Fetal HLA Research Articles

Non-invasive method for fetal trophoblast HLA typing in early pregnancy

Non-invasive method for fetal trophoblast HLA typing in early pregnancy

Differences in the Expression of KIR, ILT Inhibitory Receptors, and VEGF Production in the Induced Decidual NK Cell Cultures of Fertile and RPL Women.

Results KIR2DL1 and ILT-2 expression on idNK cells was higher in healthy women than in RPL patients. Sildenafil enhanced NKG2A expression in RPL patients. VEGF concentration was higher in fertile woman idNK cell cultures. idNK cells were more sensitive for necrosis in RPL than in fertile women. SC did not influence VEGF production or idNK cell apoptosis. Conclusions A combination of hypoxia, IL-15, and AZA promotes the conversion of pbNK into idNK cells CD56+CD16−-expressing KIR receptors and produces VEGF. Alterations in KIR2DL1 and ILT-2 expression as well as impaired VEGF production were associated with RPL. SC affects NKG2A expression on RPL idNK cells. SC had no effect on VEGF release or idNK cell apoptosis.

The combination of maternal KIR-B and fetal HLA-C2 is associated with decidua basalis acute atherosis in pregnancies with preeclampsia

Acute atherosis is an arterial lesion most often occurring in pregnancies complicated by preeclampsia, a hypertensive pregnancy disorder. Acute atherosis predominates in the maternal spiral arteries in the decidua basalis layer of the pregnant uterus. This layer forms the fetal-maternal immunological interface, where fetal extravillous trophoblasts interact with maternal immune cells to promote decidual spiral artery remodeling and maternal immune tolerance towards the fetus. Of the classical polymorphic class I HLAs, extravillous trophoblasts express only HLA-C. HLA-C is a ligand for killer immunoglobulin-like receptors (KIR) on NK- and T-cells. Genetic combinations of fetal HLA-C and maternal KIRs affect pregnancy outcome. However, the role of HLA and KIR genes in acute atherosis is unknown.We hypothesized that specific genetic combinations of fetal HLA and maternal KIR are associated with the presence of acute atherosis lesions in the decidua basalis. We genotyped HLA class-I and II loci in paired fetal and maternal DNA samples from 166 pregnancies (83 preeclamptics, 83 controls). Acute atherosis was identified in 38 of these. Maternal KIR-loci were also genotyped.We found that the combination of maternal KIR-B haplotype and fetal HLA-C2 was significantly associated with acute atherosis in preeclampsia. In preeclamptic pregnancies with acute atherosis, 60% had this combination, compared to 24.5% in those without acute atherosis (p = 0.001). We suggest that interactions between fetal HLA-C2 and activating KIRs on maternal decidual NK-cells or T-cells may contribute to the formation of acute atherosis by promoting local decidual vascular inflammation.

Establishment of a novel HLA genotyping method for preimplantation genetic diagnonis using multiple displacement amplification-polymerase chain reaction-sequencing based technique

To establish a novel HLA genotyping method for preimplantation genetic diagnonis (PGD) using multiple displacement amplification-polymerase chain reaction-sequencing based technique (MDA-PCR-SBT). Peripheral blood samples and 76 1PN, 2PN, 3PN discarded embryos from 9 couples were collected. The alleles of HLA-A, B, DR loci were detected from the MDA product with the PCR-SBT method. The HLA genotypes of the parental peripheral blood samples were analyzed with the same protocol. The genotypes of specific HLA region were evaluated for distinguishing the segregation of haplotypes among the family members, and primary HLA matching was performed between the embryos. The 76 embryos were subjected to MDA and 74 (97.4%) were successfully amplified. For the 34 embryos from the single blastomere group, the amplification rate was 94.1%, and for the 40 embryos in the two blastomeres group, the rate was 100%. The dropout rates for DQ allele and DR allele were 1.3% and 0, respectively. The positive rate for MDA in the single blastomere group was 100%, with the dropout rates for DQ allele and DR allele being 1.5% and 0, respectively. The positive rate of MDA for the two blastomere group was 100%, with the dropout rates for both DQ and DR alleles being 0. The recombination rate of fetal HLA was 20.2% (30/148). Due to the improper classification and abnormal fertilized embryos, the proportion of matched embryos HLA was 20.3% (15/74),which was lower than the theoretical value of 25%. PGD with HLA matching can facilitate creation of a HLA-identical donor (saviour child) for umbilical cord blood or bone marrow stem cells for its affected sibling with a genetic disease. Therefore, preimplantation HLA matching may provide a tool for couples desiring to conceive a potential donor progeny for transplantation for its sibling with a life-threatening disorder.

Maternal–fetal HLA sharing and preeclampsia: variation in effects by seminal fluid exposure in a case–control study of nulliparous women in Iowa

Whereas histocompatibility is critical for transplantation, HLA histoincompatibility is associated with successful pregnancy. Literature on HLA sharing and preeclampsia has been inconsistent; most studies focused on maternal–paternal rather than maternal–fetal sharing. This study examines whether maternal–fetal histocompatibility is associated with preeclampsia, and whether effects vary by semen exposure history. This case–control study of nulliparous women was designed to examine associations among HLA sharing, semen exposure, and preeclampsia. 258 preeclampsia cases and 182 normotensive controls met the eligibility criteria. HLA typing for mother and baby was performed for HLA-A, -B, -C, -DRB1, and -DQB1. We further restricted our study sample to 224 mother–baby pairs who had complete HLA typing for all five genes. Seminal fluid exposure indexes incorporated information on type of practice, frequency, contraceptive use (for vaginal exposure) and ingestion practices (for oral exposure). Multivariate models were adjusted for BMI and education. HLA-A matching, Class I matching, and combined Class I and II matching were associated with increased odds of preeclampsia. Among women with low semen exposure, effects of Class I matching were amplified (HLA-A matching, OR=6.27, 95%CI=1.04, 37.97; Class I matching, OR=4.49 per one-match increase, 95%CI=1.89, 14.50). With moderate to high semen exposure, Class II matching effects predominated (HLA-DQB1, OR=3.22, 95%CI=1.04, 9.99; Class II, OR=1.76 per one-match increase, 95%CI=1.05, 2.98; and total matches, OR=1.45 per one-match increase, 95%CI=1.02, 2.06). We found consistent evidence that maternal–fetal HLA sharing was associated with preeclampsia in a pattern influenced by prior vaginal exposure to paternal seminal fluid.

Non-random mating, parent-of-origin, and maternal–fetal incompatibility effects in schizophrenia

Although the association of common genetic variation in the extended MHC region with schizophrenia is the most significant yet discovered, the MHC region is one of the more complex regions of the human genome, with unusually high gene density and long-range linkage disequilibrium. The statistical test on which the MHC association is based is a relatively simple, additive model which uses logistic regression of SNP genotypes to predict case–control status. However, it is plausible that more complex models underlie this association. Using a well-characterized sample of trios, we evaluated more complex models by looking for evidence for: (a) non-random mating for HLA alleles, schizophrenia risk profiles, and ancestry; (b) parent-of-origin effects for HLA alleles; and (c) maternal–fetal genotype incompatibility in the HLA. We found no evidence for non-random mating in the parents of individuals with schizophrenia in terms of MHC genotypes or schizophrenia risk profile scores. However, there was evidence of non-random mating that appeared mostly to be driven by ancestry. We did not detect over-transmission of HLA alleles to affected offspring via the general TDT test (without regard to parent of origin) or preferential transmission via paternal or maternal inheritance. We evaluated the hypothesis that maternal–fetal HLA incompatibility may increase risk for schizophrenia using eight classical HLA loci. The most significant alleles were in HLA-B, HLA-C, HLA-DQB1, and HLA-DRB1 but none was significant after accounting for multiple comparisons. We did not find evidence to support more complex models of gene action, but statistical power may have been limiting.

Maternal HLA Panel-Reactive Antibodies in Early Gestation Positively Correlate with Chronic Chorioamnionitis: Evidence in Support of the Chronic Nature of Maternal Anti-fetal Rejection

Maternal tolerance of the fetus is essential for viviparity, yet anti-fetal rejection occurs in several pregnancy complications. Chronic chorioamnionitis is a feature of anti-fetal cellular rejection. There is a robust association between chronic chorioamnionitis and maternal seropositivity for anti-human leukocyte antigen (HLA) panel-reactive antibodies (PRA) at the time of delivery. This longitudinal study was performed to assess maternal HLA PRA status in early gestation and the temporal evolution of maternal HLA PRA in the context of chronic chorioamnionitis and, thereby, to determine whether HLA PRA during the course of pregnancy is useful for the detection of anti-fetal rejection. Maternal sera obtained before 16 weeks of gestation and at delivery were analyzed for HLA PRA in cases with (N = 100) and without (N = 150) chronic chorioamnionitis. IgG (but not IgM) HLA class I and II PRA positivity at delivery was higher in cases with chronic chorioamnionitis than in those without chronic chorioamnionitis. IgG HLA class I PRA positivity before 16 weeks of gestation was higher in cases with chronic chorioamnionitis than in those without (30.3 versus 13.3%; P = 0.001). Positive conversion (negative HLA PRA before 16 weeks of gestation but positive at delivery) of IgG HLA class I and II PRA was significantly associated with chronic chorioamnionitis. Fetal HLA class I antigen-specific antibodies were confirmed in 12 of 16 mothers tested who were sensitized to HLA class I antigens before 16 weeks of gestation. Positive maternal HLA PRA before 16 weeks of gestation and the temporal evolution of maternal HLA PRA are associated with the presence of chronic chorioamnionitis at the time of delivery. Maternal IgG HLA PRA has the potential to be a monitoring tool of anti-fetal rejection. Furthermore, the findings herein indicate that subsets of fetuses are exposed to alloimmune HLA antibodies for months, especially in cases with chronic chorioamnionitis.

The changing maternal “self” hypothesis: A mechanism for maternal tolerance of the fetus

Recent advances in placental biology and immunology lead us to propose a novel hypothesis for maternal tolerance of the semi-allogeneic fetus and amelioration of rheumatoid arthritis (RA) during pregnancy. The initial event in this hypothesis is extrusion of placental apoptotic syncytiotrophoblast debris recently identified to contain intracellular fetal HLA Class II molecules, into maternal blood. The second event is uptake of apoptotic syncytiotrophoblast by immature maternal dendritic cells and presentation of fetal HLA class II peptides. In addition to presenting foreign antigens, HLA molecules also present HLA self-peptides. In the setting of the non-inflammatory environment of pregnancy, this process is expected to induce peripheral tolerance of fetal antigens through T cell death, anergy or induction of regulatory T cells in the lymph nodes. This hypothesis suggests a mechanism by which the simultaneous presentation of fetal and (RA-associated) HLA peptides by tolerogenic dendritic cells during pregnancy may explain the observed amelioration of RA as a secondary benefit of fetal tolerance. After delivery, apoptotic syncytiotrophoblast debris disappears from maternal blood, autoimmunity returns and RA recurs. Thus, during pregnancy maternal immunologic self includes fetal HLA Class II as a result of apoptotic syncytiotrophoblast uptake by maternal tolerogenic dendritic cells.

The Changing Maternal “Self” Hypothesis: A Mechanism for Maternal Tolerance of the Fetus

Recent advances in placental biology and immunology lead us to propose a novel hypothesis for maternal tolerance of the semi-allogeneic fetus and amelioration of rheumatoid arthritis (RA) during pregnancy. The initial event in this hypothesis is extrusion of placental apoptotic syncytiotrophoblast debris recently identified to contain intracellular fetal HLA Class II molecules, into maternal blood. The second event is uptake of apoptotic syncytiotrophoblast by immature maternal dendritic cells and presentation of fetal HLA class II peptides. In addition to presenting foreign antigens, HLA molecules also present HLA self-peptides. In the setting of the non-inflammatory environment of pregnancy, this process is expected to induce peripheral tolerance of fetal antigens through T cell death, anergy or induction of regulatory T cells in the lymph nodes. This hypothesis suggests a mechanism by which the simultaneous presentation of fetal and self (RA-associated) HLA peptides by tolerogenic dendritic cells during pregnancy may explain the observed amelioration of RA as a secondary benefit of fetal tolerance. After delivery, apoptotic syncytiotrophoblast debris disappears from maternal blood, autoimmunity returns and RA recurs. Thus, during pregnancy maternal immunologic “self” includes fetal HLA Class II as a result of apoptotic syncytiotrophoblast uptake by maternal tolerogenic dendritic cells.

Prolonged Preterm Rupture of Fetal Membranes, a Consequence of an Increased Maternal Anti-fetal T Cell Responsiveness

A fetus, although semi-allogeneic, is usually accepted by the maternal immune system. However, complications, including alloresponsive mechanisms, are thought to be potentially detrimental for a successful pregnancy. Therefore, we compared allogeneic T cell responses of nonpregnant women with the response of healthy pregnant women and pregnant women who have various gestation-associated diseases. Peripheral blood mononuclear cells (PBMCs) of all three groups were stimulated with PBMCs from unrelated volunteers. Pregnant women had significantly reduced stimulation indices (SIs) compared with nonpregnant women. Exposing PBMCs from pregnant women to PBMCs of their own fetus led to a further significant decrease of SIs. Among the two groups of pregnant individuals, SIs of women with prolonged preterm rupture of fetal membranes (PPROM) were significantly higher when the maternal PBMCs were stimulated with PBMCs of their own fetus. This phenomenon could not be observed after stimulation with PBMCs from unrelated volunteers. In addition, an increased humoral immune response was assessed for women with PPROM in comparison with women with uncontrollable preterm labor. Our results revealed a strongly reduced allogeneic T cell response of PBMCs from pregnant women that was further down-regulated when PBMCs from their own fetus were used as stimulators. By contrast, data from women with PPROM suggest an increased maternal T cell response specifically toward the fetal HLA antigens.

Invited commentary

Invited commentary

Villitis of unknown etiology as a placental counterpart of transplantation rejection: The demonstration of cd8+ T lymphocyte and NK cell infiltration in this lesion

Human pregnancy is characterized by a transient suppression of the adaptive immune response, which is thought to allow survival of the semi-allograft. Villitis of unknown etiology (VUE) is a placental lesion in which there is destruction of chorionic villi and subsequent exposure of fetal cellular HLA (HLA-DR, -DP, -DQ) molecules to maternal leukocytes. This lesion mimics the pathologic features of allograft rejection, in which CD8+ cytotoxic T cells and NK cells are frequently found. This study was conducted to characterize the immunophenotypic profile of leukocytic infiltrates in VUE, with special emphasis on the presence of CD8+ T cells and NK cells.

Fetal HLA typing in β thalassaemia: implications for haemopoietic stem-cell transplantation

Stem-cell transplantation can cure beta thalassaemia. We aimed to assess whether fetal HLA typing done early in the pregnancy of couples who were at risk of beta thalassaemia could provide an alternative to pregnancy termination if the prospect of a bone-marrow transplantation from a family member was available. In our clinic in Sardinia, we did fetal HLA typing for 49 couples at risk of having a baby with beta thalassaemia. Two affected children were born and successfully received a transplantation from a family donor. Five non-affected fetuses were HLA compatible with an affected sibling and their cord blood was harvested for a future transplantation.

Prenatal T-cell reconstitution after in utero transplantation with fetal liver cells in a patient with X-linked severe combined immunodeficiency

Objective: Fetuses with severe combined immunodeficiency may be treated with intrauterine transplantation of fetal hematopoietic stem cells. In previous reports on intrauterine transplantation with T-cell-depleted bone marrow, repeated injections have led to partial immunoreconstitution at birth, with subnormal T-cell counts and a delayed response to mitogens. Study Design: A male fetus with X-linked severe combined immunodeficiency because of a stop mutation in the gene encoding the common γ chain of cytokine receptors was transplanted in week 14 of gestation with a single injection of 7 × 107 cryopreserved nucleated fetal liver cells (9 × 108 cells per estimated kilogram fetal weight) into the fetal abdomen. At 24 and 33 weeks of gestational age, fetal blood samples were taken to detect evidence of engraftment. Fetal mixed chimerism was determined using polymerase chain reaction amplification of a variable number of tandem repeats and was verified by genomic HLA class II typing and flow cytometry. Results: The course of pregnancy, delivery, and the first 18 months of life have been uncomplicated. At week 24 of gestation, donor HLA class II alleles were detected at a low level in the background of the recipient's fetal HLA genotype. The chimeric proportion of donor cells was about 10% at 24 weeks of gestation, increasing to 50% at 33 weeks of gestation. Whereas the T-cell fraction was still markedly reduced in week 24, it increased thereafter and was in the normal range from week 33 of gestation. In vitro response to T-cell mitogens was normal from birth. Conclusion: In utero transplantation of cryopreserved fetal liver cells in week 14 of gestation with a single injection led to complete T- and NK-cell reconstitution at birth. Signs of engraftment were found already in week 24 of gestation. We consider intrauterine transplantation a valuable experimental method and a useful adjunct to postnatal transplantation and gene therapy in the treatment of severe combined immunodeficiency. (Am J Obstet Gynecol 2002;187:475-82.)

Prenatal HLA genotyping of uncultured amniotic fluid samples contaminated with maternal blood

Objective: Allogenic transplantation of umbilical cord blood (UCB) from HLA-identical siblings is a therapeutic concept of increasing importance. Prenatal HLA typing results can provide important information as to whether the UCB should be collected. Therefore, we tested the suitability of two methods based on polymerase chain reaction (PCR) for low-resolution HLA genotyping of uncultured amniocytes contaminated with maternal blood. Study Design: Amniotic fluid samples (~10 mL, n = 4) were collected and divided into 5 equal sized portions. Subsequently, maternal blood was added to produce a series of varying degrees of artificial contamination ranging from 0% to 5% (vol/vol). Corresponding maternal blood and cord blood samples were collected and served as reference material. After DNA preparation, all samples were subjected to both PCR-SSP (sequence-specific primers) and PCR-SSO (sequence-specific oligonucleotides) typing procedures, designed for low-resolution typing of the highly polymorphic HLA-B locus. Results: Both PCR tests were able to accurately predict the fetal HLA-B type when pure amniotic fluid was used in all cases. The PCR-SSP method could still determine the fetal HLA type at 0.1% contamination but not at levels above this; in contrast, PCR-SSO failed if any degree of contamination was present. Conclusion: Fetal cells from amniotic fluid represent a reasonable source for prenatal testing of the fetal HLA genotype with either the PCR-SSP or the PCR-SSO method. Low levels of contamination with maternal blood in the amniotic fluid are tolerated by the more robust PCR-SSP method. In contrast, the PCR-SSO procedure is more sensitive to any degree of contamination, but it is the method of choice if the amount of DNA is limited because of the very low volume of specimens of amniotic fluid available. (Am J Obstet Gynecol 2002;186:1366-71.)

Prenatal HLA typing of uncultured amniocytes prior to the collection of related allogeneic cord blood.

DNA samples isolated from corresponding uncultured amniotic fluid, cord blood and maternal blood (n=5) were subjected to low resolution typing of the HLA-A, -B and -DRB loci by the polymerase chain reaction using sequence-specific primers (PCR-SSP). Furthermore, the effect of ethylene diamine tetraacetate disodium salt (EDTA) on the quality of genomic DNA isolated from amniotic fluid samples after long-term storage was evaluated. Unambiguous results of HLA typing could be achieved from all amniotic fluid samples stabilized with EDTA. PCR-SSP typing failed in DNA samples from amniotic fluid without the addition of EDTA. In all cases the fetal HLA type could be confirmed by the result from the corresponding cord blood typing. Contamination with maternal DNA led to additional weak PCR-SSP bands in one case, but data interpretation was still unambiguous. Reliable fetal HLA typing can be achieved directly from amniotic fluid and culturing of amniocytes is not required.

Lupus nephritis and the anti-phospholipid antibody syndrome in pregnancy

Lupus nephritis and the anti-phospholipid antibody syndrome in pregnancy

Pregnancy, HLA allogeneic challenge, and implications for AIDS etiology

Because of the large HLA genetic polymorphism, a human fetus usually has several paternal HLA antigens allogeneic to its mother. The maternal γ-immunoglobulin (IgG) antibody response to fetal HLA alloantigens is noncytotoxic and associated with local suppression of maternal cell-mediated immunity (CMI) at the maternal–fetal interface. When mother and fetus are syngeneic for most HLA antigens, an increased risk exists for a maternal anti-placental cytotoxic CMI responses, compromising fetal survival. Local suppression of maternal CMI by an anti-HLA IgG response may have evolved to protect the fetoplacental unit from a maternal CMI cytotoxic reaction against expressed developmental neoantigens. A negative aspect of this adaptive response is that infectious organisms bearing HLA-homologous alloantigens (e.g. human immunodeficiency virus type 1, HIV-1) may generate a systemic IgG response suppressing CMI. Findings are reviewed suggesting this is an etiologic factor in the acquired immune deficiency syndrome (AIDS).

Immunosuppressive properties of plasma and lymphocytes from the human retroplacental blood.

The retroplacental compartment in which maternal blood is in direct and continuous contact with trophoblasts derived from the fetus seems to be the place where maternal and fetal cells regulate each others' activities. To assess the role of the retroplacental compartment in the immune regulation of pregnancy, 10 healthy pregnant women were selected for evaluation of the immune activity of their lymphocytes and plasma from the retroplacental blood. The results showed that the proliferation and cytotoxic capacity of these lymphocytes, but not of maternal peripheral lymphocytes, against fetal cells were significantly inhibited in undirect mixed lymphocyte culture and direct cell-mediated cytotoxic assay, respectively. The plasma from retroplacental blood showed significant immunosuppressive properties and depressed the proliferation of maternal peripheral lymphocytes stimulated by fetal HLA as well as the cytotoxic activity of these cells against the fetal lymphocytes. The present data suggest that the retroplacental compartment seems to be an immunosuppressive barrier, protecting the fetus from maternal rejection.

Isolating fetal cells in the maternal circulation.

Fetal cells exist in maternal blood and can be utilized for prenatal genetic diagnosis. The use of polymerase chain reaction (PCR) technology on maternal blood has enabled the detection of fetal sex, Mendelian disorders (e.g. beta-globin mutations), HLA polymorphisms and fetal Rhesus (D) blood type. Enrichment for erythroblasts and trophoblasts by various density gradient and flow sorting techniques followed by fluorescence in-situ hybridization (FISH) with chromosome-specific DNA probes has allowed detection of trisomy 21, trisomy 18, Klinefelter syndrome (47,XXY) and 47,XYY. The fetal cell type that has generated the most success is the nucleated erythrocyte; however, trophoblasts, lymphocytes and granulocytes are also considered to be present in maternal blood. Fetal cells circulate in maternal blood during the first and second trimesters, with frequency increasing as gestation advances. Emphasis is thus now directed toward determining the most practical and efficacious manner for this technique to be applied to prenatal genetic diagnosis. A large scale collaboration of clinical evaluations is underway in the USA, upon completion of which assessment can be made of whether this technology can serve as an alternative to conventional invasive and non-invasive methods of prenatal cytogenetic diagnosis.