Inhibitory serum factor of Iymphoproliferative response to allogeneic cells in pregnancy

Introduction: An inhibitory serum factor of mixed Iymphocyte culture (MLC) has been associated with successful pregnancy after. Iymphocyte transfusion in women with unexplained recurrent spontaneous abortions (ASA). Objective: Investigate whether the inhibitory serum factor of MLC is essential for a successful pregnancy. Method: Sera from 33 healthy pregnant women and from 40 women with ASA were assessed by a one-way MLC in which the woman's Iymphocytes were stimulated with her partner's Iymphocytes or with third.party Iymphocytes. Results: An inhibitory serum effect (inhibition > 50% as compared to normal serum) was detected in 45% of the pregnant women who had at least 1 previous parity, in 80/0 of the primigravidea, in 29% of those with one abortion and in 58% of those with more than one abortion. Conclusion: ,MLC inhibitory serum factor does not seem to be an essential factor for pregnancy development. Therefore, it should not be considered as a parameter for the assessment of ASA patients.


INTRODUCTION
T he maternal immunologic response to the fetus has been assessed in several studies.I -4 However, the essential factors that lead to the establishment and maintenance of successful pregnancy remain unclear.It has been suggested that in a normal pregnancy there is a modulation of potentially deleterious immune responses against the fetus in order to prevent rejection reactions.5 ,6 Thus, an abnormal allogeneic response, with insufficient production of blocking factors, has been proposed as the cause of certain unexplained recurrent spontaneous

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Silvia Daher Rua Bela Cintra, 1920 apt.41 São Paulo/ SP -Brasil-CEP 01415-002 abortions (RSA).1-3,6Inorder to induc~an adequate maternal immune response, these patients have been immunized with paternal or third party donor leukocytes.3 ,7-1O The mixed lymphocyte culture (MLC). is an in vitro model for allegeneic T cell proliferati ve response that has been used to detect blocking factors in maternal serum.Several investigators reported an inhibitory serum effect upon the autologous lymphoproliferative response to the partner's lymphocytes in women with successful pregnancy and its absence in RSA patients.3 ,5,6Therefore, it has been suggested that the absence of this factor may predispose to abortion. 1 ,2,1~Moreover, its determination has been adopted as a parameterío select 3 ,9,11 and to assess RSA patients that receive leukocyte transfusions.2 ,9,12-14 This factor has been observed in transfused RSA patients, 2,9,11,15 but çontroversial results have been reported regarding its association with a subsequent successful pregnancy.2,1O,12,13,[15][16][17] The MLC inhibitory serum factor has been assessed mostly in non-pregnant women with a successful obstetric
An inhibitory activity on the mixed lyrnphocyte culture involving the maternal lyrnphoproliferative response to partner's cells was observed in the sera of 45% (9/20) ofthe pregnant women who had at least one parity and in only 8% (1/13) ofthe primigravidae (p < 0.05) (Fig. 1).The frequency of the inhibitory factor in the sera ~f pregnant women with one previous pregnancy (3/6) did not differ from that observed in pregnant women with more than one previous pregnancy (6/14).Fifteen patients were assessed during the frrst, 14 during the second and four during the third trimester of pregnancy and the serum factor was detected with the same frequency in the three trimesters of pregnancy.The presence of an inhibitory factor in the serum was defined as a decrease of at least 50% of the counts in cultures with the experimetnal serum' as compared to the mean counts observed with the controI serum.
3. Phytohemagglutinin (PHA) stimulated cultures: PeripheraI blood mononuclear cells (lxl0 5 ) were incubated in culture rnedium with PHA (Difco, Detroit, USA) at a concentration of 10 rng/mI, for three days.The cultures were set up in triplicates, with 20% of experimental or controI sera.Otherwise, the method was the same as the one described for the MLC.
4. Statistical analysis: Data were analysed by the Fisher's exact test.and a p value < 0,05 was taken as the leveI of significance.

MATERIAL AND METHODS
history and in RSA patients.IO ,1l,13,14,16,18  In the present study we have assessed the sera of healthy pregnant women and of non-transfused RSA patients, in order to determine whether this factor is essential to the development of a normal pregriancy and to investigate whether its absence could be considered a relevant parameter for the diagnosis and therapeutic orientation of RSA patients.

Patients:
The study comprised 33 pregnant women with normal obstetric histories and 40 non-pregnant women with recurrent spontaneous abortions (RSA).Thirteeen of the pregnant women were primigravidae and the others had 1 to 6 previous pregnancies.
They were assessed in different trimesters of pregnancy.RSA was defined as three or .moresuccessive miscarriages in the first trimester of pregnancy in women without evidence of uterine abnormalities, genital-tract infections, hormonal deficiencies or autoantibodies.Chromosome abnormalities were not detected in the patients or their partners.RSA patients were classif~ed as primary or secondary aborters on the basis of their obstetric histories.The women who had never had a viable pregnancy were considered to be primary aborters, whereas women who had already had a live birth or stillbirth with more than 20 weeks of gestation were classified as secondary aborters.
None of the women assessed in this study had a history of previous blood transfusion.Informed consent to participate in the study was obtained from all couples.This study was conducted at the Immunology Laboratory of tlie Allergy, ClinicaI Immunology and Rheumatology Division -Department of Pediatrics of UNIFESP.
2. Mixed lymphocyte culture: Mononuclear cells were separated from peripheraI blood using a Ficoll-Hypaque density gradiente The one-way MLC test was performed with 1 x 10 5 responder ceIIs and 1 x 10 5 Mitomycin C treated stimulator cells in RPMI-1640 \Slgma, St. Louis, Mo) culture medium supplemented with HEPES, L-glutamine, non-essential amino acids, sodium bicarbonate and gentamicin.
The cultures were carried out in triplicates, in the presence 20% controI serum (pooI of male donor sera) or 20% serum being assessed for the presence of an MLC inhibitor factor.All sera were heat inactivated, at 56°C for 30 minutes.The cells were incubated at 37°C in a 5% CO 2 atmosphere for 6 days.Eighteen hours before termination, the cultures were labeled with 2 mCi of [3H]thymidine (specific activity frequencies of sera with a suppressive effect were observed among pregnant women (45%), secondary aborters (40%) and non-pregnant women with at least one previous pregnancy (43%).Only 1 out of 9 (11 %) of the nulliparae sera (p = 0,11 when compared to the above mentioned groups) and none of the male sera presented a suppressive effect (Fig. 2).Among RSA patients, 29 % ofthe primary and 58% of the secondary RSA patients presented the inhibitory serum factor (Fig. 1), with an overall incidence of 38%.The difference in incidence of inhibitory factor between primary and secondary aborters was not statistically significant (p = 0.09).However, the incici.ence of the inhibitory factor was significantly higher among secondary (p = 0.01), but not among primary aborters, when compared to primigravidae women.No significant differences were detected regarding the incidence of this serum factor in the primary or secondary RSA patients when they were compared to pregnant women with at least one previous pregnancy.
A suppressive effect of the serum on the maternal lumphoproliferative response to third party lymphocytes was observed in ,40% of the non-primigravidae pregnant women, in 21 % of the primary aborters and in 25 % of the secondary aborters.With few exceptions-, the sera that inhibited the response to third party cells also inhibited the response to partner's cells (Table 1).
The suppressi ve effect of sera from different sources was also investigated in a one-way MLC performed with cells from two HLA (human leukocyte antigens) class II incompatible donors, not including cells from the partner or from the individuals whose sera were being tested.Similqr The investigation of a factor in the serum of women with a successful pregnancy history and from RSA patients capable of inhibiting the in vitro proliferative response of maternal to paternal cells (MLC) has been the focus of many studies published over the last few years.Regarding RSA patients, the absence of this factor was considered ,by Mclntyre et aI. 3 as a characteristic of primary aborters for whom immunization with the partner's leukocytes would be indicated.On the other hand, according to these same authors, in secondary aborters, a group that would benefit from heparin therapy, this factor is only present in a fraction of the cases.In the present study an inhibitory activity of the serum was detected in 29% of the primary aborters and in 58% of the secondary, clearly showing that the absence of this factor cannot be.used as a distincti ve marker for primary aborters.
Another aspect that has drawn attention to the MLC inhibitory serum factor has been its potential to predict the outcome of a pregnancy folIowing the immunization with parentalleukocytes.Some authors have found that the development of this factor could be considered predictive of a successful pregnancy 2,4,5,8,11,12, while others have not found such a correlation.10, [14][15][16] We reasoned that if this factor is biologicalIy important for the development of a successful pregnancy, it should regularly be detected in the serum from pregnant women.The present data show that this is not the case, since the inhibitory factor was detected in only 1 out of 13 primigravidae, alI of whom have already delivered a healthy baby.Furthermore, a similar incidence of the inhibitory factor was observed in pregnant women who had at least one previous parity (45%) and in secondary aborters (58%).We believe that these results, taken together, support the contention that the inhibitory factor should be regarded as an epiphenomenon of pregnancy rather than an important factor for fetal survival, as has already been suggested. 5,18  The nature of the MLC inhibitory factor is not yet fully clarified.It has been suggested that the blocking activity may be related to antibodies directed against HLC antigens or against trophoblast antigens that cross-react with HLA antigens, or could represent anti-idiotype antibodies directed to the T celI receptors (TCR) involved in alIorecognition.Our study was not specifically designed to study the nature of the inhibitory factor, but we have addressed the question concerning its specificity in blocking the response of the pregnant or RSA women against their partners' celIs.Results have shown that the majority of the sera that inhibited the cultures with the partner cells also showed inhibition in cultures with other cell combinations (Table 1 and Fig. 2), confirming observations of other authors. 2 ,6 It is interesting to note that no inhibitory activity was detected in any of the mal e sera tested, as already reported in medicalliterature. 6 When the women's sera were assayed in PHA-stimulated cell cultures, only a few (three out of 29 pregnant and one out of 20 RSA) showed an inhibitory effect.Thus it appears that the inhibitory factor somehow interferes with allorecognition or, alternatively, that is is not powerful enough to block the response to mitogens that represent a stronger stimulus than allogeneic cells.

CONCLUSION
Our data suggest that the MLC inhibiting factor should be regarded as a consequence of pregnancy rather than an essential factor for fetal survi vaI.Therefore, we believe that this test, which is costly, is not informative either regarding the decision to immunize RSA women with parental leukocytes or as a predictor of a successful pregnancy in women submitted to immunization.
It is therefore our opinion that the only immunological test that should be performed in RSA patients that are being considered for entering an immunization protocol is the search for lymphocytqtoxic antibodies against the cells (crossmatch) that will be used for immunization.A positive crossmatch should contraindicate the immunization in order to avoid hypersensitization and also because, as noted by some authors, 10 a positive crossmatch is already an indication that an immune response to paternal antigens has occurred.

2 .Figure 2 -
Figure 2 -Percentage of sera that presented an inhibitory effect in a mixed Iymphocyte culture (MLC) performed with Iymphocytes from HLA c1ass II incompatible donors.* with normal obstetric history.

%
of sera that presented MLC inhibitory factor

Table 1
F -female; P -partner; T -third party Fm, Pm, Tm -mytomicin treated cells * Percentage of women who presented inhibitory serum tactor both in cultures with Pm and with Tm as stimulator cells ** Percentage of women who presented inhibitory serum tactor both in cultures with P and with T as responder cells.