In Vitro Studies on Allotype Suppression IV. Abrogation of Waning Suppression by Normal Immunoglobulin of the Suppressed Allotype

The mechanism of allotype suppression in rabbits has been investigated by studying the in vitro immune responses of spleen cells from rabbits in advanced stages of spontaneous recovery from suppression. Whereas the spleen cells from highly suppressed rabbits can be released from their suppressed state only if treated with a combination of antibodies against the non-suppressed type and immunoglobulin (Ig) of the suppressed type, treatment with either of these two components alone suffices to overcome suppression when cells of more poorly suppressed spleen donors are used. The demonstration that suppression can be abrogated by normal Ig of the suppressed type alone, when cells are obtained from rabbits in the final phases of suppression, lends further support to the previously suggested concept that the probable role of this normal Ig in the release phenomenon may be that of neutralizing an effector of allotype-specific repression. possibly involving suppressor cells.     

In Vitro Studies on Allotype Suppression IV. Abrogation of Waning Suppression by Normal Immunoglobulin of the Suppressed Allotype

The mechanism of allotype suppression in rabbits has been investigated by studying the in vitro immune responses of spleen cells from rabbits in advanced stages of spontaneous recovery from suppression. Whereas the spleen cells from highly suppressed rabbits can be released from their suppressed state only if treated with a combination of antibodies against the non-suppressed type and immunoglobulin (Ig) of the suppressed type, treatment with either of these two components alone suffices to overcome suppression when cells of more poorly suppressed spleen donors are used. The demonstration that suppression can be abrogated by normal Ig of the suppressed type alone, when cells are obtained from rabbits in the final phases of suppression, lends further support to the previously suggested concept that the probable role of this normal Ig in the release phenomenon may be that of neutralizing an effector of allotype-specific repression. possibly involving suppressor cells.     

In vitro studies on allotype suppression. IV. Abrogation of waning suppression by normal immunoglobulin of the suppressed allotype.

The mechanism of allotype suppression in rabbits has been investigated by studying the in vitro immune responses of spleen cells from rabbits in advanced stages of spontaneous recovery from suppression. Whereas the spleen cells from highly suppressed rabbits can be released from their suppressed state only if treated with a combination of antibodies against the non-suppressed type and immunoglobulin (Ig) of the suppressed type, treatment with either of these two components alone suffices to overcome suppression when cells of more poorly suppressed spleen donors are used. The demonstration that suppression can be abrogated by normal Ig of the suppressed type alone, when cells are obtained from rabbits in the final phases of suppression, lends further support to the previously suggested concept that the probable role of this normal Ig in the release phenomenon may be that of neutralizing an effector of allotype-specific repression, possibly involving suppressor cells.     

Expression of b 4 and b 5 chi light chain allotypes by B and pre-B cells in allotype-suppressed and neutralized b4b5 rabbits.

In previous studies, we described a primitive lymphoid cell found in fetal liver and in the bone marrow of older rabbits which contained cytoplasmic IgM but lacked surface IgM detectable by immunofluorescence. In heterozygous b⁴b⁵ rabbits, the pre-B cells in which we could detect these kappa chain allotypes appeared to exhibit allelic exclusion. In the present study, we investigated the effects of allotype suppression and its neutralization on the expression of the b4 and b5 allotypes by B and pre-B cells from the spleens and bone marrow of b⁴b⁵ rabbits. We found that in young allotype suppressed rabbits, pre-B cells of the suppressed allotype persist in bone marrow when B cells of the suppressed allotype are absent or severely depleted. The persistence of pre-B cells of the suppressed type supports the view that pre-B cells differ in their responsiveness to external influences such as anti-Ig compared to B lymphocytes. Injection of serum with b5 immunoglobulin into b⁴b⁵ animals suppressed 14-23 days previously for b5 was followed by the appearance of increased proportions of b 5 B cells in spleen within 24 h. Surviving pre-B cells are a likely source of these rapidly appearing B cells as well as of the B cells bearing surface immunoglobulin of the suppressed allotype which appear during the recovery phase of allotype suppression.     

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.     

Effects of IgM Allotype Suppression on Serum IgM Levels,B-1 and B-2 Cells,and Antibody Responses ir Allotype Heterozygous F1 Mice

IgM allotype heterozygous F1 mice were independently suppressed for Igh6a or Igh6b to evaluate the contribution of B-1 and B-2 cells to natural serum IgM levels and Ab responses. B-2 B cells expressing IgM of the suppressed allotype were evident in the spleens of suppressed mice 4 to 6 weeks after cessation of the suppression regimen, whereas B-1 B cells of the suppressed allotype were undetectable for up to 9 months. Although serum IgM of the suppressed allotype was initially depleted in mice suppressed for either allotype, by 7 months of age, there were detectable levels of IgM of the suppressed allotype in the serum; however, the levels were significantly below that found in nonsuppressed mice. When mice were immunized with either the T-independent or T-dependent form of phosphorylcholine, those suppressed for either allotype, and consequently depleted of B-1 B cells of that allotype, did not respond with phosphorylcholine-specific IgM of the suppressed allotype. In contrast, when mice were immunized with α1-3 dextran, the Igh6a allotype-suppressed mice were able to produce dextran-specific IgM of that allotype. These results show that allotype-bearing B-1 cells of both allotypes can be effectively suppressed by this suppression protocol and this produces long-lasting effects on B-1 cell levels and serum IgM of the suppressed allotype. These observations reflect the derivation of the majority of B-1 cells from fetal-neonatal precursors, which cannot be replaced by newly emerging B-2 cells of adult origin. Their ablation by antibody treatment results in permanent alterations to the adult B-cell repertoire.     

Suppression of VH, C gamma and C mu gene products in rabbits by maternal immunization against IgM allotype and detection of somatic recombinant molecules in these animals

Suppression of the mu-chain allotype Ms16 was obtained in young heterozygous Ms16/Ms17 rabbits by immunizing the Ms17 mother against the paternal Ms16 allotype. Examination of the concentration of the VH and CH allotypes of paternal origin (a2,Ms16 and e14) in the offspring, revealed not only Ms16 suppression but also VH and C gamma allotypic suppression. The degree of suppression for the C gamma markers (4-25-fold decrease) was much less than that for the C mu or VH markers (50-150-fold decrease). The excess C gamma marker (e14) was found to be present for the major part on molecules possessing the VH allotype derived from the homologous allelic chromosome. The pattern of persistence of molecules with the suppressed C gamma marker in the serum is consistent with the idea that these molecules arise by somatic recombination and that the gene order is VH, C mu and C gamma.     

Allotype suppression in the chicken III. Analysis of the recovery from suppression by neonatally injected or maternal antibodies

Injection of M-1 (Cμ), G-1 (CμgM) heterozygous chickens on the day of hatch with anti-IgM-1 antiserum induced allotype suppression from which chickens recovered over a period of approximately 4 months. The suppression of the serum IgM-1 levels was matched by a decrease in the number of splenic and peripheral blood B cells bearing the relevant IgM-1 allotype, and a compensatory increase in the number of cells bearing the alternative nonsuppressed IgM-1 allotype. However, the proportion of IgM-1-bearing bursal cells was only marginally altered. The recovery from suppression was due to B cell recruitment and could be abrogated by bursectomy. Allotype suppression induced in ovo or maintained by repeated injections of anti-IgM-1 anti-serum resulted in chronic suppression and depletion of the relevant peripheral as well as bursal IgM-1-bearing cells. Antibody titers of the relevant allotype in partially suppressed chickens generally correlated with serum allotype levels without clonal restriction in antibody response of the suppressed allotype.     

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.     

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.     

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.     

A mouse monoclonal antibody specific for an allotypic determinant of the Igha allele of murine IgM: genetic and functional analysis of Igh-6a epitopes using anti-IgM monoclonal antibodies.

An IgG1 mouse monoclonal antibody (MAb) specific for a mouse IgM allotypic determinant in the a, c, f, g, h, and j haplotypes was derived from a fusion of SP2/O-Ag14 mouse myeloma cells with C57BL/6 mouse spleen cells (Igh-Cb) immune to TC31, a MAb of the IgMa allotype. MAb from one hybridoma derived from this fusion (designated DS1) was demonstrated to bind in an ELISA to immunoglobulin bearing the IgMa allotype (TC31, MOPC104E), but not to immunoglobulin bearing the IgMb allotype (C.BPC112). Fluorescein-conjugated DS1 was shown to bind to the surface of BALB/cByJ splenic B cells, but was shown to have negligible binding on C57BL/6J cells. Similarly, DS1-conjugated Sepharose beads were able to stimulate in vitro proliferation of BALB/c, but not C57BL/6 splenic B cells. DS1 was unable to bind to spleen cells from BALB/c allotype congenic strains, BAB/14 (Igh-Cb) and C.AL-20 (Igh-Co), demonstrating that DS1 recognizes a determinant under the control of a gene linked to the Igh-C gene complex. Using sera from recombinant inbred lines, the determinant defined by DS1 was shown to be linked to the Igh-1 locus. Furthermore, the determinant was localized to the CH1 domain of the mu heavy chain. Sera from BALB/cByJ, NMRI, CBA/J, SEA/GnJ, RIIIs/J, and CE/J mouse strains were shown to bind to DS1 in an ELISA, while sera from A/J, SJL/J, NZB/B1NJ, AKR/J, C57BL/6J, and C57BL/10SnJ mouse strains did not bind to DS1. From these data we propose that DS1 is reactive with specificity Igh-6.1, which was originally defined by an allotypic antiserum developed by Black et al. (Immunogenetics 7:213, 1978).     

Homogeneity of antibodies produced by clones in vitro

Peripheral blood lymphocytes from primed rabbits were stimulated with sheep red cells (SRC) in microcultures. In this method, if a limiting number of lymphoid cells is used, only a small percentage of the cultures responds with production of antibody. We have analyzed the antibody produced in the responding cultures from the point of view of allotype and electrophoretic mobility. In rabbits heterozygous at the b locus, the anti-SRC antibody in single cultures was shown to express either one or the other allele. In a few of the cultures the antibody produced did not express any b locus allotype and was therefore assumed to be carrying lambda-type L-chains. In high voltage agar electrophoresis, the antibody activity of most of the positive cultures was localized in a single band, the position of which varied from culture to culture. These results show that the antibody produced in single microcultures is the product of single B cell clones. The homogeneity for allotype observed in the antibody produced by single microcultures is strong evidence that allelic exclusion, once achieved, is maintained throughout clonal proliferation.     

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.     

Humoral and cellular aspects of immunoglobulin allotype suppression in the rabbit: II. Effect of b locus suppression on immunoglobulin receptor-bearing lymphocytes

Rabbits born as b locus genotypic b4, 6 heterozygotes, but exposed in utero or neonatally to anti-b6 antibody, show chronic phenotypic suppression of the synthesis of the b6 allele in serum immunoglobulin. The suppression has been quantified over an extended period in relation to the synthesis of the alternative b locus allele (b4), a locus determinants, and total IgG. b locus suppression does not alter the synthesis or proportions of a locus (a1 and a3) determinants. The abrogation of suppression which can be effected neonatally by injection of immunoglobulin appears to be an antibody-neutralizing event since it requires specifically b6 determinants and cannot be achieved with non-b6 (5,9) complexes formed in vivo. Neutralization results in the rapid establishment of a b6 immunoglobulin-synthesizing cell population. Two assays, the mixed rosette and lymphocyte transformation tests with anti-allotype sera, have been used to examine the influence of suppression and suppression-neutralization on the distribution of allotypic determinants on immunoglobulin receptor bearing lymphocytes in the peripheral circulation and lymphoid tissues. In b6-suppressed rabbits there is an absence of b6-carrying lymphocytes in all the lymphoid tissues examined. In suppression-neutralized rabbits, however, such cells are present in numbers which correlate reasonably well with the proportion of b6 in the serum immunoglobulin. In rabbits in which no b6 was detectable at any time in the serum to 100 days of age, intensive immunization with b6 did not result in production of antibody to the b6 determinant. These findings are discussed in relation to proposals of the mechanism of induction and maintenance of chronic suppression in the rabbit.     

Papain-resistant (f) and papain-sensitive (g) subclasses of rabbit sLgA. Allotypic specificities on different domains of the g subclass of alpha-chains (alpha g)

Secretory IgA (sIgA) from rabbits homozygous for the f71 and g75 allotypic specificities of the f and g subclasses, respectively, was digested into four fractions: sIgA, Fc_2α, Fab_2α and Fab_α. The subclass and allotypic characteristics of each fraction were determined by quantitative radioprecipitation analyses. The f71 allotypic specificities were found predominantly in the undigested sIgA (with some in the Fab_α fragment) and the g75 allotypic specificities were in the Fc_2α and Fab_2α fractions. Previous data from our laboratory indicated that the f72 and f73 sIgA allotypes of the g subclass are resistant to cleavage and that the g74 sIgA allotype of the g subclass is cleaved by papain. The present and previously reported data support the hypothesis that resistance or sensitivity of rabbit sIgA to papain digestion is a function of the subclass rather than the individual allotypic specificities. Quantitative radioprecipitation analyses of the Fc_2α and Fab_2α fragments showed that the “g” allotypic specificities are composed of multiple determinants, some of which reside on the Fd part of the α-chain and some of which reside on the Fc part of the α-chain. Anti-allotype antisera have been prepared which are specific for the allotype determinant(s) present on each of the fragments. Of special interest is the ability to serologically distinguish the C_H1 domain on the Fd fragment of a rabbit α-chain.     

Long-term antibody synthesis in vitro. VI. Anti-allotype sera as probes of clonal products in affinity maturation.

A new experimental system is described for measuring the allotypic product of rabbit B cells during long-lasting in vitro antibody responses. The immunoenzymatic assays described allow determination of several parameters mapping in different regions of the same molecule, which can be measured and combined to yield a multidimensional picture of the time-course dynamics of antibody synthesis. The rabbit immune system responding to Escherichia coli beta-D-galactosidase was sample and disassembled by (a) culturing lymph node microfragments and (b) sorting out from among all anti-enzyme antibodies only those activating a mutant enzyme, AMEF, which bore the b4 or b9 allotype. A considerable simplification of the response was achieved in the microcultures as documented by cultures of heterozygous cells which produced only one allotype and by the fact that each culture showed a distinctive pattern when antibody titre, association constant, heterogeneity index, L-chain type, and k-chain allotype were considered together. This array of patterns was not an artifact but the result of disassembling a representative sample of the rabbit immune system into small components, since the b4/b9 ratio obtained by averaging the results of all cultures from a heterozygous rabbit lymph node was the same as the serum ratio. Despite the Poisson distribution of the responder microcultures, none of them was monoclonal; i.e. no antibodies homogeneous by all parameters tested were observed, This finidng supports the notion that in normal lymphoid tissue in its native tridimensional arrangement, one T cell can trigger several B cells clustered in one antibody-forming unit. This natural arrangement would ensure the monospecificity of the cluster (dictated by the T cell) while allowing for variation in affinity (depending upon the array of B cells in the unit). Accordingly our findings would results from the fact that as the size of the microfragments was reduced, the cells diluted out first were T cells, but as long as one of them was present, several B-cell clones were triggered. The b4/b9 pattern of any given culture remained constant over several months, but the ratio kappa/lambda underwent changes. An increase in molecules with non kappa-chains (which could not be reacted with anti-kappa-chain allotype antisera) was usually associated with a parallel decrease in antibody affinity. This occurred by the end of the antibody cycle and might be related to the regulation of antibody synthesis by T-cell suppressor factors.     

Humoral and cellular aspects of immunoglobulin allotype suppression in the rabbit. IV. Kinetics of induction.

The injection of anti-b6 antibody into neonatal heterozygous b4b6 or b5b6 rabbits causes allotype suppression. Animals receiving a weight-related dose of antibody on day 2 or day 5 of life showed complete long-term chronic suppression of b6 immunoglobulin. Delay of injection until day 8, day 10 or day 11 of life resulted in more variable suppression, with animals showing either short- or long-term complete suppression or partial suppression. There was some correlation between levels of anti-b6 present in the circulation at day 21 and the degree of suppression obtained. Allotype suppression could be partially neutralized by injection of the paternal type immunoglobulin on days 19, 28, 30 or 43; if the injection was delayed until day 54, 66 or 79 there was no effect and chronic suppression proceeded as in control rabbits not receiving neutralizing immunoglobulin. The results indicate that the critical period for the induction of allotype suppression is between days 11 and 43 of life.     

Localization by immunofluorescence of gamma-globulin allotypes in lymph node cells of homozygous and heterozygous rabbits

The cellular production of two rabbit γ-globulin allotypic specificities, A4 and A5, determined by allelic genes was investigated by the fluorescent antibody method. The 7S γ-globulin fractions of precipitating antisera were conjugated to fluorescein isothiocyanate and to lissamine rhodamine B sulphonyl chloride. Frozen sections of lymph nodes from eighteen rabbits, A4—A4 and A5—A5 homozygotes and A4—A5 heterozygotes, were studied after exposure to the fluorescent antibody conjugates. The conjugates, each specific for antigenic determinants of 7S γ-globulin, reacted specifically with the cytoplasm of plasma cells and intrinsic cells of the germinal centres. The rabbit anti-A4 conjugate reacted only with lymph node cells of A4—A4 and A4—A5 rabbits; the rabbit anti-A5 conjugates reacted only with cells of A5—A5 and A4—A5 rabbits; the horse anti-rabbit γ-globulin conjugates reacted with cells of all three genotypes. By a variety of techniques, identical cellular localization of the two allotypes, A4 and A5, was found in the A4—A5 heterozygotes. Less than 1 per cent of the cells in any heterozygous lymph node section contained one allotype without the other.