Paternal Allotype Research Articles

Maternal response to paternal trophoblast antigens.

What is the function of the immunoglobulin (Ig) G antibody bound to trophoblast in normal pregnancy, and what is the antigen? IgG was acid eluted from term human placental microvesicles and reacted with the antigen, R80K, left on the vesicles. The eluted antibody was used to detect the antigen on monocytes, lymphocytes, and lymphoblastoid cell lines. The eluted antibody is highly polymorphic, but monoclonal antibodies (mAbs) were made against conserved regions of the molecule. These also reacted with the murine equivalent of the human R80K and were used in inhibition studies of natural killer (NK) cell killing and the mouse abortion models, CBA x DBA2 F1 resorption in CBA females, the endotoxin-induced resorption model, and a sonic stress-induced murine resorption model. All 600 syncytiotrophoblast microvesicle preparations of human term placenta had IgG antibody bound, elutable at pH 3.0. The eluted antibody reacted with about 15% of unrelated human placentae. In horses mares make detectable antibody early in pregnancy, at about the time of implantation. The IgG antibody was bound to an 80-kDa protein (R80K) also detected on B lymphocytes and monocytes. In HLA homozygous lymphoblastoid B cell lines, which reacted with one or more eluted antibodies, had a pattern of cytotoxicity independent of HLA Class I; and as a single 80-kDa peptide chain, R80K did not resemble HLA molecules. Genetic studies in horses show that of the two paternal allotypes of R80K detectable by placental alloantibodies, only one, usually the grandpaternal one, is present in all the placentae of a sibship. Two of 26 eluted human antibodies had affinity for K562 and inhibited killing by human peripheral blood NK cells. One mAb, BA11, against a conserved site on R80K inhibited killing of K562, and also reacted with the murine R80K homologue. BA11 inhibited murine NK cell killing and virtually completely inhibited three NK cell-dependent mouse resorption models. R80K protein is a target molecule for NK cell activity expressed on all placentae. It has a polymorphic alloantigenic determinant completely covered with maternal antibody in all successful term pregnancies. In murine NK cell-dependent models of abortion, a mAb against a monomorphic determinant present in human and murine R80K prevents abortion very effectively. It seems that the R80K molecule must be covered with antibody to prevent NK attacks on trophoblast.

Anomalous inheritance of a paternally derived trophoblast antigen.

Recurrent spontaneous abortion occurs in 1 in 500 random matings and usually results in abortion of all pregnancies. If absence of antibody to a paternally derived antigen caused abortion, the woman would be expected to make antibody to the other paternal antigen and abort only half her pregnancies. Microvesicles were prepared from equine placentae. Acid-eluted IgG antibody was eluted from the polymorphic R80K antigen and used to type the residual R80K antigen on vesicles or on peripheral blood leucocytes. In several equine sibships all the half-sibs had the same paternal R80K alloantigen. In the extended horse family descended from the stallion Nearco, three allotypes were found. The allele present was usually the grandpaternal one, but exceptions are seen. Whichever allele is transmitted all the progeny have the same alloantigen (probability of this occurring by chance = 2(-45)). Because only one paternal allotype is present in all progeny, lack of antibody to the R80K antigen would result in loss of all pregnancies, not one half.

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.

Feedback regulation of murine Ly-1 B cell development.

Studies presented here, conducted with allotype homozygotes, demonstrate the existence of a feedback mechanism that regulates development of Ly-1 B cells from immature progenitors. In the preceding study (P. A. Lalor et al., Eur. J. Immunol. 1989. 19:501), conducted with allotype heterozygotes, we showed that treating neonates with monoclonal antibody to the paternal allotype IgM depletes roughly half of the neonatal B cell population (i.e. those expressing the paternal IgM allotype) and that paternal allotype Ly-1 B cells specificically remain depleted for the life of the animal. Here we show that treating allotype homozygotes with the same antibody depletes all (rather than half) of the B cells and that, under these conditions, relatively normal numbers of Ly-1 B cells reappear shortly after the treatment antibody disappears. The recovery, we also show, is prevented by restoring allotype-congenic Ly-1 B cells to the treated homozygotes, i.e. by reconstituting treated neonates with allotype-congenic peritoneal cells, sorted Ly-1 B cells or a monoclonal population of Ly-1 B "tumor" cells. These findings in essence reveal a feedback mechanism through which mature Ly-1 B cells prevent further Ly-1 B cell development from Ig- precursors. This feedback regulation is independent of Ig secretion by the mature Ly-1 B cells, since the monoclonal Ly-1 B "tumor" population that prevents endogenous Ly-1 B development does not secrete Ig. Furthermore, it appears to be independent of Ly-1 B surface Ig specificity, since a monoclonal population is sufficient to block all Ly-1 B cell development. This mechanism appears to operate normally to fix the composition of the Ly-1 B population, which survives through self-replenishment in adults, in accord with conditions that influence Ly-1 B development during neonatal life.

Permanent alteration of the murine Ly-1 B repertoire due to selective depletion of Ly-1 B cells in neonatal animals.

Studies presented here demonstrate that paternal allotype Ly-1 B cells are permanently depleted following neonatal treatment with antibodies to the paternal IgM allotype. Paternal allotype conventional B cells, in contrast, are temporarily depleted by treatment with either anti-IgM or anti-IgD allotype antibodies and return rapidly to normal frequencies once the antibody treatment disappears. These differences are explained by basic developmental differences between Ly-1 B and conventional lineage B cells. That is, the conventional B cell population is replenished from Ig- precursors throughout life and, therefore, is only temporarily affected when depleted in neonates. The Ly-1 B cell population, in contrast, develops from Ig- progenitors during the prenatal and neonatal life but survives because it is exclusively self-replenishing in adults. Therefore, elimination of a population of Ly-1 B cells from neonates is tantamount to removing it forever. These findings suggest that while conventional B cells turn over rapidly and have an effectively unlimited repertoire, Ly-1 B cells express a repertoire whose composition is strongly influenced by neonatal conditions that favor or select against the retention of cells producing certain antibody molecules. Thus, Ly-1 B cells play a unique role in the immune system in that they retain indefinitely the history of the neonatal animal's immunological experience.

Monoclonal antibody to an IgD allotype induces a new type of allotype suppression in the mouse.

Studies presented here show that perinatal exposure to anti-IgD allotype antibodies induces a persistant IgG-allotype suppression in the mouse that differs markedly from either the short-term or chronic allotype suppressions induced by antibodies to IgG or IgM allotypes. This novel form of allotype suppression induced by injecting neonatal BALB/c x SJL mice with monoclonal antibody to the paternal Igh-5b (IgD) allotype drastically reduces paternal allotype production during the first 6 mo of the affected animal's life and simultaneously stimulates compensatory production of maternal allotype IgG. In addition, it interferes with the development of B cells carrying the paternal IgD allotype and impairs the development of memory B cells destined to give rise to paternal allotype IgG-producing cells. Thus, its properties make it more like allotype suppression as described in the rabbit than like the known forms of allotype suppression in the mouse. As shown here, Igh-5b-bearing (5b+) B cells are completely depleted from the neonate after anti-5b exposure and only gradually appear as the animal ages. The recovery of the 5b+ population to near normal size (by approximately 14 wk of age) substantially preceeds recovery of the ability to generate normal-size memory B cell populations. Paternal allotype levels in serum remain well below normal until the anti-5b-exposed animals reach approximately 6 mo of age and then climb rapidly, finally stabilizing at levels comparable to levels in controls of the same age. The elevated maternal allotype levels characteristic of the suppression period began falling somewhat earlier and are clearly stabilized within the normal range in 6-mo-old animals. Thus, perinatal exposure to anti-5b compromises B cell development and IgG production throughout early adulthood but has little apparent effect in older animals. Perinatal exposure to antibody to the paternal IgG2a allotype (Igh-1b) or IgM allotype (Igh-6b), in contrast, induces a chronic allotype suppression that has relatively little affect on IgG production in young adults but severely suppresses allotype production in older animals. Furthermore, this type of (chronic) suppression does not influence maternal allotype production and does not interfere with memory B cell development. These differences, illustrated here by data from parallel sets of animals exposed either toi anti-5b or anti-1b, raise a series of intriguing questions concerning the mechanisms regulating B cell development and expression and the nature of the neonatal (B) cell populations with which the suppression-inducing antibodies react.

B-cell influences on the induction of allotype suppressor T cells

Allotype suppressor T-cell (Ts) populations that persist for the life of the animal arise in (BALB/c x SJL)F(1) hybrids exposed perinatally to antibody to the paternal (Ig-1b) allotype on IgG(2a)-isotype immunoglobulin H chains. These Ts suppress Ig-lb production by depleting the supply of allotype- specific helper T cells (Th) required, in addition to carrier-specific Th, for the latter stages of Ig-1b memory B-cell differentiation. In this publication, we show that specific Ig-1 allotype Ts are induced by perinatal exposure to antisera which interfere with normal B-cell maturation, i.e., by antibodies reactive with surface IgM on immature precursors of IgG(2a), memory cells. Antibodies to IgM (Ig-6) allotypes carried on precursors induce specific suppression for the IgG2, allotype produced by progeny of the target precursor. Anti-Ig-6a and anti-Ig-6b induce Ts that specifically suppress Ig-1a and Ig-1b, respectively. Heterologous (goat) anti-IgM induces suppression for both IgG(2a) immunoglobulins (Ig-1a and Ig-1b). Ts activity in these antiprecursor-Ig-suppressed mice is expressed in adoptive transfer assays and, as with anti-Ig-1b-induced Ts, is rendered ineffective by cotransfer of adequate numbers of T cells but not B cells from nonsuppressed mice. The Ts induction, in contrast with Ts expression, is reversed by the introduction of appropriate adult B-cell populations from nonsuppressed donors. Taken together, these data suggest that the development of mature B cells plays a central role in the early establishment of the balance between helper cells and suppressor cells that determines whether Ts or Th will dominate in regulating Ig-1b production in adult animals.

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.

Maternal allotype dominance and allelic exclusion in the B lineage cells of the newborn rabbit.

Newborn rabbits from parents which differ at the b allotype locus (kappa chain), show a strong maternal allotype dominance, especially in the bone marrow. This concerns not only the membrane but also the cytoplasm, and it cannot be simply due to a passive uptake of maternal immunoglobulins. The spleen precedes the bone marrow for a high level of expression of the paternal allotype. In the thymus, cells which contain cytoplasmic immunoglobulin are found at low frequency, but their absolute number represent a substantial contribution to the B lineage cell pool. A small proportion of such thymus cells do not show allelic exclusion of kappa chain allotypes.

Release from maternally-induced allotypic suppression in rabbit by Nocardia water-soluble mitogen.

The in vitro synthesis of allotypes of b4/b5 offspring obtained from b4/b4 mothers immunized against paternal allotype b5/b5 was studied in comparison to similar offspring that had escaped from suppression and normal heterozygous b4/b5 rabbits. Nocardia water-soluble mitogen-a rabbit B-cell mitogen which is known to induce the differentiation of small lymphocytes into plasma cells and polyclonal activation of Ig, was able to break in vitro the allotypic suppression induced in vivo.

Induction of IgG in young nude mice by lipid A or thymus grafts.

Postnatal serum concentrations of IgG2a of paternal allotype, measured in congenitally thymusless nude mice, increase with kinetics and titers comparable to their normal congeneic counterparts. Lipid A, the mitogenic part of LPS, stimulates IgG synthesis in nude mice when it is given 7 days after birth. IgG concentrations at 15 days of age are 6- to 8-fold higher than in untreated control nudes; this is considerably lower, however, than in normal mice, which show up to 45-fold higher IgG2ab levels after lipid A treatment. A thymus graft from nearly congeneic donors of the same age, transplanted at 4 days after birth, also stimulates long-lasting IgG synthesis in the nude recipients. If the grafted nudes are injected with lipid A 3 days later, IgG synthesis is further stimulated 8- to 16-fold. The data are discussed in relation to the thymus dependency of IgG production and the conditions for lipid A stimulation.

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.

Induction of IgG by lipid A in the newborn mouse.

IgG of paternal allotype first becomes detectable in the serum of (BALB/c x C57BL/6)F(1) mice between day 12 and 14 after birth and reaches adult levels at an age of 5 wk. Since in mice there is a transfer of maternal IgG molecules through the placenta and via milk, F(1) heterozygous at the allotype locus were used and the concentrations of IgG with paternal allotype were measured. This was done by a sensitive method capable of detecting IgG concentrations as low as 5 x 10(-4) of normal adult serum levels. It is based on the quantitative inhibition of allotype-specific facilitation of hemolysis. When lipid A or Salmonella bacteria were injected into neonatal mice, a stimulation of IgG synthesis was observed. Thus IgG levels were enhanced 10-30-fold compared to the nontreated mice. No increase in IgG levels was obtained in adult mice after treatment with lipid A. Whether the newborns were injected at birth, on day 2, 4, or 7, IgG was first demonstrable in the treated mice at an age of 6-11 days. The increase in IgG levels was not paralleled by a demonstrable antibody activity against lipid A, SRBC, and LPS. Thus the bulk of newly induced IgG is probably a statistical distribution of different specificities.

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.

Active suppression of immunoglobulin allotype synthesis. II. Transfer of suppressing factor with spleen cells.

The mechanism of chronic allotype suppression in (SJL x BALB/c)F(1) mice has been investigated by means of cell transfer studies. These mice are phenotypically negative for serum Ig-1b, the paternal allotype determinant on gammaG(2a) immunoglubulin, as a result of perinatal exposure to maternal anti-Ig-1b. When spleen or bone marrow (B) cells from suppressed mice were injected into irradiated BALB/c "indicator" hosts, detectable levels of Ig-1b were demonstrated in the sera of a majority of the recipients early after transfer. These results indicate that Ig-1b-producing cells or their precursors are present in the lymphoid tissues of suppressed mice, even though they are not expressed. Within 5-7 wk, it was no longer possible to detect Ig-1b in the sera of these hosts, although cells producing another paternal allotype (Ig-4b) were shown to persist. Control BALB/c mice, injected with spleen and B cells from normal mice, continued to produce high levels of immunoglobulin carrying this allotype. The disappearance of serum, Ig-1b occurred most frequently in the recipients of suppressed spleen cells. Similar results were obtained using a mixture of spleen cells from normal and suppressed mice. Ig-1b production in the recipient mice ceased within a few weeks, even though the majority of cells in the mixture were obtained from normal (nonsuppressed) donors. The data are interpreted as evidence that chronic allotype suppression in mice is actively maintained by cells which are resident in the lymphoid tissues, splenic cells being the most effective. These cells are capable of proliferating in a new host and exerting their suppressive influence on Ig-1b-producing cells and/or their precursors.

Active suppression of immunoglobulin allotype synthesis. I. Chronic suppression after perinatal exposure to maternal antibody to paternal allotype in (SJL x BALB-c)F 1 mice.

Long-term (chronic) allotype suppression, previously reported only in rabbits, is shown here to occur in at least one strain combination of mice as well. Close to 50% of the offspring of SJL (Ig(b)) males mated to BALB/c (Ig(a)) females immunized against the paternal allotype were found to be suppressed for Ig-1b (gammaG(2a)) at 6 months of age. These mice are called "chronically" suppressed. The percentage of offspring in this strain combination suppressed for the paternal allotype at 8 wk of age is the same as that seen in an earlier strain combination tested, [(C57 x BALB/c)F(1)], in which all mice recover from suppression by 10-12 wk. After 8 wk, two distinct patterns of long-term (chronic) suppression emerge in (SJL x BALB/c)F(1) mice: a small number of these mice never produce detectable amounts of Ig-1b throughout their lives, while the majority produce detectable Ig-1b sporadically, sometimes over a period of several weeks, the level of which eventually falls below detectability. Attempts to "cure" suppression by destroying the existent lymphoid population and forcing endogenous repopulation in chronically suppressed animals were unsuccessful. Furthermore, attempts to restore Ig-1b production by injection of cells from syngeneic Ig(a)/Ig(b) donors into irradiated, chronically suppressed recipients were also unsuccessful, although the same cell inocula, when injected into irradiated BALB/c (Ig(a)/Ig(a)) mice produced high levels of gamma globulin carrying the allotype. These results suggest that long-term allotype suppression resulting from perinatal exposure of offspring to specific anti-allotype antibody (anti-Ig-1b), is not due merely to an absence of Ig-1b-producing cells or their progenitors, but appears to be an active process, which dominates physiologically over normal production.