Journal of Reproductive Immunology. 20 (1991) 115--128 115 Elsevier Scientific Publishers Ireland Ltd.

JRI 00720

The alloantibody response of pregnant women and its suppression by soluble HLA antigens and

anti-idiotypic antibodies

Elaine Reed a, Alan E. Beer b, Hilda Hutcherson c, Donald West King a and Nicole Suciu-Foca a

°Department of Pathology, College of Physicians and Surgeons of ('olumhia UniversiO'. New York. N Y I(X)32, hUniversity of Health Sciences. Tile Chit'ago Medical School. North Chicago, IL 60064 and "Department of Obstetrics and Gynecology, Colh,ge q/ Physicians anti Surgeons ~]" ('olumhid Ultirersitl',

Nen' York, NY I(X)32 (U.S.A.)

(Accepted for publication 21 January 1991)

Summary

The aim of this study was to investigate the time course of maternal allosensitiza- tion to fetal HLA antigens during normal human pregnancy and to explore mechanisms of suppression of ant i-HLA alioantibodies. We found that the mother

produces antibodies against some but not all o f the mismatched HLA antigens of the fetus as early as the 8th week of pregnancy. These antibodies (Abl), however, are often complexed with soluble HLA alloantigens and become detectable when im-

mune complexes are dissociated. Soluble HLA antigens of fetal origin are present in the maternal circulation throughout the entire pregnancy beginning at 8 weeks. In some women the production of anti-anti-HLA antibodies (Ab2) became evident

as early as the first trimester, while in others Ab2 was documented during the second or third trimester. Analysis of antibody specificity showed that some healthy primipara develop antibodies reactive with self HLA antigens. Although the allo-

and autoantibody responses appear to be modulated by soluble HLA antigens, cyclic variations in the level of alloantibodies, as well as the mother 's selective response to some, but not all, paternal HLA antigens, are best explained by the development of

anti-idiotypic antibodies.

Correspondence to." Dr. Elaine Reed, Department of Pathology, College of Physicians and Surgeons of Columbia Universityl P&S 14-403, 630 West 168th Street, New York, NY 10032. U.S.A.

0165-0378/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

116

Key words: anti-idiotypic antibodies to HLA," anti-HLA; autoantihodies: so/u- ble HLA antigen; HLA specl.'[ic antigen-antibody immune complexes." maternal tolerance.

Introduction

The mechanisms by which the pregnant mother tolerates the semi- allogeneic fetus is still a puzzle for the immunologist. Various non-exclusive mechanisms, based on concepts developed in transplantation and cancer im- munology have been proposed (Johnson, 1989). Transplantation im- munology models suggest that prolonged exposure of the mother to fetal tissues may result in acquired immunological tolerance to the fetus, and/or that the immune response to paternal histocompatibility antigens is actively suppressed. Concepts evolving from cancer immunology are based on the finding that the extravillous cytotrophoblast which is present at the fetal- maternal interface, expresses no other HLA antigens than the class 1 MHC antigen, HLA-G, which is monomorphic, and thus unlikely to provoke an immune response (Johnson, 1989; Loke, 1989; Kovats et al., 1990; Ellis et al., 1990). It is possible that HLA-G antigens elicit "self" recognition signals securing immune survival of invasive trophoblasts and evasion from rejec- tion. Both the transplantation and cancer immunology models share the postulate that a breakdown of maternal-fetal tolerance may lead to im- munologic abortion.

It is known, however, that during pregnancy as many as 50'¼, of multigravidae may develop cytotoxic antibodies to paternal HLA-class I and class II antigens (Antczak, 1980). The source of allosensitization, however, is still obscure since neither the trophoblast or preimplantation embryo ex- press HLA antigens (Desoye et al., 1988). It is likely that anti-HLA sensitiza- tion is caused by the passage of fetal lymphocytes into the maternal circulation, an event known to occur as early as at 15 weeks of gestation (Antczak, 1980). The reciprocal passage of maternal lymphocytes into the fetal circulation can also take place as demonstrated by the finding that labeled leukocytes injected into pregnant women can be traced in fetuses and cord blood (Desai et al., 1964; Adinolfi et al., 1969). Depending on the stage of immunologic maturity, the fetus seems able to respond to the antigens provided by the passenger leukocyte, either by immunity or tolerance. This is indicated by the finding that the non-inherited maternal HLA antigens elicit antibodies in some individuals and tolerance in others (Antczak, 1980; Chardonnens, et al., 1980; Class et al., 1988).

The above considerations suggest that the placental barrier does not pre- vent sensitization in either direction and that active suppression mechanisms are involved in the maintenance of maternal-fetal tolerance.

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In previous studies we have shown that suppression of alloimmune responses is provided by anti-idiotypic, anti-anti-HLA antibodies and by seemingly clonotypic antibodies which recognize alloantigen receptors on T- lymphocytes (Suciu-Foca, et al. 1983, Reed, et al. 1983, 1984, Suciu-Foca, et al. 1985, Bonagura, et al. 1986, 1987). To explain the suppression ofalloreac- tivity in pregnant women we have studied the development of maternal humoral responses to fetal HLA antigens and the possible role of anti- idiotypic (anti-anti-HLA) antibodies and soluble HLA antigens in the inhibi- tion of anti-HLA antibodies.

We now report that pregnant women develop anti-HLA antibodies during the first trimester and that such antibodies are blocked by soluble HLA an- tigens and anti-idiotypic autoantibodies.

Materials and Methods

Blood donors The population studied consisted of 21 healthy primigravidae. Samples of

blood were obtained from the pregnant women during the first, second and third trimester. Umbilical cord blood from the baby was obtained at de- livery.

HLA typing Blood donors were typed for HLA-A, B, C and DR antigens using

monospecific anti-HLA alloantisera as previously described (Reed et al., 1987). The typing method consisted of complement mediated lympho- cytotoxicity, using 5,6 carboxyfluorescein diacetate and ethidium bromide for discriminating between viable and dead lymphocytes.

Evaluation of anti-HLA antibodies Sequential samples of sera obtained from the study subjects were screened

for lymphocytotoxic antibodies against HLA antigens. Screening was per- formed on purified T and B lymphocytes from an HLA reference panel of 70 members. The percent of panel reactive antibodies (PRA) and their specificity was determined for each serum. Assignment of antibody specificities was based on correlation coefficient > 0.7 with distinct HLA an- tigens. An automated Leitz microscope, operated by an Arden System Com- puter was used for scoring and analyzing the reactions.

Additional target cells used for evaluating anti-HLA antibodies included lymphocytes from the mother, father and child. To determine the isotype, specificity and titer of these anti-lymphocytic antibodies indirect im- munofluorescence studies were performed on a FACStar cytofluorograph (Becton-Dickinson). Target lymphocytes were incubated with test sera, washed and stained with FITC-conjugated goat-anti-human IgG or IgM (Tago, CA) (Reed et al., 1987).

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Evaluation of soluble HLA antigens The presence of soluble HLA antigens of fetal origin in the mother's serum

was tested by determining whether the serum blocked the cytotoxic activity of murine MoAb specific for HLA antigens inherited by the fetus from the father. In this study two MoAb were used: BB7.2 (IgG 2A) which recognizes HLA-A2 and NM (IgM) specific for HLA-A3.

Serial dilutions (1:2 to 1:512) of murine MoAb in McCoy's medium (1 /A) were incubated with 1 /~1 of test serum for 1 h at 22°C in Terasaki plates. HLA-A2 or A3 positive lymphocytes were stained with carboxyfluorescein diacetate and plated in each well at 3 x 103//,tl. After 1 h of incubation, 5 #1 of rabbit complement was added. The incubation was continued for 1 h. Ethidium bromide was added to each well and the plates were read on the automated microscope. The serum was considered to be positive for soluble HLA antigens if it inhibited the cytotoxic activity of serial dilutions of anti- HLA MoAb. Serum depleted of soluble HLA antigens by magnetic im- munoaffinity caused no inhibition of murine MoAb to human HLA. To determine whether a serum specimen contained soluble HLA antigen we tested its ability to block the reactivity of MoAb with lymphocytes prior and following depletion of soluble HLA. The percent inhibition caused by solu- ble HLA was calculated by the formula:

Control - Test Inhibition -

Control × 100

where the value for "control" represents the percent of target cell lysis observed when the MoAb was mixed with HLA antigen-depleted human serum and "test" represents the percent lysis observed when MoAb was mix- ed with the non-depleted serum specimen.

The depletion of soluble HLA antigens from human serum was performed as previously described (King et al., 1989; Suciu-Foca et al., 1990a; Suciu- Foca et al., 1990b). Briefly, 150-#1 aliquots of sera were incubated with 10 /~1 of Magnisort M chromium dioxide particles coupled with high affinity mouse-anti-HLA-Class I antibodies (B9.12.1 MoAb, Pel Freeze, WI) and with 50/~1 of Dynabeads HLA cell Prep II (Dynal Inc, Great Neck, NY) coupled with MoAb specific for HLA-class II. Following 1 h of incubation at 4"C, with continuous mixing, the beads were removed using a magnetic field, and the serum was collected and used for testing.

Determination of immune complexes consisting of soluble HLA antigens bin- ding anti-HLA antibodies

To determine whether maternal sera contain immune complexes (IC) of

119

soluble HLA antigens and anti-HLA antibodies, aliquots of sera depleted of HLA were tested for anti-HLA antibodies in parallel with their non-depleted counterparts (King et al., 1989; Suciu-Foca et al., 1990a; Suciu-Foca, 1990b). The PRA and the anti-HLA antibody specificity found in the antigen depleted and non-depleted specimens were compared, Sera which, following antigen depletion showed increased PRA and/or antibodies to additional HLA specificities, were considered to contain HLA antigen/antibody com- plexes.

Anti-idiotypic antibodies to HLA To determine whether sera which were negative for anti-HLA antibodies

(Abl) following soluble HLA depletion contained anti-idiotypic antibodies (Ab2) we used a slight modification of our previously described procedure (Suciu-Foca et al., 1983; Reed et al., 1987). Briefly, maternal sera containing anti-HLA antibodies (Abl) were diluted serially from 1/2 to 1/256. As a dilu- ent we used HLA depleted serum from a healthy HLA matched male (con- trol) or HLA depleted serum from the same woman, i.e. autologous to Abl. The latter represents the serum tested for Ab2. Only sera which did not bind to target cells by immunofluorescence cytofluorometry were tested for Ab2. Also, to exclude the possibility that murine MoAb from the beads used for depletion have leaked into the serum, target cells incubated with test serum were stained with FITC goat anti-mouse Ig. Sera free of mouse Ig were tested for Ab2. Target lymphocytes were incubated with each of the diluted specimens of serum for 30 min at 4°C, washed, and then stained with FITC- conjugated F(Ab')2 fragment of goat-anti-human lgG. After 30 min of in- cubation at 4°C, cells were washed and analyzed on the FACStar. The final titer of Abl was established from the highest serial dilution binding to 50% of the target cells. A serum was considered to contain Ab2 if it blocked bin- ding of Ab I in two serial dilutions by more than 50% and if the mean channel of fluorescence shifted by more than 10 channels.

Results

To explore the immune response of pregnant women to self and fetal (paternal) HLA antigens we have tested anti-HLA antibodies in sequential samples of sera obtained during the first, second and third trimester of pregnancy from 21 primigravida. Sera were tested by complement-dependent lymphocytotoxicity for alloantibodies against HLA antigens represented in the HLA-reference panel of lymphocytes. Immunofluorescence cytofluoro- metry was used to confirm reactivity with autologous lymphocytes, paternal T and B lymphocytes or umbilical cord blood lymphocytes.

In these 21 cases of normal pregnancy there were 50 possible mismatches

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for HLA-A, B and DR antigens between mother and child. Study of first trimester sera revealed the presence of lymphocytotoxic antibodies in 11 primiparous women (Table 1). The percent reactive antibodies (PRA) found by testing the sera on the HLA reference panel ranged from 2% to 35%. Some lymphocytotoxic antibodies were detectable only when B cells were used as targets, because of the increased expression of HLA-class I and presence of HLA-class II antigens on such cells. Distinct anti-HLA antibody specificities were found in only two first-trimester sera (case Nos. 1 and 4).

Since anti-HLA antibodies may be blocked by soluble HLA antigens from the fetus, we depleted the sera of soluble HLA and retested them on the pan- el. This procedure resulted in a better definition of antibodies both in terms of frequency and specificity. Thus, an increase in PRA was observed in eight of the eleven cases (cases Nos. 1, 2, 5, 7, 8, 9, 10, 11) and antibodies to distinct HLA of the father were recognized in 7 cases (cases Nos. I, 2, 4, 7, 8 ,9 , 10).

Study of the second and third trimester sera showed that of the ten women who had no anti-HLA antibodies in the first trimester only one developed antibodies at a later time (case No. 12) while the other nine (case No. 13-21) remained negative. Testing of second trimester sera showed that 6 of the 11 women with antibodies in the first trimester (cases Nos. 2, 4, 5, 7, 8, 11) had a decreased PRA in the second trimester (Table 1 and Fig. l). In the third trimester a further decrease was observed in 4 cases (case Nos. 3, 4, 7, 8), while in 3 cases (cases Nos. 2, 6, 11 ) there was a slight rise in the PRA. Com- parison of the PRA prior and following antigen depletion showed differences which were consistent with the presence of antigen/antibody complexes in the sera. In most cases the PRA observed following antigen depletion was higher than that found before depletion indicating that some immune complexes have been dissociated. However, with progression of pregnancy there was a decrease in PRA below the level found when antigen depleted sera from the first trimester were tested (Fig. 1). Three women (cases Nos. l, 2 and 12) developed antibodies which reacted with a self HLA-DR antigen, such as DR2, D R w l l and DRw6, respectively (Table l). These autoantibodies were detected only after having depleted the soluble HLA antigens from the sera. The autoreactive nature of the lymphocytotoxic antibodies detected in these sera was confirmed by cross-matching the sera with autologous lymphocytes.

In an attempt to monitor the release of fetal HLA antigens in the circula- tion, we used two marker antigens, HLA-A2 and HLA-A3. The presence in maternal sera of the marker antigen was traced by use of murine monoclonal antibodies. Only cases in which HLA-A2 or A3 were inherited by the fetus from the father, and in which the mother was HLA-A2 or HLA-A3 negative were informative for these studies.

Evaluation of sequential sera from 6 such cases (cases Nos. 1, 2, 6, 7, 8,

121

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13) showed that in all these women soluble HLA alloantigens of fetal origin were detectable throughout pregnancy starting as early as 8 weeks (Fig. 2). The shedding of soluble HLA-A2 or A3 alloantigen in the circulation was not related chronologically to the production of anti-HLA-A2 or A3 an- tibodies since in all but one case (case No. 6) the mother failed to produce anti-HLA-A2 or A3 antibodies, as ascertained by cytotoxicity and im- munofluorescence testing. The lack of anti-HLA-A2 or A3 antibodies may indicate that in such cases soluble HLA antigens were tolerogenic rather than immunogenic and/or that the anti-HLA antibody response was blocked by anti-idiotypic anti-anti-HLA-antibodies (Ab2).

123

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To explore the possibility that Ab2 suppress the alloantibody response, we selected cases in which the women had formed a distinct anti-HLA alloan- tibody which was absent from other serial bleedings. Sera from six women (cases Nos. 1, 3, 4, 6, 7, 8) were informative for this study. The presence of Ab2 was ascertained from the blocking activity of antigen depleted, Abl-negative sera. Autologous sera with anti-HLA activity and homologous HLA antisera with identical antibody specificity were used as a source of Abl. Blocking was determined by indirect immunofluorescence cytofluorometry using as targets, lymphocytes which expressed the antigen recognized by Abl . The results from a representative experiment are shown in Fig. 3. Of the 6 women tested, four (cases Nos. 1, 3, 4, 8) showed Ab2 in at least one serum (Table I). In case No. i, the mother carried twins and her second trimester serum showed Ab2 specific for anti-HLAiA2 alloan- tibodies. Although this woman never showed antibodies to the HLA-A2 an- tigen carried by one child, purified IgG obtained after antigen depletion from the second trimester serum blocked specifically anti-HLA-A2 alloantisera. Blocking was not determined by residual soluble HLA antigen, since the IgG fraction of this serum inhibited anti-HLA-A2 alloantisera but not murine MoAb to HLA-A2. Serum obtained during the third trimester from this woman contained Ab2 to HLA-AI carried by the other child; this serum blocked autologous and homologous sera containing anti-HLA-A1 an- tibodies (Fig. 3).

Case No.. 3 showed Ab2 to HLA-DR2 in the first trimester sera. The Ab2

124

positive serum inhibited ant i -DR2 antibodies present in sera from the second trimester. Case No. 4 developed during the third trimester Ab2 to HLA-B7 which was present in sera obtained during the first trimester. In case No. 8 the woman exhibited, during the first and second trimester, Ab2 to anti- HLA-A2 antibodies. In the remaining two cases (case No. 6 and No. 7) no Ab2 were found.

Discussion

Maternal tolerance to the fetus is an immunological paradox which has pre-occupied transplant biologists over the last decades. Cellular exchange

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125

between mother and fetus has been considered with respect to blood cells as well as to fragments of syncytial trophoblast breaking away from the surface of placental villi (Billington, 1969). It has been established that trophoblast material does not induce an inflammatory reaction in the maternal lung where considerable amounts are known to lodge during normal human pregnancy (Billington 1969). Such chronic exposure of the mother to fetal tissues has led to the suggestion that this phenomenon of deportation has biologic significance as it may contribute to the induction of maternal tolerance to the semi-allogeneic conceptus. There is no convincing evidence however, that tolerance to the fetus is acquired. Furthermore, sensitization to fetal antigens encountered during a first pregnancy, should render tolerance to subsequent pregnancies more difficult to induce, a situation which in fact is not found. Recent evidence that the trophoblast expresses an unusual non-polymorphic form of HLA-class I molecules (HLA-G) (Kovats et al., 1990; Ellis et al., 1990) supports the concept that the trophoblast, is at best, neutral with respect to its HLA immunogenicity.

However, since anti-HLA sensitization takes place, tissues other than trophoblasts, such as fetal lymphocytes probably serve as a source of alloantigen.

In an attempt to evaluate mechanisms of suppression of anti-HLA im- munity we have sought factors which are likely to block the reactivity of cir- culating anti-HLA-antibodies. Our present study shows that maternal sera contain antibodies reactive with paternal HLA-class I and class II antigens as early as 8 weeks of gestation. Concomitant or prior to the development of these antibodies we have detected fetal HLA antigens in the maternal cir- culation as free or immune complexed forms. Following immunoaffinity depletion of HLA antigens from maternal sera we were able to demonstrate previously masked allo- and autoantibodies. The magnetic immunodepletion technique uses high affinity murine anti-HLA antibodies to displace human anti-HLA antibodies from the HLA/anti-HLA immune complexes found in the maternal serum. This displacement reaction releases the complexed maternal anti-HLA alloantibodies into the supernatant, allowing their detec- tion (King et al., 1989; Suciu-Foca, 1990a; Suciu-Foca, 1990b). Such antibody-induced displacement reactions, based on affinity differences be- tween different antibodies, has been used in the past to explore the confor- mational flexibility of HLA antigens as well as for the study of low affinity antibodies from immune sera (Parham, 1984; Kim et al., 1974). The depletion of soluble HLA antigen from the serum facilitates the identification of anti- idiotypic antibodies. These Ab2, which are complementary to anti-HLA-Abl fit the description of Ab2 gamma, i.e. of antigen-inhibitable anti-idiotypes recognizing a paratope-associated idiotype (Adorini et al., 1990).

We found that while more than 50'V,, of pregnant women develop anti-

126

HLA antibodies during the first trimester, there is a decline in the frequency and titer of Abl during the second trimester. This decline is best explained by the development of Ab2. Antibodies to fetal HLA antigens may be unable to harm the fetus since they are complexed to soluble HLA antigens and/or suppressed by anti-idiotypic antibodies. Similar conclusions have been reached by studies on pregnant ra t s , in which it was found that alloan- tibodies with binding sites occupied by alloantigen are detectable both early and late postpartum (Amsden et al., 1988). In rats, however, the earliness of the antibody response as well as the presence of Ab2 escaped detection, most likely because of immune complexes.

It is possible that persistent antigen may be of importance for maintenance of immunologic tolerance to the fetus, although this could not explain the nature of the barrier opposing rejection of the fetus during early pregnancy, i.e. before chronic exposure to high levels of circulating antigen takes place. Furthermore, data from heart and kidney allograft recipients indicate, that HLA-alloantigen complexed to antibody is predictive of rejection rather than tolerance (Suciu-Foca et al., 1990a, 1990b).

The argument that the immunoregulatory role of immune complexes depends on whether the antigen or the antibody is in excess, can certainly be made, although it is unlikely that the fate of pregnancy depends on this ratio (Terres et al., 1972; Laissue et al., 1971; Henney et al., 1970; Caulfield et al., 1987). The relative contribution of Ab2 versus antigen to the suppression of Abl, cannot be ascertained at the present time, since quantitative measurements await the development of more sensitive tools.

Of particular interest is the.finding that healthy pregnant women develop quite often autoantibodies against HLA antigens. This observation may be explained by the binding of self or non-self peptides to self MHC and by subsequent alteration(s) of its immunogenicity (Matis et al., 1987; Suciu- Foca et al., 1984a; Suciu-Foca et al., 1984b; Adorini et al., 1990). Molecular mimicry between self MHC and exogenous antigens is evidently not exclud- ed, nor can the possibility be dismissed that the immunological surveillance mechanism is somehow depressed during pregnancy (King et al., 1989).

On the other hand, the development of anti-HLA autoantibodies may serve a protective function, since such antibodies can block the access of anti- MHC reactive T cells to their target. Again, the detection of anti-HLA au- toantibodies relies heavily on the dissociation of immune complexes formed by the antibody with soluble antigen (King et al., 1989; Suciu-Foca et al., 1990a; Suciu-Foca et al., 1990b).

While underscoring the complexity of the immunological events that take place during pregnancy our investigation also brings into focus the need of exploring T cell-mediated regulatory events. Since antibody responses re- quire the contribution of helper T cells, understanding the network regula-

127

tion of T-cell responses becomes mandatory. The existence of autoreactive anti-idiotypic T-cells with suppressor function has been demonstrated by Autologous Mixed Lymphocyte Culture studies (Suciu-Foca et al . , 1982; Rohowsky et al., 1983). It is conceivable that immunization with idiotopes of alloreactive T cells may lead to enhancement of the suppressor mechanism and, possibly, to the development of effective means for preventing spon- taneous abortion (Suciu-Foca et al., 1983).

Acknowledgments

This work has been supported by the following grants from the National Institutes of Health: POL-HL36581-03, R01-HD22920-01AI and R01- A125210-03. The authors acknowledge the secretarial help of Mrs. Lillian Acosta.

References

Adinolfi, M. and Humphrey. J. (1969) Immunology and Development, pp. 2 7 ~ 1 . Spastics Intern. Med. Publications, London.

Adorini, L., Barnaba, V., Bona, C., Celada, F.. Lanzavecchia. A., Sercarz, E., Suciu-Foca. N. and Wekerle, H. (1990) New perspectives for immunointervention in autoimmune diseases. Immunol. To- day, II, 383--386.

Amsden, A.F., Smith, R.N. and Chirakalwason, N. (1988) The alloantibody response in the allogeneically pregnant rat Vl. Detection in maternal serum of specific antigen-antibody complexes. J. immunol. 141, 2295---2302.

Antczak. D.F. (1980) Maternal antibody responses in pregnancy. Curr. Opin. Immunol. I, 1135--1140. Billington, W.D. (1969) In: Immunology and Development, Chapter 10, (Adinolfi, M. and Wood. C.B.S.

eds.), pp. 89--113. Spastics Int. Med. Publ. Bonagura, V., Rohowsky-Kochan, C., Reed, E., Ma, A. and Suciu-Foca, N. (1986) Pertubation of the

immune network in herpes gestations. Hum. Immunol. 15, 211--219. Bonagura, V., Rohowsky-Kochan. C., Reed, E., Ma, A. and Suciu-Foca, N. (1987) Anti-clonotypic au-

toantibodies in pregnancy. Cell. Immunol. 108, 356--365. Caufield, M. and Shaffer, D. (1987) Immunoregulation by antigen/antibody complexes. I. Specific im-

munosuppression induced in vivo with immune complexes formed in antibody excess. J. Immunol. 138, 368O.

Chardonnens, X. and Heannet, M. (1980) Immunolobiology of pregnancy: Evidence for a fetal immune response against the mother? Tissue Antigens 15, 401~406.

Class, F.H.J., Gijbels, Y.. Van Der Velden-De Munck, J. and Van Rood, J.J. (1988) Induction of B cell unresponsiveness to non-inherited maternal H LA antigens d uring fetal life. Science 24 I, 1815-- 1817.

Desai, R.D. and Creger, W.P. (1964) Maternal feta! passage of leukocytes and platelets in man. Blood 2 I, 665.

Desoye, G., Dohr, G.A., Motter, W., Winter, R., Urdl, W., Pusch. H., Uchanska-Ziegler, B, and Ziegler, A. (1988) Lack of HLA-class I and class II antigens on human preimplantation embryos, J. Immunol. 140, 4157--4159.

Ellis, S.A., Palmer, M.S. and McMichael, A.J. (1990) Human trophoblast and the choriocarcinoma cell line BeWo express a trisencated HLA-class I molecule. J. Immunol. 144, 731--735.

Henney, C. and Ishizaka, K. (1970) Studies on the immunogenicity of antigen antibody precipitates. !1. The suppressive effect of anti-hapten antibodies on the immunogenicity of dinitrophenylated human gamma G globulin. J. immunol. 104, 1540.

128

Johnson, P.M. (1989) Immunology of reproduction. Curr. Opin. Immunol. I, 1117 1118. Kim Tai, Y., Werblin, T.P. and Siskind, G.W. (1974) Distribution of antibody-bindng affinity. I11. Detec-

tion of low affinity antibody in the presence of high affinity antibody. J. Immunol. 112, 2002 2008. King, D.W., Reed, E. and Suciu-Foca, N. (1989) Complexes of soluble HLA antigens and anti-HLA au-

toantibodies in human sera: possible role in maintenance of self-tolerance. J. lmmunol. Res. 8, 249 262.

Kovats, S., Main, E.K., Librach, C., Stubblebinc, M., Fisher, S.J. and Demars, R. (19901 A class 1 an- tigen, HLA-G., expressed in human trophoblast. Science 248, 220 223.

Laissue, J., Cottier, H., Hess, M.W. and Stoner, R.D. (1971) Early and enhanced germinal center forma- tion and antibody response in mice alter primary stimulation with anligen-isologous antibody com- plexes as compared with antigen alone. J. Immunol, 107, 822.

Loke, Y.W. (1989) Trophoblast antigen expression. Curr. Opin. lmmunol, 1, 1131~ 1134. Matis, L.A., Sorger, S.B., McEIligott, D.L., Fink, P.J. and Hedrick, S.M. (19871 The molecular basis of

alloreactivity in antigen-specific, major histocompatibility complex-restricted T-cell clones. Cell 51, 59--69.

Parham, P. ( 19841 Changes in conformation with loss of alloantigenic determinants of a histocompatibili- ty antigen (HLA-B71 induced by monoclonal antibodies. J. Immunol. 132, 2975 2980.

Reed, E., Bonagura, V., Kung, P., King, D.W. and Suciu-Foca, N. (19831 Anti-idiotypic antibodies to HLA-DR4 and DR2. J. Immunol. 131, 2890--2984.

Reed, E., Rohowsky-Kochan, C. and Suciu-Foca, N. (1984) Analysis of 9W antisera detecting DR4 and DR2 associated epitopes by use of anli-idiotypic antibodies. In: Histocompatibility Testing (Albert, E.D., Baur, M.P. and Mayr, W.R., eds.), pp. 422 424. Springer Verlag, New York.

Reed, E., Hardy, M., Benvenisty, A., Lattes, C., Brensilvcr, J., McCabe, R., Reemstma, K., King, D.W. and Suciu-Foca, N. (1987) Effects of antiidiotypic antibodies to HLA on graft survival in renal- allograft recipients. N. Engl. J. Med. 316, 1450 1455.

Rohowsky-Kochan, C., Suciu-Foca, N., Kung, P., Tang, T.F., Reed, E. and King, D.W. (19831 Sup- pressor T-cell generated in autologous MLR with activated T lymphoblasts. Transplant. Proc. 15(1), 765--770.

Suciu-Foca N., Rohowsky-Kochan, C., Kung, P. and King, D.W. (1982) ldiotypic-like determinants on human T lymphocytes alloactivated in mixed [ymphocytc culture (MLCI. J. Exp. Med. 156, 283 -288.

Suciu-Foca N., Reed, E., Rohowsky-Kochan, C., Kung, P. and King, D.W. (1983) Anti-idiotypic anti- bodies o anti-HLA receptors induced by pregnancy. Proc. Natl. Acad. Sci. U.S.A. 80, 830 834.

Suciu-Foca N., Rohowsky-Kochan, C., Kung, P. and King, D.W. (1984a) Idiotypic receptors for soluble antigens on human T-lymphocyte clones. Hum. lmmunol. 93, 34 47.

Suciu-Foca N., King. D.W., Reemtsma, K. and Kohler, H. (1984b) Autoimmunity and sell" antigens. Concepts lmmunopathol. I, 173-189.

Suciu-Foca N., Rohowsky-Kochan, C., Reed, E,, Haars, R., Bonagura, V., King, D.W. and Reemstma. K. (1985) [diotypic network regulations of immune rcsponses to HLA. Fed. Proc. 44, 2483 2487.

Suciu-Foca N., Reed, E., Marboe, C., Yu Ping Xi, Sun Yu-Kai, Ho, E., Rose, E., Reemtsma, K. and King, D.W. (1991al. Role of anti-HLA antibodies in heart transphmtation. Transplantation, 51, 716 724.

Suciu-Foca, N., Reed, E.. D'Agati, V.D., Ho, E., Cohen, D.J., Benvenisty, A.I. McCabc, R., Brensilver. J.M., King. D.W. and Hardy, M.A. (1991b) Soluble HLA-antigcns, anti-HLA antibodies and anti- idiotypic antibodies in the circulation of renal transplant recipients. Transplantation, 51, 593--602.

Terres, G., S.L., Morrison, G,S., Habich, and R.D. Stoner (1971) Appearance of an early "primed state" in mice following the concomitant injections of antigen and specific antiserum. J. Immunol. 108. 1743.