1 Department of Immunology, Kitasato University School of Medicine, 1151 Kitasato, Sagamihara 228-8555, 2 Department of Obstetrics and Gynecology, National Okura Hospital, 2101 Okura, Setagaya 157-0074 and 3 Department of Immunology, Kitasato University School of Allied Health Science, 1151 Kitasato, Sagamihara 228-8555, Japan
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Abstract |
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Key words: anti-idiotypic antibody/HLA/immunotherapy/recurrent spontaneous abortion/TCR
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Introduction |
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The immunological regulation which maintains pregnancy is reported to involve both cellular and humoral regulation. It has been reported that suppressor cells found in the decidua suppress the maternal immune response to the fetus (Daya et al., 1985; Clark et al., 1986
). HLA-G antigens expressed on trophoblast cells are reported to inhibit natural killer cell activity (Ellis et al., 1990
; Kovats et al., 1990
; Mandelboim et al., 1997
; Rouas-Freiss et al., 1997
). Anti-paternal cytotoxic antibodies have been detected with high frequency in the sera of normal pregnant women (Jensen, 1962
; Payne, 1962
; Ahrons, 1971
; Doughty and Gelsthorpe, 1971
; Regan et al., 1991
). Furthermore, anti-T-cell receptor (TCR) idiotypic antibodies capable of specifically inhibiting maternal autologous immune responses have been demonstrated in normal pregnant women (Sucui-Foca et al., 1983; Singal et al., 1984
).
As immunotherapy for RSA, women are immunized with their partner's lymphocytes (paternal lymphocytes) in an attempt to enhance the production of immune-suppressing antibodies. Although high success rates of maintaining pregnancy have been achieved using this therapy (Taylor and Faulk, 1981; Mowbray et al., 1985
; Reznikoff-Etievant et al., 1988
; Daya and Gunby, 1994
; Agrawal et al., 1995
; Carp et al. 1997
), the mechanisms of this therapeutic effect have yet to be elucidated. Some investigators have suggested that the RSA patient's ability to produce anti-paternal cytotoxic (Mowbray et al., 1985
; Reznikoff-Etievant et al., 1988
; Lubinski et al., 1993
; Agrawal et al., 1995
) and anti-autologous activated T-cell antibodies (Sugi et al., 1991
; Lubinski et al., 1993
) correlates with successful immunotherapy. However, none of these studies have determined the specificity or identified the possible target molecules of such antibodies. In this study, we examined whether immunotherapy for RSA induces anti-TCR idiotypic antibodies against autologous TCR in the sera of RSA patients. The study group comprised RSA patients who successfully maintained pregnancy after receiving this treatment as well as patients who had an unsuccessful pregnancy after immunotherapy. The results indicate that such antibodies were produced in successful, but not in unsuccessful, immunotherapy cases.
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Materials and methods |
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Serum samples
Serum samples were collected from each RSA patient before and after immunotherapy, at various times during pregnancy, and at delivery. The sera were subjected to complement inactivation at 56°C for 30 min and were stored at 20°C until required for the experiments.
HLA typing
Twenty-seven different HLA-DRB1 genotypes were determined by DNA typing using the polymerase chain reaction and hybridization with sequence-specific oligonucleotides established in our previous study (Obata et al., 1991).
Mixed lymphocyte reaction (MLR) inhibition test
The presence of anti-TCR idiotypic antibodies was examined by determining the inhibitory activity of the maternal serum against the MLR of the paternal lymphocytes (stimulator cells) with the pre-immunotherapy autologous maternal lymphocytes (responder cells). In the MLR test, the responder cells (2.5x106) were treated with test serum (0.5 ml) at room temperature for 30 min, followed by incubation with rabbit complement (2.5 ml) at 37°C for 1 h. Then the responder cells were washed with culture medium three times and the proliferation assay was carried out in triplicate using the serum-treated responder cells and 5x104 irradiated (3000 rad) stimulator cells in 200 µl RPMI-1640 supplemented with 20% (v/v) pooled human male serum (complete medium), in 96-well round-bottomed microtitre plates at 37°C in a 5% CO2 incubator. On day 5, the cells were incubated with 1 µCi [3H]-thymidine (6.7 µCi/mM) for 18 h, were harvested and 3H incorporation was determined by liquid scintillation counting. As the control of the MLR experiments, the responder cells were treated with pooled human male serum and rabbit complement. Lymphocytes from other donors having HLA types either common or unrelated to those of the RSA patients and their husbands were also used as responder or stimulator cells to examine the specificity of the anti-TCR idiotypic antibodies.
Absorption test
Serum sample 7S-9, which was obtained at 9 weeks of gestation (Figure 2B), was used for this study. Serum 7S-9 (1 ml) was absorbed with patient 7 autologous T-lymphoblasts (1x107) that had been induced by MLR using various donor lymphocytes as stimulator cells (husband, HLA-DRB1*0901/1502; KT14, HLA-DRB1*0901/0901; Pitout, HLA-DRB1*0701/0701) in complete medium at 37°C in a 5% CO2 incubator for 9 days. After absorption, fresh pre-immunotherapy patient 7 maternal lymphocytes were treated with either absorbed or non-absorbed 7S-9 serum and rabbit complement, and used as responder cells in a second set of MLR, in which irradiated paternal lymphocytes were used as the stimulator cells.
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Results |
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For the two other successful patients, 8 and 26, a serum sample collected at 20 weeks of gestation from patient 8 showed significant MLR inhibition of 29.6% (P < 0.01), but none of sera collected from patient 26 showed MLR inhibition (Table II). The five women who aborted despite receiving immunotherapy (21, 10, 7, 50 and 38) showed no MLR inhibition.
Specificity of MLR inhibition
The specificity of MLR inhibition was investigated using serum 7S-9, which was obtained at 9 weeks of gestation (Figure 2B) from patient 7, which was considered to be a representative sample. Patient 7 maternal lymphocytes were treated with autologous serum 7S-9 and complement and used as responder cells in MLR, in which irradiated lymphocytes from her husband and various unrelated donors were used as stimulator cells. The MLR was inhibited significantly by 41.9, 76.3 and 44.6% when patient 7 maternal lymphocytes were stimulated with lymphocytes from her husband (DRB1*0901/1502), donor O.M. (DRB1*1502/1502), and donor KT14 (DRB1*0901/0901) respectively. Each of these donors had one HLA-class II antigen in common with the patient's husband (Figure 3
). By contrast, no MLR inhibition was observed when patient 7 maternal lymphocytes were stimulated with lymphocytes from donor KT11 (DRB1*1302/1302) who shared no HLA-class II antigens with patient 7's husband. These results suggest that 7S-9 serum specifically inhibited autologous T-cells that recognized the husband's HLA-DR molecules (i.e. DRB1*0901 and 1502), but not those that recognized the other DR molecules, such as DRB1*1302. Furthermore, we investigated whether 7S-9 serum could inhibit MLR by non-autologous T-cells. T-cells from donor 8F (DRB1*0802/1201) were treated with 7S-9 serum and complement and were used as responder cells for MLR. As shown in Figure 3,
7S-9 serum did not show any inhibition against 8F T-cells even when they were stimulated with cells from patient 7's husband. Thus, 7S-9 serum appears to contain an antibody that specifically reacts with an antigenic structure that is characteristic of the autologous T-cells, but not of non-autologous T-cells, recognizing the particular allogenic HLA-DR molecules. It is reasonable to suggest that this antigenic structure on the T-cells is a TCR idiotype and, therefore, that 7S-9 serum contains antibodies against the TCR idiotypes.
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Discussion |
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Anti-TCR idiotypic antibodies were detected in four of the five patients whose pregnancies were maintained, but in none of the five patients whose pregnancies resulted in abortion. This result indicates that the presence of the anti-TCR idiotypic antibodies correlates strongly with successful pregnancy. Although in this study we focused our analysis on the antibodies against the maternal T-cells, the reactor cells against the fetus, the antibodies against the paternal HLA, the target of the maternal T-cells, may be another important humoral factor to be considered. In our previous study, in which we tested a large number of samples, anti-paternal HLA antibody was induced in a significant number of women who had successful pregnancies compared with that of women with unsuccessful pregnancies, after immunotherapy (K.Ito, unpublished). This finding indicates that the presence of anti-HLA antibody, together with the anti-TCR idiotypic antibody detected in this study, is another indicator for successful pregnancy in patients given immunotherapy.
Some reports have disputed the efficiency of immunotherapy (Cauchi et al., 1991; Ho et al., 1991
; Gatenby et al., 1993
; Perino et al., 1997
). Christiansen (1996), in a review, stated that recurrent miscarriage is caused by some immunological or non-immunological factors, and therefore specific therapies are needed in order to prevent recurrent miscarriage. Although it is questionable whether all our RSA patients required this immunotherapy, it is interesting that paternal lymphocyte immunization induced anti-TCR idiotypic antibody in successful cases, but not in unsuccessful ones. In successful cases, paternal lymphocytes may act as an immunogen to enhance the maternal immunoresponse, and to induce antibodies as an immunological regulator for maintaining pregnancy. To clarify the correlation between the presence of anti-TCR idiotypic antibody and successful pregnancy, future studies of larger numbers of RSA patients are required.
The aetiology of RSA is still not clear. One possible mechanism is that the RSA patient has some kind of defect in the immunological regulation that protects the fetus from maternal attack. Immunotherapy with paternal lymphocytes has been performed in an attempt to modulate this immunological regulation in RSA patients, and a high success rate of maintenance of pregnancy has been obtained. Our previous results and those of other investigators have suggested that the anti-paternal HLA antibody produced by the immunotherapy is involved in the successful maintenance of pregnancy. These antibodies would mask the fetal HLA antigens and prevent them from being attacked by the maternal T-cells. The anti-TCR idiotypic antibody that has been reported to be present in the sera of normal pregnant women (Sucui-Foca et al., 1983; Singal et al., 1984) and which was detected in this study would provide another mechanism for the immunotherapy effect. After immunization with paternal lymphocytes, maternal T-cells recognizing paternal HLA antigens (one of the HLA antigens of the fetus) would expand and serve as immunogens to produce anti-TCR idiotypic antibodies. The anti-TCR idiotypic antibody would then bind specifically to the TCR and suppress the maternal immunoresponse against the fetus, allowing the fetus to escape the maternal immunological attack.
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Acknowledgments |
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Notes |
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5 Present address: Department of Obstetrics and Gynecology, Jikei University School of Medicine, 3258 Nishishinbashi, Minato-ku 105-0003, Japan
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References |
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Submitted on April 20, 1998; accepted on October 14, 1998.