Maternal cell-mediated sensitisation to paternal HLA may occur, but is not a regular event in normal human pregnancy

Maternal cell-mediated sensitisation to paternally-inherited fetal HLA was studied in 10 primigravid and 9 multigravid women. One woman in each group was found to have circulating cytotoxic effector cells specific for paternal lymphocytes, but both had a normal pregnancy outcome. These results show that sensitisation is not a regular event in pregnancy and that there must therefore be a block in the afferent (recognition) phase of the mother's immune response. Where sensitisation does occur there must also be a block on the efferent (effector) arm of the response to prevent rejection of the fetus. The restricted distribution of major histocompatibility complex antigens on trophoblast and the function of the placenta as a barrier between maternal and fetal immune compartments can explain the occurrence of both types of blockade.     

Potentiating maternal immune tolerance in pregnancy: A new challenging role for regulatory T cells

The maternal immune system needs to adapt to tolerate the semi-allogeneic conceptus. Since maternal allo-reactive lymphocytes are not fully depleted, other local/systemic mechanisms play a key role in altering the immune response. The Th1/Th2 cytokine balance is not essential for a pregnancy to be normal. The immune cells, CD4+CD25+Foxp3+, also known as regulatory T cells (Tregs), step in to regulate the allo-reactive Th1 cells. In this review we discuss the role of Tregs in foeto-maternal immune tolerance and in recurrent miscarriage as well as their potential use as a new target for infertility treatment. Animal and human experiments showed Treg cell number and/or function to be diminished in miscarriages. Murine miscarriage can be prevented by transferring Tregs from normal pregnant mice. Tregs at the maternal–fetal interface prevented fetal allo-rejection by creating a “tolerant” microenvironment characterised by the expression of IL-10, TGF-β and haem oxygenase isoform 1 (HO-1) rather than by lowering Th1 cytokines. Tregs increase placental HO-1. In turn, HO-1 may lead to up-regulation of TGF-β, IL-10 and CTLA-4. In vivo experiments showed Tregs sensitisation from paternal antigens to be essential for maternal–fetal tolerance. Tregs increase throughout pregnancy and diminish in late puerperium. Recent data also support the capacity of Tregs to block maternal effector T cells, thereby reducing the maternal–fetal pathological responses to paternal antigens. These findings also permit us to consider new strategies for improving pregnancy outcomes, i.e., anti-TNF blockers and granulocyte-colony stimulating factors as well as novel approaches to therapeutically exploiting Treg + cell memory.     

Lymphocyte alloantigens of the horse. II. Antibodies to ELA antigens produced during equine pregnancy

Evidence is presented for a reproducible maternal immune response to histocompatibility antigens during equine pregnancy. Mares were stimulated as a result of pregnancy to produce cytotoxic antibodies to paternal lymphocyte alloantigens. The majority of these antibodies were directed against antigens of the equine lymphocyte antigen (ELA) system, which is the major histocompatibility complex (MHC) of the horse. In 16 experimental pregnancies produced using 12 mares and 4 stallions which had been typed for ELA antigens, there was correlation between ELA incompatibility between sire and dam and the appearance of antibody in the serum of the dam as a result of pregnancy. In ELA incompatible pregnancies, antibody was first detected very early in pregnancy, usually by day 60 in a 336-day gestation. The appearance of antibody following shortly the development of the endometrial cups, which can be seen macroscopically for the first time between days 38 and 40. The strength (titre) of the cytotoxic antibody response appeared to vary with the paternal ELA antigens expressed by the fetus. Circulating antibody to 'third party' alloantigens generated during prior pregnancies did not alter the kinetics of the appearance of antibody in a current pregnancy. Evidence for an anamnestic response was obtained in second pregnancies in which the histocompatibility difference between mare and fetus was the same as in the first pregnancy. However, this accelerated response did not occur before the time of formation of the endometrial cups. Serum samples from approximately 90% of parturient mares on horse farms in New York State contained antibody reactive with paternal lymphocyte alloantigens.     

Mechanisms of action of regulatory T cells specific for paternal antigens during pregnancy

OBJECTIVE: To investigate whether pregnancy-induced regulatory T cells are generated specifically for paternal antigens or expanded by hormonal changes and to study regulatory T cell-related mechanisms during pregnancy. METHODS: We used murine models of normal, abortion-prone, and pseudopregnancy to characterize regulatory T cells and hormones by methods such as flow cytometry, molecular biology techniques, and chemiluminescence. Antigen specificity was studied in experiments in which animals were vaccinated with paternal antigens or adoptively transferred with regulatory T cells. To analyze regulatory T cell-mediated mechanisms, we used neutralizing antibodies against IL-10 or TGF-beta. RESULTS: Regulatory T cells are activated by male antigens, and minor antigens are protected by linked immunosuppression. Our data exclude the possibility that regulatory T cell expansion during pregnancy is exclusively driven by hormonal changes. An increase in systemic regulatory T cell levels in pseudopregnant females after mating with vasectomized males but not after pseudopregnancy induced mechanically confirms generation of regulatory T cells specific for paternal antigens. As for the mechanisms, neutralizing IL-10 abrogates the protective effect of regulatory T cells, whereas blockage of TGF-beta does not provide the same effect. CONCLUSION: Our data confirm that regulatory T cells act in an antigen-specific manner during pregnancy and strongly suggest that IL-10 is involved in regulatory T cell-mediated protection of the fetus. These data contribute to the knowledge of the basic mechanisms regulating immune tolerance during pregnancy, a major biologic question with important medical implications. LEVEL OF EVIDENCE: II.     

Maternal humoral immune response to incompatibility at multiple minor histocompatibility loci in mice

Mice of the H-2b haplotype were mated with males of the same MHC haplotype, but differing at multiple minor histocompatibility loci. Mice were bled during each pregnancy and at 2-day intervals post-partum. The sera were assayed by indirect immunofluorescence for evidence of a humoral immune response to paternal minor histocompatibility antigens. Alloantibody was first detected in the post-partum period following the third pregnancy, and was also detected during the fourth pregnancy. Thereafter, alloantibody levels dropped and by the post-partum period following the fifth pregnancy, fell to control values. Assays on a panel of cells from mice of different inbred strains revealed specificity of the alloantibody to H-3.1, H-4.1 and H-7.1 antigens. A conventional dye exclusion cytotoxicity test revealed the pregnancy-induced alloantibody did not exhibit complement-dependent cytotoxicity. These findings are discussed in relation to the regulation and functional significance of the humoral immune response in allogeneic pregnancy.     

Immune reactivity of allogeneically pregnant mice to paternal MHC antigens on fetal and placental cells assessed by second set rejection of ascites tumor cells

In vivo immunogenicity of fetus- and placenta-derived cells as well as the immune reactivity of pregnant mice to fetal cells were examined for graft rejecting response (GRR). Systemic administration of small numbers of fetal cells but not placental cells from allogeneically pregnant mice (10(6) cells per mouse) or adult allogeneic spleen cells (10(4) cells) sensitized mice for second-set rejection of an ascitic tumor bearing paternal major histocompatibility complex (MHC) antigens. Despite this fact and the known positive humoral response, pregnant and parous mice are not even minimally sensitized with fetal MHC antigens for GRR transplacentally. Nevertheless, any pregnancy-related systemically active control, which would selectively prevent the mother from being sensitized for GRR by limiting numbers of semi-allogeneic fetal cells, was not demonstrable in either allogeneically or syngeneically pregnant mice. Irrespective of pregnancy, mice did not, however, respond to repeated administration of very small numbers of allogeneic spleen cells (5 X 10(2) cells per mouse) for graft rejection. These findings support the notion that deviation of maternal immunity to fetal antigens away from harmful GRR is mediated principally by local mechanisms which inhibit fetal cells from gaining access to the mother for GRR, and additionally by the innate inability of mice to respond to very small numbers of allogeneic cells that might escape past the local maternal-fetal barrier.     

Active suppression of host-vs-graft reaction in pregnant mice. IV. Local suppressor cells in decidua and uterine blood

The mammalian fetus bears a wide variety of antigens against which the maternal immune system can respond. Although some of these antigens are transplantation antigens, the type of immune response mounted by the mother seems incapable of mediating graft rejection. We have previously demonstrated suppressor cells in the lymph nodes draining the uterus (DLN) that regulate the immune response in allogeneically pregnant C3H/HeJ and CBA/J mice. The suppressor cells were shown to be small lymphocytes (sedimenting at 3 mm/h at unit gravity) resistant to anti-T cell serum + complement that elaborated a soluble suppressor activity and selectively inhibited the generation of cytotoxic T lymphocytes (CTL) reactive with paternal alloantigens. Suppression could be induced in the DLN by syngeneic pregnancy or pseudopregnancy, and behaved as an anatomically localized activity during pregnancy. We now report that during first allogeneic pregnancy, the most potent suppressor cell activity is found in lymphocytes in uterine venous blood and in decidual lymphocytes. This suppressor cell population also sediments at 3 mm/h and is associated with production of a soluble suppressor factor. Substantial suppressor cell activity can also be obtained from the deciduomata of pseudopregnant mice. Local suppressor cell activity within the uterus may play an important role in ensuring the immunological success of the fetal allograft.     

Inhibition of lymphocyte proliferation and activation: a mechanism used by equine invasive trophoblast to escape the maternal immune response

At days 36-38 of gestation, the equine invasive trophoblast cells migrate into the endometrium of the pregnant mare to form the endometrial cups. During their migration, they become surrounded by maternal CD4+ and CD8+ T lymphocytes, and stimulate a cytotoxic antibody response to the paternal major histocompatibility complex class I antigens that they express. Nevertheless, endometrial cup cells remain viable at the site of uterine invasion up to days 80-100 of gestation, suggesting the participation of immunomodulatory mechanisms to the maternal cellular immune response. To determine the effects of the invasive trophoblast cells on lymphocyte proliferation, an in vitro co-culture system was developed using isolated equine invasive trophoblast cells and peripheral blood lymphocytes. Fetal fibroblast cells from the same conceptuses were used as controls. The presence of invasive trophoblast cells or their pre-conditioned medium inhibited 50% or more of lymphocyte proliferation, while fetal fibroblasts had no effect. The invasive trophoblast cell inhibitory factor needed to be present constantly to affect lymphocyte proliferation, and it was ineffective if lymphocytes had been previously stimulated to proliferate. The lymphoproliferative inhibitory mechanism affected lymphocyte subpopulations similarly. In addition, lymphocyte expression of cytokine mRNA including IFNgamma, IL-2, IL-4, and IL-10 was affected compared to controls. The implication of these observations in vivo may explain, in part, the apparent equine maternal immune acceptance of the presence and development of endometrial cup cells.     

Fetus specific T cell modulation during fertilization, implantation and pregnancy

Recently there is an increasing interest in aspects of a more specific immunoregulation during pregnancy. Understanding these mechanism might have a broader application not only for reproductive immunology but also in general for biology and medicine. Especially the induction, already before conception, of feto-specific T cells with a possibly regulatory function gives a biological explanation of local immunotolerance at the maternal fetal interface, supporting the epidemiological evidence of a feto/paternal-specific immuneregulation. Understanding the expression of specific HLA-classes on trophoblast and the crosstalk of these antigens with various cell types, specifically modulated in the decidua, resulting in the secretion of cytokines and (angiogenic) chemokines has given us a more and more detailed understanding of this regulation. This regulation could be induced by fetal cells circulating in the mother (microchimerism) and from the interaction with fetal subcellular fractions as exosomes, but also from paternal antigens present in seminal fluid. Molecular interaction between paternal and fetal antigens and receptors in endometrium and the decidua are discussed. This review highlights besides uNK cells, especially the function of CD4+ and CD8+ T cells with a regulatory function in the context of recurrent miscarriage and pre-eclampsia. Besides HLA, also male-specific minor histocompatibility antigens and the genetic background for these pregnancy complications are discussed.     

Indications of an altered immune balance in preeclampsia: a decrease in in vitro secretion of IL-5 and IL-10 from blood mononuclear cells and in blood basophil counts compared with normal pregnancy

It has been suggested that maladaptation of the maternal immune response during pregnancy might be a causal factor for preeclampsia. This study was designed to examine the systemic immune status at both the innate level and the adaptive level in pregnancies complicated by preeclampsia (n=15) and normal pregnancies (n=15). Spontaneous and in vitro-induced secretion of IL-5, IL-6, IL-10, IL-12, IL-13 and TNF-alpha, in response to paternal blood cells and the vaccination antigens purified protein derivate of tuberculin (PPD) and tetanus toxoid (TT), was detected in cell culture supernatants from blood mononuclear cells by ELISA. Preeclamptic women showed reduced numbers of basophil granulocytes in the blood (p=0.004) and lower spontaneous secretion of IL-5 from blood mononuclear cells (p=0.016). In addition, paternal antigen-induced secretion of IL-10 was decreased in preeclampsia compared with normal pregnancy (p=0.012). No further differences between preeclampsia and normal pregnancy were found for any stimuli or cytokines. The present findings of reduced basophil numbers and lower spontaneous in vitro secretion of IL-5 in preeclampsia compared with normal pregnancy indicate a decrease in systemic Th2 immunity in preeclampsia. Furthermore, the decrease in paternal antigen-induced secretion of the immunosuppressive cytokine IL-10 in preeclampsia indicates a fetus-specific decrease in immunosuppression mediated by blood mononuclear cells. Whether these systemic changes are a cause or a consequence of preeclampsia remains to be elucidated.     

Activating T regulatory cells for tolerance in early pregnancy — the contribution of seminal fluid

A state of active tolerance mediated by T regulatory (Treg) cells must be functional from the time of embryo implantation to prevent the conceptus from maternal immune attack. Male seminal fluid and ovarian steroid hormones are implicated in regulating the size and suppressive function of the Treg cell pool during the peri-implantation phase of early pregnancy. Evidence that antigens and cytokine signals in seminal fluid regulate the maternal immune response includes the following: (1) the Treg cell-inducing cytokine TGFβ and male alloantigens are present in seminal fluid; (2) seminal fluid delivery at coitus is sufficient to induce a state of active immune tolerance to paternal alloantigen, even in the absence of conceptus tissue; (3) female dendritic cells can cross-present seminal fluid antigens to activate both CD8⁺ and CD4⁺ T cells, and (4) mating events deficient in either sperm or seminal plasma result in diminished CD4⁺ CD25⁺ Foxp3⁺ Treg cell populations at the time of embryo implantation. Ongoing studies indicate that the cytokine environment during priming to male seminal fluid antigens influences the phenotype of responding T cells, and impacts fetal survival in later gestation. Collectively, these observations implicate factors in the peri-conceptual environment of both male and female origin as important determinants of maternal immune tolerance. Defining the mechanisms controlling tolerance induction will be helpful for developing new therapies for immune-mediated pathologies of pregnancy such as miscarriage and pre-eclampsia.     

Review: Fetal antigens--identity, origins, and influences on the maternal immune system

Pregnancy induces priming of the maternal cellular and humoral immune systems. The paternally-inherited fetal antigens that influence maternal T and B cells include both major and minor histocompatibility antigens - the same antigens that are problematic in allotransplantation. Animal models have facilitated our understanding of the lymphocyte responses to fetal antigens, and our appreciation of the parallel response in pregnant women is increasing. The physiologic properties of the placenta as well as trafficking of cells between mother and fetus allow ample opportunity for sampling of fetal proteins by the maternal immune system. Here, the current state of knowledge of fetal antigen-specific lymphocyte responses in pregnancy is reviewed.     

Immunoregulatory effects of human cord blood T lymphocytes on mixed lymphocyte reaction

Fetuses of outbred mammalian species can survive despite disparities of participants' histocompatibility antigens. During pregnancy, maternal immune response is supposed to be suppressed by various immune suppressive mechanisms. In order to know whether fetal lymphocytes can suppress the maternal immune reaction, we investigated the suppressive effect of human newborns' T lymphocytes. Newborn T cells were isolated from the umbilical cord blood by means of the sheep red blood cell rosette forming method and treated with mitomycin C. The newborn T cells were added as regulator cells to one way mixed lymphocyte reaction (MLR) using maternal lymphocytes, paternal lymphocytes and unrelated donor's lymphocytes. In a control study, adult T cells were treated in the same manner. In any combination of the MLR, the response was substantially suppressed by the addition of cord T cells in comparison with the response by the addition of normal adult T cells. The suppression was dose-related with an increasing number of regulator cord T cells. The suppressive activity of cord T cells showed radiation resistance until 1,000 rads irradiation. In the kinetics study, cord T cells were found to suppress the recognition phase of MLR. These results suggest that cord T lymphocytes suppress the mother's immunological reaction to the fetal antigens.     

In vitro susceptibility of mouse placental trophoblast to cytotoxic effector cells

The susceptibility of mouse placental cells to hyperimmune cell killing directed against paternal combined H-2 and non-H-2 antigens was investigated using [3H]uridine as a terminal label in an in vitro cell-mediated microcytotoxicity test. The stage of development of the conceptus from which the short-term placental cell monolayers were prepared determined their susceptibility to immune cell lysis. Cultures from whole placentae taken at 9 days post-coitum (p.c.) were not significantly affected whereas similar monolayers prepared at 10.5 days p.c. or later underwent extensive destruction. Embryonic fibroblasts were susceptible at all stages examined from 9-16 days p.c. The onset of susceptibility correlates with the reported appearance of H-2 antigens on the placenta during ontogeny. All cultures of dissected populations of 13-day p.c. placental spongiotrophoblast were susceptible but only 70% of those of labyrinthine trophoblast. It is suggested that of the two major trophoblastic components of the mouse placenta the spongiotrophoblast expresses antigens that render it vulnerable to maternal immune attack whilst the labyrinthine trophoblast is only weakly or non-antigenic, with the observed killing being due largely to contamination of these cultures by antigenic foetal mesenchymal elements. Since failure to express appropriate target antigens cannot be the reason for the in vivo survival of the spongiotrophoblast it must be presumed that some form of immunoregulatory mechanism(s) is responsible for the maintenance of the foeto-placental allograft.     

Increased circulating paternal antigen-specific IFN-gamma- and IL-4-secreting cells during pregnancy in allergic and non-allergic women

INTRODUCTION: Allergic women have been reported to give birth to more children than non-allergic women, speculatively explained by the former's predisposition for Th2 polarization, possibly favoring pregnancy. AIM: The aim of this study was to test the hypothesis that allergy is associated with more Th2-deviated responses to paternal antigens throughout pregnancy. METHODS: Blood samples were collected on six occasions during pregnancy and two occasions postpartum (pp). Of the 86 women initially included, 54 women had a normal pregnancy and completed the sampling procedures. Eleven women fulfilled the strict criteria for allergy (allergic symptoms and circulating IgE antibodies to inhalant allergens) and 23 were strictly non-allergic (non-sensitized without symptoms). The numbers of blood mononuclear cells secreting IFN-gamma and IL-4, spontaneously and in response to paternal alloantigens, were compared between the groups. RESULTS: The numbers of spontaneously as well as paternal antigen-induced IFN-gamma- and IL-4-secreting cells were similar in allergic and non-allergic pregnant women on all occasions. A similar increase in the numbers of both IFN-gamma- and IL-4-secreting cells were found in allergic and non-allergic women during pregnancy, both regarding spontaneous and paternal antigen-induced secretion. CONCLUSIONS: This study does not support the hypothesis of a more pronounced Th2-deviation to paternal antigens in allergic pregnant women compared with non-allergic pregnant women, as measured by number of cytokine-secreting cells. The observed increase of both IFN-gamma- and IL-4-secreting cells during normal pregnancy may be interpreted as a Th2-situation, since the effects of IL-4 predominate over the effects of IFN-gamma.     

Inadequate tolerance induction may induce pre-eclampsia

The fetus is semi-allograft to the maternal host; therefore, a system of tolerance must be present during pregnancy. Epidemiological findings support a relationship between pre-eclampsia and the failure of tolerance induction. For induction of major histocompatibility complex (MHC) class I-specific tolerance, long-term exposure to seminal fluid, which contains paternal soluble MHC class I antigens, may induce paternal MHC class I-specific tolerance. Furthermore, soluble HLA-G1, which induces the deletion of CD8(+) T-cells, and the combination of maternal killer-immunoglobulin-like receptors (KIR) on NK cells and fetal HLA-C, which affects the balance between inhibition and activation signals of NK cells, regulatory CD8(+) T cells, and regulatory NK cells, may play very important roles in the induction of MHC class I-specific tolerance. On the other hand, exposure to sperm, which express paternal HLA-DR, and trophoblastic debris, which contain intracellular fetal HLA-DR, may induce paternal MHC class II-specific tolerance. In this process, CD4(+)CD25(+) regulatory T (Treg)-cells play central roles. In this review, we discuss the relationship between the risk of pre-eclampsia and tolerance induction.     

The alloantibody response of pregnant women and its suppression by soluble HLA antigens and anti-idiotypic antibodies.

The aim of this study was to investigate the time course of maternal allosensitization to fetal HLA antigens during normal human pregnancy and to explore mechanisms of suppression of anti-HLA alloantibodies. We found that the mother produces antibodies against some but not all of the mismatched HLA antigens of the fetus as early as the 8th week of pregnancy. These antibodies (Ab1), however, are often complexed with soluble HLA alloantigens and become detectable when immune 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.     

CD4(+)CD25+ T regulatory cells in murine pregnancy

Mammalian pregnancy is thought to be a state of immunological tolerance and immunological pregnancy complications may result from incomplete allo-tolerance. We reported recently a higher frequency of Th1 cytokine-producing T cells specific against paternal antigens in abortion-prone mice compared to normal pregnant mice. Since Th2 cells were shown to be not essential for normal pregnancy; alloreactive Th1 cells must be differently regulated. In this context, T regulatory cells (Treg) were proposed to play an essential role. Normal pregnant mice show an expansion of CD4(+)CD25+ and IL-10+ Treg cells at the periphery compared to non-pregnant animals. Further, we reported significantly lower frequencies of Treg in abortion-prone mice. Interestingly, CD4(+)CD25+ Treg cells from normal pregnant mice were able to prevent fetal rejection. Accordingly, down-regulated levels of Treg were also reported during human miscarriage. The putative mechanisms involved in Treg-induced tolerance in mice and humans are discussed in this review.     

Inadequate tolerance induction may induce pre-eclampsia

The fetus is semi-allograft to the maternal host; therefore, a system of tolerance must be present during pregnancy. Epidemiological findings support a relationship between pre-eclampsia and the failure of tolerance induction. For induction of major histocompatibility complex (MHC) class I-specific tolerance, long-term exposure to seminal fluid, which contains paternal soluble MHC class I antigens, may induce paternal MHC class I-specific tolerance. Furthermore, soluble HLA-G1, which induces the deletion of CD8⁺ T-cells, and the combination of maternal killer-immunoglobulin-like receptors (KIR) on NK cells and fetal HLA-C, which affects the balance between inhibition and activation signals of NK cells, regulatory CD8⁺ T cells, and regulatory NK cells, may play very important roles in the induction of MHC class I-specific tolerance. On the other hand, exposure to sperm, which express paternal HLA-DR, and trophoblastic debris, which contain intracellular fetal HLA-DR, may induce paternal MHC class II-specific tolerance. In this process, CD4⁺CD25⁺ regulatory T (Treg)-cells play central roles. In this review, we discuss the relationship between the risk of pre-eclampsia and tolerance induction.     

Antigen presenting capacity of murine decidual tissue in vivo

Antigen presenting cells (APC) within murine decidual tissue in vivo have been shown to process the soluble antigen ovalbumin after intravenous administration and to present it in a form recognizable by immune T lymphocytes. In vivo antigen pulsed decidual APC stimulated T cell proliferation as efficiently as splenic APC and in an MHC restricted manner. In addition, anti-class II antibody plus complement treatment significantly reduced decidual antigen presenting capacity in vitro. These findings show that class II positive cells within the decidua can present antigen effectively in vivo and may therefore serve as APC for the presentation of fetal antigens to the maternal immune system during pregnancy.