha-(1-3) glucosidic linkage of some dextrans (Dex) carry the idiotype of the BALB/c myeloma protein J558. Both specific antibody and idiotype are inherited in a dominant fashion, linked to the immunoglobulin (Ig) (heavy chain) allotype Igla of BALB/c mice (Eur. J. Immunol. 1975. 5: 775). In F1 hybrid mice from the parent strains SJL and BALB/c, we were able to suppress the expression of anti-Dex antibodies by immunizing prospective SJL mothers to the J558 idiotype. The state of suppression in the progeny was ascertained by immunization with Dex, and tests for the following were carried out: (a) antibodies specific for Dex; (b) inhibition of such antibodies (if present) by antiidiotypic serum to protein J558; (c) presence of the J558 idiotype; and (d) concentration of lambda1 chains (which are associated with the 558 idiotype) in the serum. SJL mothers, once immunized, conferred suppression upon several successive litters, spanning a period of 4-5 months. Suppression in F1 progeny animals lasted for 16 weeks or more. Spleen cells from suppressed F1 mice which had neither been treated with Dex nor with J558 protein, were able to confer suppression to further F1 newborn mice.     

Mouse monoclonal antibodies induced by anti-allotype antibody display internal images of the rabbit VHa1 allotype: direct visualization by immunoelectron microscopy

We have previously shown that injection of adult rabbits with anti-immunoglobulin (Ig) antibody (Ab) induces the expression of genetically unexpected Ig markers, i.e., a1 allotypic determinants. We now show that these rabbit Ig markers can also be induced in mice by a similar treatment; in the latter case the a1 determinants are located in the antigen-combining site and thus represent "internal images". Two monoclonal antibodies (mAb) were developed from mice treated with anti-allotype Ab. These mAb were reactive with all homologous and heterologous anti-a1 Ab but not normal Ig; efficiently inhibited the binding of rabbit a1 Ig to anti-a1 Ab; and elicited the production of anti-allotype Ab when injected into normal mice. To determine whether the a1-like determinants on these mAb were located in the antigen-combining site, immunoelectron microscopy was utilized to directly visualize Ab complexes. Complexes composed of intact Ab and anti-a1 Fab fragments yielded uniform binding patterns which were identical to those produced by anti-idiotypic reactions. In each case, an identical tip-to-tip binding configuration was observed with a single Fab fragment attached at an approximate 180 degree angle to the V region terminus of each Ab arm. In contrast, rabbit a1 Ig bound as many as two anti-a1 fragments per Ig arm; these fragments were attached laterally and at right angles to the intact molecule. These mAb thus provide the first direct evidence that Ab2 beta determinants are located in, or near, the antigen-combining site.     

Suppression of anti-DNA antibody production in MRL mice by treatment with anti-idiotypic antibodies.

Antibodies against idiotypic determinants carried by a monoclonal polyspecific natural autoantibody were raised in rabbits and in syngeneic BALB/c mice. These anti-idiotypic antibodies were administered to newborn and to pregnant BALB/c mice and to MRL-lpr/lpr mice. Serial measurements of the idiotypes, naturally occurring autoantibodies, and antibodies obtained after antigenic stimulation were performed in the sera of the injected mice and in the offspring of pregnant mice. No idiotypic suppression was noted in newborn injected mice. Transient suppression of idiotypes recognized by the syngeneic anti-idiotypic antibody was noted in the offspring of pregnant mice injected with the rabbit polyclonal anti-idiotypic antiserum. No changes in naturally occurring autoantibodies or in antibodies appearing after antigenic stimulation were noted in BALB/c mice. In contrast, a significant decrease of spontaneously occurring anti-DNA antibodies was found in MRL-lpr/lpr mice treated with rabbit polyclonal anti-idiotypic antiserum. Furthermore in these mice a slight decrease of anti-TNP antibodies was also observed. These results suggest that anti-idiotypic antibodies directed against natural autoantibodies may play a regulatory role in the immune system; this role is more easily appreciated in mice suffering from immune dysregulation.     

Rabbit allotype a locus subspecificities of homogeneous anti-streptococcal antibodies.

Rabbits were immunized with group A-variant (Av) streptococcal vaccine to elicit antisera containing antibodies of "restricted heterogeneity" specific for Av polysaccharide. Antibodies that migrated as single bands in polyacrylamide gel isoelectric focusing were isolated on agarose block electrophoresis and preparative isoelectric focusing. These homogeneous antibodies were analyzed for content of the a locus markers by inhibition of allotype-anti-allotype radioimmunoassays. Both single-band preparations and preparations containing 2--5 bands were deficient in content of the a locus markers. These analyses were strongly influenced by the different anti-allotype antisera used. Single-band preparations, deficient in a 2 or a 3 markers, were further used to produce anti-allotype antisera, and these sera were assayed for reaction with pooled a2 or a3 molecules. The results, in contrast to the a1 allotype, failed to demonstrate common determinants on either a 2 or a 3 molecules indicating that each a allotype consists of a set of different Ig molecules. The data suggest different evolutionary pathways of the a 1, as opposed to the a 2 and a 3 molecules.