1 Department Immunogenetics, National Institute of Haematology and Immunology, Budapest, H-1113, 2 Department OB/GYN, Semmelweis University, Faculty of Health Sciences, Budapest, H-1135, 3 Department Haemapheresis, St Laszlo Hospital, Budapest, H-1097, and 4 Department Haemapheresis, National Blood Service, Budapest, H-1113, Hungary
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Abstract |
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Key words: cytotoxic T-lymphocyte precursors/immunological background/recurrent spontaneous abortion
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Introduction |
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The mechanism by which the haplo-identical fetus (a kind of natural allograft) is not rejected is extremely complex and is relatively little understood, although several components thought to be involved in the process have been studied extensively (Johnson and Vince, 1996). The protective immunological mechanisms possibly responsible for the maintenance of pregnancy, as well as a summary of data suggesting immunological mechanisms responsible for pregnancy loss, have been reviewed (Hill and Anderson, 1990; Hill, 1992
; Hill et al., 1995
). Extensive studies have been carried out on classical human leukocyte antigens (HLA) (Beer et al., 1985
; Verp et al., 1993
) and the non-classical HLA-G molecule (Carosella et al., 1999
), as well as the characteristics of the complex trophoblast leukocyte cross reactive antigens/CD46/membrane co-factor protein antigen system (Thaler et al., 1989
; Petranyi Gy et al., 1995b). It has become evident that normal pregnancy is characterized by a dominant harmless humoral immune response and a lack of strong maternal cell-mediated anti-fetal immunity (Wood, 1994
; Voisin and Raghupathy, 1995
, Bermas and Hill, 1997
).
Although less is known about cell-mediated immune reactivity, several lines of evidence point to the role of some immunocompetent effector cell populations in the pathogenesis of unexplained repeated miscarriages (Lachapelle et al., 1996; Bermas and Hill, 1997
; Marzusch et al., 1997
; Tranchot-Diallot 1et al., 1997
; Vassiliadou et al., 1999
). An increased cell-mediated immune reactivity could reflect a rejection-like phenomenon (Johnson et al., 1986
; Raghupathy, 1997
). The association of natural killer cell activity in the human decidua (Lachapelle et al., 1996
; Vassiliadou and Bulmer, 1996
) and peripheral blood (Katano et al., 1997
) with pregnancy outcome have been assessed.
The purpose of this study was to gain more information about the possible causes of unexplained RSA, and in particular, whether an immunological cause might be identified in certain cases by use of a suitable method. The question we have raised is whether cytotoxic T-lymphocytes (CTL) are involved in the course of unexplained recurrent pregnancy failure and whether their activity and the frequency of their precursors are informative in the phenomena. If anti-paternal cytotoxic T-lymphocytes are generated, they might have a central role in pregnancy outcome, as CTLs are primary mediators of allograft rejection.
Cell-mediated lympholysis (CML) and limiting dilution analysis (LDA) have been performed to study the anti-paternal cytotoxic T-cell response and also to estimate the frequency of CTL precursors (CTLpf) according to the established methods (Lefkovits and Waldman, 1984; Kabelitz et al., 1985
). Identification of the number of cytotoxic precursor cells against the various HLA class I and minor H antigens in humans serves as a quantitative measurement of T-cell reactivity. As cell-mediated immune reactivity induced by transfusion (Vandeckerckhove et al., 1989
) and rejection in transplantation (Kaminski et al., 1989
) results in increased CTLp frequency, it was plausible to investigate partner specific CTLpf in patients with unexplained RSA.
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Materials and methods |
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Determination of cytotoxic T-lymphocyte precursor frequency (CTLpf) by limiting dilution analysis (LDA)cell-mediated cytotoxicity (CML)
Lymphocytes were isolated by Ficoll-Uromiro density gradient centrifugation from the sterile heparinised blood samples collected from patients with RSA some months after their last abortion. Lymphocytes were obtained from healthy non-pregnant women a few months after undergoing elective termination (in the first trimester) of apparently normal pregnancies and healthy multigravidae women served as controls. Peripheral blood mononuclear cells were isolated from the healthy male partners of patients and controls, as well as from third party people, were used for mixed lymphocyte reaction in the studies. A limited number of the wife's responder (R) cells (5x104 781 per well) were co-cultured with a constant number (5x104) of Mitomycin C (Sigma, Steinheim, Germany) treated (25 µg/ml) stimulator (S) cells from the husband and third party cells (in some cases). Twenty four replicate microcultures for each responder cell concentration were set up in 96 well round-bottomed microtitre plates. The RPMI 1640 culture medium (Gibco Brl, Paisley, UK) was supplemented with gentamycin (50 µg/ml), glutamine (2 mmol/l), sodium pyruvate (1%), 15% pooled human blood group AB serum and 10 IU/ml human recombinant interleukin-2 (rec IL-2, Boehringer Mannheim, Mannheim, Germany). The microcultures were incubated at 37°C in a humidified atmosphere of 5% CO2 thermostate, and fed with 100 µl fresh medium on days 2 and 6.
51Cr release cytotoxicity assay
On day 9 the generated effector cells were tested for cytotoxicity against Phytohaemagglutinin (PHA; Wellcome, Murex Diagnostics, Dartford, UK) blast cells of the original stimulator, in a standard 51Cr release assay. Briefly, PHA transformed original stimulator cells (5x106) were labelled with 400 µCi 51Cr (sodium chromate; Amersham, UK) for 90 min in 37°C waterbath and washed. From each microculture 100 µl of supernatant were removed, and after shaking the plates, 5000 (in 100µl medium) labelled target cells were added. After a 4 h incubation at 37°C in a CO2 incubator, 100µl of supernatant was removed from each well for gamma-ray counting. Total 51Cr release was assessed by incubating labelled blasts with a 1% solution of Triton-X 100 in distilled water, and spontaneous release by incubating the blasts in culture media and stimulator cells only.
Statistical analysis of CTLpf
It was assumed that the alloreactive CTL precursors are randomly and uniformly distributed in the series of cell dilutions, and that the number of CTLp per well had a Poisson distribution. For limiting dilution assays, microcultures were considered positive if their 51Cr release exceeded mean spontaneous release (24 wells containing irradiated stimulator cells and PHA blasts, but no responder cells) ± 3 SDS. From the ratio of negative wells at each responder cell concentration, the number of CTL precursors in the original cell population can be estimated, according to a standard method (Taswell et al., 1981). CTLpf with 95% confidence intervals was calculated using the jacknife version of the maximum likelihood method using computer software (Strijbosch et al., 1987
).
According to established criteria (Sharrock et al., 1987) concerning the alloreactive T-cell repertoire in man, CTLpf values are considered as follows: very high (CTLpf >1:10 000), high (1:10 0001:20 000), average (1:20 000 1:60 000), low (1:60 0001:100 000) and very low (CTLpf <100 000). Similar results could be achieved based on our investigations with healthy HLA mismatched responder stimulator pairs (only selected data presented).
Mixed lymphocyte culture reaction (MLC)
Responder cells of randomly selected women with RSA (n = 12 ) and controls (n = 10) were co-cultured with Mitomycin c-treated stimulator cells from their partners and HLA-DR different pooled third party cells for 5 days. MLC reactions were set up in triplicate, simultaneously with autologous microcultures. The proliferation of responder cells was measured by 3H-Thymidine uptake during DNA-synthesis, and evaluated by the stimulation index (SI). The stimulation index 1 (SI1) shows the degree of proliferation after allostimulation (RwomanxSpartner) in comparison with the autologous (RwomanxSwoman) level, whilst SI2 shows the proliferation against third party, HLA-DR different pooled stimulator cells. SI1 and SI2 are calculated according to the following formula:
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HLA typing
HLA-A,B typing was performed by the standard NIH microlymphocytotoxicity method (Ray, 1979). HLA-DR and DQ antigens were determined on B-enriched lymphocyte suspensions separated from peripheral blood by the long incubation time cytotoxicity test (van Rood et al., 1975
).
Erythrocyte antibody inhibition assay (EAI)
EAI was performed according to previously published methods (Padanyi et al., 1990), to measure the Fc
RII blocking activity in RSA patients. Briefly, a suspension containing B-cells from the male partner was incubated with inactivated and ultracentrifuged serum from the female partner. Human Rh(D)+ erythrocyte suspension (2%), coated previously with anti-D IgG was then added. The number of rosette forming cells was counted, and the percentage of EA rosette inhibition was calculated. Erythrocyte antibody rosette inhibition percentage (EAI%) was calculated by the following formula:
EAI% = 100x(1-EA% in test sera / EA% in control sera).
The CTLpf, MLC and EAI data of patient and control groups were analysed by Student t-statistics, and the significance of difference between the two groups was identified.
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Results |
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Significantly higher cytotoxic effector cell function could be observed in patients with RSA, mainly in the high effector/target ratio range (2070%) (individual data not shown). The autologous control microcultures showed no notable non-specific cytotoxicity, results ranged between 05%.
In our previous studies investigating HLA-typed, non-pregnant, randomly selected normal volunteers, we had confirmed the finding that the degree of HLA class I mismatch between responder and stimulator pairs considerably influences CTLpf. In the case of these control group members, responder-stimulator pairs with the highest HLA-antigen mismatch (4_HLA_A,B_2_HLA_DR) gave CTLpf values with a mean of 1:18 453 (54 CTLprecursors/106 responder cells). In RSA patients, about four to five times higher partner specific CTLpf could be measured, compared with this highly mismatched responder-stimulator control group. These data indicate that CTLpf values in the recurrent spontaneous aborter group are extremely high. This finding cannot be explained only by an existing HLA mismatch.
Our preliminary data with third party cells acting as stimulators show high CTLpf in five out of seven (71%) cases of RSA (mean third party CTLpf = 1:12 908, SEM = 1900, n = 7). In four out of the five cases, the CTLpf value is in the very high category (mean: 1:5209, SEM = 1828, n = 5). Further detailed investigations are needed to determine the specificity of these CTL precursors and the importance of the phenomena.
Alloantigen response in normal pregnancy and recurrent abortion
When mixed lymphocyte culture reactions were performed with responder cells of women with RSA, and stimulator cells of their partners, a high alloantigen-specific response against the partner could be measured. Partner alloantigen-induced proliferation in cultures from habitual aborter patient cells were highly positive in all but one case (SI1RSA mean: 17.75, SEM: 3.0, n = 12). Significantly (P < 0.001) higher SI indexes were observed in the RSA group than in healthy non-pregnant women undergoing elective terminations of apparently normal pregnancies (SI control mean: 3.2, SEM: 1.3, n = 10 ) (Table IV). A significantly (P < 0.004) higher SI was observed in patients with RSA (SI1RSA mean: 33.5, SEM 7.7, n = 12), when tested against third party pooled lymphocytes versus controls (SI control mean: 6.3 SEM 1.3, n = 10).
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Discussion |
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Our data indicate that CTLpf values in the recurrent spontaneous aborter group with nothing other than immunological cause of pregnancy loss are extremely high, and cannot be explained only by an existing HLA mismatch.
No information is available on the precursor frequency in recurrent miscarriage patients before the first conception, and it was not determined conclusively whether the CTL activity predates the first miscarriage. Furthermore, this study does not rule out the possibility that the CTLpf activity against third party cells also occurs. In most of the investigated cases we found a high or very high CTLpf against third party cells in RSA patients. Our preliminary data concerning CTL precursor frequencies against third party cells in the case of women with recurrent spontaneous abortions might suggest that the high CTLpf is a main characteristic for unexplained RSA with an immunological background, and is probably not partner specific, a possible sensitization to paternally derived antigens in women with unexplained RSA cannot be stated. In order to understand the results, it is important to take into consideration that CTLp frequency of healthy individuals against a panel of HLA class I mismatching antigens was found to be rather low (Breur-Vriesendorp et al., 1991). A wide range of CTLpf against different targets (cord blood, paternal and third party lymphocytes) has been found both in primigravid and multiparous women, reflecting the wide range of frequencies found in random populations (Manyonda, 1993b
).
The high rate of paternal leukocyte-induced proliferation of recurrent spontaneous aborters' lymphocytes, found in mixed lymphocyte cultures, begs comparison with the findings of Bermas and Hill (1997) where proliferative responses to recall and alloantigens were tested in non-pregnant women with a history of three or more spontaneous abortions against relevant controls (Bermas and Hill, 1997). Our results show a low level of blocking antibodies in cases of women with RSA, which is in line with the hypothesis that deficiency in maternal blocking factors play a role in pregnancy loss (Rocklin et al., 1982
; Petranyi Gy et al., 1995a; Malinowski et al., 1997
).
Exposing the possible mechanisms that induce recurrent miscarriages might be helpful in the selection of suitable therapies. Women with a lack of allo-immune recognition might be immunised with paternal lymphocytes or thrombocytes, or receive i.v. immunoglobulin (IVIG) treatment (Beer et al., 1985). Detailed studies have revealed differences in some humoral immune response parameters, like the incidence of antinuclear and anticardiolipin antibodies, absence of serum factors suppressing lymphocyte proliferation in response to phytohaemagglutinin (PHA) and paternal alloantigens of non-pregnant women with unexplained RSA (Malinowski et al., 1997
). For women with autoimmune serological abnormalities, other treatments, like pre-conception administration of aspirin, heparin and/or prednisolone are chosen (Coulam, 1995
).
On the basis of our investigations we state that the estimation of anti-paternal cytotoxic T-lymphocyte precursor frequency seems to be a helpful and reliable clinical test for the indication of an immunological background in unexplained RSA. This finding might be of great importance in the selection of patients for immunotherapy (with paternal or third party leukocytes or thrombocytes, or syncytiotrophoblast plasma membrane preparations, or administration of IVIG) in order to facilitate a successful pregnancy outcome. Immunotherapy might be an effective treatment in affected couples, (Stephenson et al., 1998; Sticker et al., 2000
) especially in those cases where the immunological cause of RSA could be proven. Until now there has remained a lack of agreement on suitable diagnostic tests defining immunological cause in unexplained RSA, although there have been some proposals on this topic involving measurement of NK cell activity (Aoki et al., 1995
) and proliferation to recall antigens (Bermas and Hill, 1997
).
Although a great variety of hypotheses have been proposed to explain how the haplo-identical fetus escapes immunological rejection (Gurka and Rocklin, 1987; Hill and Anderson, 1988
; Hill, 1990
; Aksel, 1992
), and how some hormonal and biochemical factors and other immuno-regulatory mechanisms (Szekeres-Bartho et al., 1990
) might be involved in the maintenance of pregnancy, the mechanisms leading to recurrent pregnancy loss have not yet been described. Recent studies with decidual lymphocytes (Marzusch et al., 1997
), regulation of antigen expression (Kodama et al., 1998
; Komatsu et al., 1998
; Vassiliadou et al., 1999
), and regulation of cytokine expression (Lea et al., 1995
; Tranchot Diallot et al., 1997
) may contribute to understand pregnancy maintenance and success. Women with unexplained RSA manifest an abnormal T-helper 1-type response following exposure to trophoblast extracts (Wegman et al., 1993
; Hill et al., 1995
).
We have concluded that RSA with an immunological background corresponds with extremely high CTL precursor frequencies, which might be of more than a passing interest. Further experiments are necessary to substantiate the present findings in order to clarify their role in the mechanism of RSA, such as further characterisation of these precursor cells and definition of their specificity.
On the basis of this very high rate of correlation between high CTLpf values and immunological cause of RSA, we suggest that CTLpf assay might be a useful diagnostic test for selecting RSA patients with an immunological background, for potential immunotherapy.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 13, 2000; accepted on February 23, 2001.