Allotype suppression in the chicken II. Suppression in homozygous chickens with antiallotype antibody and allotype-disparate B cells

Injection of heterozygous (M-1³/M-1^b, G-1^g/G-1ⁱ) B 14-line chickens with antisera directed against either IgM-1a or IgM-1b induced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes, whereas a mixture of anti-M-1a and anti-M-1b antibodies failed to produce allotype suppression. Injection of anti-M-1 antiserum into M-1 homozygous chickens induced only a transient delay of a few days in the appearance and rise of serum IgM-1 levels. However, suppression of host allotypes was induced by injecting M-1, G-1 homozygous neonatal or embryonal recipients with anti-M-1 antisera together with B locus-histocompatible allotype-disparate spleen, bone marrow or bursal cells. The active cell type were donor B cells, which established chimerism in the injected hosts, whereas peripheral blood T lymphocytes from agammaglobulinemic donors were ineffective. Allotype suppression was attributed to a homeostatic control mechanism which is exerted by normal B cells (but not T cells) over B cell recruitment in anti-M-1 antibody-treated, immature hosts.     

Allotype suppression in the chicken. IV. Deletion of B cells and lack of suppressor cells during chronic suppression

Embryonally induced allotype suppression in M-1,G-1 heterozygous chickens was stable for at least 18 months after hatching. Suppression was established rapidly since injection of antigen only 4 days after anti-IgM-1 antiserum failed to abrogate its effect. Injected chickens had undetectable serum levels (i.e. less than 40 micrograms/ml) of the suppressed IgM-1 and low (0.3-0.6 mg/ml) levels of the linked IgG-1 allotype. This correlated with a complete depletion of cells bearing the relevant IgM-1 allotypes and a compensatory increase in the alternative nonsuppressed IgM-1 allotype-bearing cells in the spleen, peripheral blood and bursa. Cell transfer studies suggested that suppression could not be attributed to allotype-specific suppressor cells.     

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.     

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.     

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.     

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.     

Allotype suppression in an adoptive transfer system in adult mice: the specificity and feedback effects of a monoclonal IgG3 anti-(Igh-1b) allotype antibody.

Using an adoptive transfer system in mice, an allotype-specific suppression has been induced by a monoclonal IgG3 anti-Igh-1b (Hyb 5.7) reagent. Suppression was specific for IgG2a (Igh-1b) and led to a compensatory increase of the Igh-1a response in irradiated mice reconstituted with allotype heterozygous (Igha/b) spleen cells. Suppression, which was not antigen-specific, lasted for at least 1 month after anti-allotype treatment.     

Expression of rabbit Ig allotypes in litters of mothers immunized against a paternal allotype.

Sera of successive littermates of mothers producing anti-allotype antibodies (Ab) were analysed for altered a locus or b locus allotype expression. We measured the allotype concentration in sera of 66 individuals (17 litters) of seven mothers producing anti-a1 Ab, and 63 individuals (15 litters) of seven mothers producing anti-b4 Ab, in an enzyme-linked immunosorbent assay (ELISA). We confirmed that the ability to induce allotype suppression in utero increases with the number of antigen boosts applied to the mother, even though the Ab titre in the maternal serum may be decreased. All individuals of a litter expressed the allotype in about equal concentration. This contrasts the results we obtained when newborn rabbits were injected with anti-allotype antiserum. Injection of the same amount of anti-allotype antiserum into nine offspring of two mothers caused allotype suppression in only five individuals, showing no effect in the others. No suppression was observed when IgG-depleted antiserum was injected into newborn rabbits. As expected, maternal antibodies to a paternal allotype do not affect the Mendelian distribution of the progeny phenotypes.     

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.     

Allelic exclusion of M1 (IgM) allotype on the surface of chicken B cells.

The membrane expression of M1 (IgM) and G1 (IgG) allotype markers on peripheral blood lymphocytes was examined by immunofluorescence. In homozygous chickens 15% lymphocytes stained with either anti-M1 or with polyspecific rabbit anti-Ig serum, suggesting that M1 is expressed on the surface of probably all B lymphocytes. In heterozygous M1a/M1b individuals antisera against either allelic antigens reacted with 50% of the total surface Ig positive cells as evidence for the allelic exclusion of surface M1 expression. Donor allotype synthesis was determined from serum allotype levels in congenic cyclophosphamide-treated recipients of lymphoid cells. Treatment of cells with anti-M1 serum prior to transfer inhibited both M1 and G1 allotype synthesis. The treatment of heterozygous (M1a/M1b, G1a/G1e) cells with anti-M1b serum inhibited specifically the synthesis of M1b and G1e allotypes controlled by linked genes from the same parental chromosome. Thus, B cells which had been the targets for anti-M1 antibody mediated suppression are also subject to allelic exclusion.     

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.     

Neutralization of allotype suppression in rabbits

Data are presented which show that allotype suppression by maternal antibody can be neutralized either by foster nursing or by intraperitoneal injections of serum with paternal allotype and that the phenomenon occurs with allotypes of both the a and b loci.     

Surface immunoglobulin of rabbit peripheral blood lymphocytes: detection of allotypes by fluorescence and rosetting

Specific anti-allotype reagents were prepared to detect heavy and light chain allotypes on surface immunoglobulin-positive (sIg⁺) cells from a³ a³ b⁴ b⁴, a² a² b⁵ b⁵ homozygous and a² a³ b⁴ b⁵ heterozygous rabbits. Total sIg⁺ peripheral blood lymphocytes (PBL) were detected with fluorescein-labeled goat anti-light chain and sheep anti-rabbit Ig reagents. The total sIg⁺ cells detected with these reagents and the sum of a and b allotypes individually measured with specific fluorescent anti-allotype reagents (a2 + a3 or b4 + b5) were more or less the same when measured by fluorescence-activated cell sorter (FACS) II flow microfluorometry. The data provided no evidence for single cells expressing both allelic allotypes (allelic inclusion). We found that FACS and rosetting techniques were generally equally sensitive. However, we detected a greater proportion of total b4⁺ plus b5⁺ cells by rosetting than by fluorescence in some PBL preparations from heterozygous b⁴ b⁵ rabbits. This was not seen with artificial mixtures of b⁴ b⁴ and b⁵ b⁵ cells. The nature of these cells is not yet known. Conceivably, they were not scored as lymphocytes by light-scatter analysis on the FACS and hence were not counted, but were indistinguishable from lymphocytes by microscopy.     

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.     

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.     

Antibody-mediated allotype suppression in adult mice: the role of antigen, effector isotype and regulatory T cells

It has been reported (Contemp. Top. Immunobiol. 1974. 3:41) that allotype-specific T suppressor cells can be induced after monoclonal anti-allotype treatment of neonatal (BALB/c X SJL)F1 (Igha/b) mice. Here we show that (BALB/c X CB20)F1 adult-derived spleen cells (SC) are, by contrast, potently suppressed by monoclonal allotype-specific reagents, (when transferred into irradiated BALB/c recipients) in the absence of primary T suppressor cell induction. Such suppression is only induced in activated B cells [exposed to lipopolysaccharide or sheep red blood cells (SRBC)], and is probably dependent on the isotype of the anti-allotype sera administered. For example, two independently produced IgG1 monoclonal reagents raised against the Igh-1b allotype were poorly suppressive or nonsuppressive, whereas an IgG3 and an IgG2a monoclonal antibody induced a 90% suppression of the target allotype in transferred adult SC. It was found that suppression was not due to a depletion of antigen-specific T cell help since: (a) the addition of SRBC-educated T cells did not break suppression and (b) suppressed SC were as good a source of T cell help as normal SC, in the response of virgin or memory B cell (Thy-1-depleted) responses to SRBC in vivo. Suppression was maintained in suppressed cells which had been rechallenged with SRBC after transfer into a second irradiated recipient, but was not induced in normal SC when these were admixed with an equal number from this suppressed SC population. These findings point to a possible mechanism for the regulation of B cell expression, through the formation of an antibody-Ig receptor complex at the surface of the B lymphocyte. After complexing the target cell is either deleted or inactivated. The response to SRBC was reduced or ablated for at least 70 days after treatment with a single dose of anti-allotype serum.     

Allotypic suppression of adult mouse spleen cells: cell differentiation, class restriction and allotypic exclusion

Allotypic suppression of adult mouse spleen cells has been investigated by treating the cells with anti-allotype serum directed against the Ig-1 specificities of the γG_2a immunoglobulin class. Suppression of precursor, memory and primed cells in the anti-sheep red blood cell response, assayed in terms of the number of plaque-forming cells producing γM, γG1 and γG_2b immunoglobulins has been investigated. The results are discussed in terms of the stage of differentiation of cells when class restriction and allotypic exclusion occurs. It is concluded that γM or γG commitment occurs at an early precursor stage, and that γG class and allotype restriction occurs sometime after contact of precursor cells with antigen.     

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

The 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 (V_H) 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.     

Kinetics of escape from suppression of Ig heavy chain allotypes in multiheterozygous rabbits.

Three rabbits of genotype a1n81f73g74/a2n82f71g75 which had been injected at birth with anti-a l (VH) antiserum and which were previously shown to be suppressed for the paternal allotypes a 1, n81, f73 and g74 at 8 weeks of age, were monitored over a 2-year period for the concentration of suppressed and nonsuppressed allotypes in their sera. In all three suppressed animals, the f73 (C alpha) and g 74 (C alpha) allotypes were expressed again at a much greater rate than the a1 (VH) and n81 (C mu) allotypes. In one suppressed animal, the a l (VH) allotype was re-expressed at a much greater rate than the n81 (C mu) allotype and reflected primarily the reappearance of a l IgG. Thus, the escape from allotype suppression in this animal was in the order IgA, IgG, IgM which is the reverse of the order of appearance of these Ig classes during ontogeny. While the al(VH) and n81 (C mu) allotyes remained suppressed, the f73 (C alpha) and g74 (C alpha) allotypes were re-expressed to the same concentration as in the unsuppressed controls, and no compensatory decrease of the f71 and g75 allotypes occurred. During the re-expression of the f73 and g74 allotypes, the ratio of the concentrations of f73/g74 remained approximately constant.