[Current debates on immunology of preeclampsia. Report of the sixth international workshop of Reunion Island (Indian Ocean, December 2008)]. / Débats actuels sur l'immunologie de la prééclampsie. Comptes rendus du sixième colloque international de La Réunion (décembre 2008).

Hypertensive disorders of pregnancy (HDP) represent globally 10% of human births and their major complication, preeclampsia, 3 to 5%. The etiology of these HDP remains still uncertain, however major advances have been made these last 25 years. The Sixth International Workshop on Reproductive Immunology, Immunological Tolerance and Immunology of Preeclampsia 2008 celebrated its 10th Anniversary in Reunion-island (French overseas Department in the Indian Ocean). Over this decade, these six workshops have contributed extensively to immunological, epidemiological, anthropological and even vascular debates. The defect of trophoblastic invasion encountered in preeclampsia, intra-uterine growth retardation and to some extend also preterm labour has been understood only at the end of the 1970's. On the other hand, clinical and epidemiological findings at the end of the 20th century permitted to apprehend that "preeclampsia disease of primiparae" may in fact well be the disease of first pregnancies at the level of human couples. Among the important advances, immunology of reproduction is certainly the topic where knowledge has literally exploded in the last decade. This paper relates some major steps in comprehension of this disease and focuses on the interest to follow these immunological works and their new concepts. It seems, at the beginning of the 21st century, that we are possibly closer than ever to understand the etiology of this obstetrical enigma. In this quest, the immunology of reproduction will certainly come out as one of the main players.

Human decidual NK cells: unique phenotype and functional properties -- a review.

Human decidual NK cells are massively recruited at the site of embryonic implantation (decidua basalis). They differ in many ways from their peripheral blood NK cell counterparts in terms of gene expression, phenotype and functionality. The major subpopulation of decidual NK cells is CD56(bright) whereas the minor subset is CD56(dim), contrasting with the peripheral blood NK cells whose major subpopulation is CD56(dim). Decidual NK cell cytolytic function is much reduced despite the presence of several activating receptors and the essential machinery required for lysis. Decidual NK cells produce a number of cytokines that are not normally secreted by peripheral blood NK cells. Human decidual NK cell potential functions at the maternal-fetal interface are not yet clearly established but several hypotheses are being evaluated, including control of extravillous invasion, control of uterine vascular remodeling, and local anti-viral activity.

Less HLA-G expression in Plasmodium falciparum-infected third trimester placentas is associated with more natural killer cells.

During pregnancy, maternal immune tolerance of the fetal semi-allogeneic graft is partly the consequence of extravillous trophoblast HLA-G expression and its interaction with natural killer (NK) cells. Plasmodium falciparum malaria is frequently associated with maternal and fetal complications. Local HLA-G expression and the number of NK cells were evaluated immunohistochemically in P. falciparum-infected and uninfected placentas (15 each) collected in a seasonal malaria-hypoendemic area. In control placentas, HLA-G was almost always expressed in extravillous trophoblast whereas, in infected placentas, it was significantly more weakly expressed in extravillous trophoblast but was also detected in intervillous space macrophages. NK cells were evaluated in intervillous and intravillous spaces and in basal plate. NK cells were always more abundant in basal plate than in intervillous and intravillous spaces in infected or control placentas. For each area, more NK cells were seen in infected than control placentas. These data suggest that HLA-G down-regulation and more NK cells in placentas may be among the mechanisms involved in poor birth outcome associated with P. falciparum infection.

HLA class I chromosomal region, genes, and products: facts and questions.

Among the various areas of recent investigation in the field of human MHC class I antigens, the following have been selected for discussion in this review: (1) classical HLA class I genes: are they ubiquitously expressed?, what are the special features of their polymorphism?, are HLA-C molecules functional?, (2) non-classical HLA class I gene products: how restricted is their tissue distribution?, do they exhibit a little polymorphism?, what is their function, if any? (3) non-HLA genes recently detected in the HLA class I chromosomal region: are some of them involved in immunological function and development?, (4) other novel coding sequences present, or possibly present, in the region: the hemochromatosis gene, grc region and associated tumor suppressor genes, housekeeping genes, human equivalent of the murine H-2M region and Ped gene; (5) transcriptional regulation: are there cis-regulatory elements, including locus control region(s), located elsewhere than in the promoters? are CpG methylation, gene imprinting, chromatin structure, DNA rearrangement also implicated? what are the transcription factors involved and how do they interact with each other? is there HLA class I locus-, allele-, or isoform-specific regulation? is class I gene expression dysregulated in human tumors? The answers to these questions are crucial for the development of the future directions for research.

Identification of pro-EPIL and EPIL peptides translated from insulin-like 4 (INSL4) mRNA in human placenta.

Recently, a new member of the insulin gene superfamily termed insulin-like 4 (INSL4) was identified in the human placenta and uterus. The present study investigated whether placenta translates INSL4 mRNA into a putative peptide named early placenta insulin-like (EPIL). Among antibodies elicited against the C chain of pro-EPIL, one antibody (AB7381) was specifically directed against the C chain 59-88 portion, and among those elicited against the A and B chains of EPIL, one antibody (Ab1661) was directed against the A chain 115-139 and the B chain 23-52 portions. Immunohistochemistry based on antibody 7381 to pro-EPIL and antibody 1661 to EPIL demonstrated that the cytotrophoblast from early placenta preferentially expresses the pro-EPIL peptide, whereas the EPIL peptide is expressed by both the cytotrophoblast and the syncytiotrophoblast. At term, the pro-EPIL peptide was detected in villous cytotrophoblast cells, whereas the EPIL peptide was not detected. Moreover, in vitro experiments performed on term placenta showed that the steady state levels of INLS-4 mRNA in the cytotrophoblast are 10 times (one log unit) lower than in the differentiated villous syncytiotrophoblast cells. Taken together, these findings reveal that expression of EPIL peptides in the villous cytotrophoblast is different from that displayed by the syncytiotrophoblast. Finally, these data are the first demonstration that INSL4 mRNA are translated into pro-EPIL and EPIL peptides.

Is antigen presentation the primary function of HLA-G?

Human histocompatibility leukocyte antigen (HLA)-G is an antigen-presenting molecule. This review discusses the possibility that this might not be its primary function. HLA-G indeed modulates innate immunity by interacting with immunoglobulin-like receptors and by regulating HLA-E expression and its subsequent interaction with CD94/NKG2 receptors. HLA-G also down-modulates both CD8(+) and CD4(+) T-cell responsiveness.

Detection of radiolabelled bone marrow cells bearing surface immunoglobulins by combined autoradiography and immunoperoxidase.

A method is described for the simultaneous detection of radiolabelled bone marrow cells bearing surface immunoglobulins by combined autoradiography and immunoperoxidase. Bone marrow cells from normal CBA mice prelabelled in vivo with 125IUDR or exposed in vitro to [3H] thymidine were incubated with rabbit anti-mouse immunoglobulins under capping conditions, washed, cytocentrifuged and treated with methanol and hydrogen peroxide to destroy endogenous peroxidase. Cells were then covered with peroxidase-conjugated goat anti-rabbit immunoglobulins, washed, treated with diaminobenzidine and hydrogen peroxide and finally covered with autoradiographic stripped film and exposed for different times. Peroxidase-positive cells were typically capped and those radiolabelled had autoradiographic silver grains overlying the nucleus.

Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes.

A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK- (H-2Kk) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (micrometer 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5-20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2Kk molecules at the surface of transformed cells (mean of 350,000 molecules/cell).

Epitopic analysis of detergent-solubilized HLA molecules by solid-phase radioimmunoassay.

A solid-phase radioimmunoanalysis of plasma membrane molecules solubilized in the presence of detergent was developed. From a crude cell lysate, membrane molecules were specifically immobilized by solid-phase adsorbed monoclonal antibodies. Analysis of these molecules was easily performed with radiolabeled monoclonal antibodies of different specificities. This technique was used to analyze the HLA-DR and HLA-A,B,C molecules expressed by RAJI cells. It was possible (a) to determine which epitopes are expressed on the same molecules; (b) to define subsets of HLA molecules according to the epitopes which they express, and (c) to perform quantitative analysis of HLA molecules on a crude cell lysate.

Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques.

Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.

Evidence for a polymorphism of HLA-G gene.

HLA-G gene polymorphism was analyzed by RFLP using seven restriction enzymes and an HLA-G locus-specific probe. Hybridization of 55 DNAs digested with three enzymes (Taq I, Pst I, and Bgl II) revealed two polymorphic bands in each case. RFLP patterns obtained with Taq I and Pst I corresponded to the same allelic polymorphism and differed from the Bgl II polymorphism. Combining both polymorphisms enabled determination of four alleles. Allelic frequencies were calculated: 40% of the subjects tested had allele 1, 36% had allele 2, 22% had allele 3, and 2% had allele 4. Analyzing the complete HLA class I phenotype revealed strong linkage disequilibrium with the HLA-A locus. The polymorphism described is located in the 3' flanking region of the gene. Moreover, extended HLA-A haplotypes were constructed by combining the HLA-G polymorphism with other class-I-sequence polymorphisms.

The HLA-G gene is expressed at a low mRNA level in different human cells and tissues.

Recently, HLA-G transgenic mice were shown to exhibit transgene transcription in several extraembryonic tissues. To determine whether HLA-G mRNAs are also expressed in other human tissues, we have undertaken Northern blot and RT-PCR assays using HLA-G locus-specific probe and primers. These studies demonstrate that the HLA-G gene is transcribed in a variety of cells and adult tissues obtained from different individuals (peripheral blood leukocytes, placenta, skin, spleen, thymus, prostate, testicle, ovary, small intestine, colon, heart, brain, lung, liver, and kidney), as well as in fetal tissues (heart, lung, liver, and kidney). The HLA-G mRNA level observed in most tissues is orders of magnitude lower than the level of classic class I genes in the same tissues. RT-PCR studies have demonstrated that alternative splicing of the HLA-G primary transcript is different from tissue to tissue and could be regulated in a tissue-specific fashion. Sequencing of keratinocyte transcripts has confirmed previous observations: (a) three different alternative splicing transcripts are produced (a full-length transcript, an mRNA lacking exon 3, and a transcript devoid of exon 3 and 4) and (b) HLA-G polymorphism is limited in the coding regions. In view of this wide HLA-G tissue distribution, a new hypothesis dealing with possible HLA-G function is proposed.

The full length HLA-G1 and no other alternative form of HLA-G is expressed at the cell surface of transfected cells.

In contrast to HLA class Ia, the HLA-G class Ib transcripts can be alternativeley spliced to yield several isoforms including four potentially membrane-bound variants, namely HLA-G1, -G2, -G3 and G4. It is so far unclear whether each of these splice variants lacking one or two external domains is properly translated and expressed at the cell surface. We used targeted Enhanced Green Fluorescence Protein (EGFP)-HLA-G fusion cDNA to track HLA-G isoform expression in living murine (L-human beta2m) and human (JAR) transiently transfected cells. It was demonstrated that the four HLA-G1, -G2, -G3, and -G4 isoforms were translated in these transfectants by the means of (i) Western blotting analysis, using an anti-EGFP mAb; (ii) intracellular double labeling flow cytometry analysis, using the EGFP natural fluorescence and phycoerythrin-labeled HCA2 anti-HLA-G mAb; and (iii) immunocytochemistry on isolated acetone fixed transfectants with the use of different anti-HLA-G mAbs. Cell surface flow cytometry analysis using the HCA2 mAb revealed that only the HLA-G1 isoform was expressed as a membrane-bound protein. Two color confocal microscopy performed on fixed, permeabilized cells further showed that the EGFP green fluorescence co-localized with anti-calnexin rhodamine fluorescence in the four HLA-G isoform transfectants but only in HLA-G1 transfectant was the green EGFP fluorescence also detectable at the outer part of the cells, suggesting that the HLA-G2, -G3, and G4 were retained in the endoplasmic reticulum. Such intracellular retention of the three shorter forms of HLA-G suggest that they may play a role in regulating cell surface expression either of the full length HLA-G1 form or of HLA-E.

Transformation of LMTK- cells with purified HLA class I gene. VI. Serological characterization of HLA-B7 and AW24 molecules.

Serological characterization of HLA-B7 and HLA-AW24 class I molecules following transfection of murine LMTK- cells with purified HLA class I genes was performed using human alloantisera. Induction by murine alpha interferon of the expression of class I molecules was required to obtain unambiguous identification of these molecules which appear serologically identical to the HLA-B7 and HLA-AW24 molecules expressed at the surface of human peripheral blood lymphocytes of 20 unrelated individuals. Analysis of the transformed cells with 8 different anti-HLA class I monoclonal antibodies results in the definition of 3 separate clusters of antigenic determinants shared by all HLA class I molecules. These studies further suggest the existence of locus-specific serological reactivities associated either with the HLA-A or with the HLA-B and C gene products.

Acquisition of a stimulatory activity for Vgamma9/Vdelta2 T cells by a Burkitt's lymphoma cell line without loss of HLA class I expression.

Daudi Burkitt's lymphoma cells activate Vgamma9/Vdelta2 T cells through TCR ligation by an unknown antigen. This activity is for a large part revealed by their lack of HLA class I antigen expression, allowing their escape from KIR downregulation. We characterize here a culture variant of the Burkitt's lymphoma line Raji, RJ-A3, which is able to promote as efficiently as Daudi cells the outgrowth of Vgamma9/Vdelta2 T cells in cocultures in spite of unchanged HLA class Ia/Ib antigen expression. RJ-A3 is resistant to lysis by most Vgamma9/Vdelta2 lines and clones, even those lacking CD9-4/NKG2 and p58, p70 p140 KIR molecules. However, one Vgamma9/Vdelta2 line which can efficiently kill RJ-A3 do so in a TCR-dependent manner since killing is modulated by anti-TCR antibodies. The CDR3 sequences of the T cell clones amplified with Daudi and RJ-A3 reveal that some clones can be expanded with both lines while others are expanded preferentially with one or the other but not both. This indicates differences in the antigenic determinants of the two Burkitt's lines. The occurrence of this Raji variant line demonstrates that the stimulatory phenotype for Vgamma9/Vdelta2 cells can be acquired by some tumors independently of the loss of class I antigens and comforts the hypothesis of an anti-tumoral function for the Vgamma9/Vdelta2 T cell population.

Soluble HLA-G1 at the materno-foetal interface--a review.

HLA-G differs from the other MHC class I genes. This includes a unique promoter region, a restricted constitutive tissular distribution, the translation of different membrane-bound and soluble isoforms, a shortened cytoplasmic tail and a minimal polymorphim. Soluble HLA-G1 is an immunosuppressive molecule inducing apoptosis of activated CD8(+) T cells and down-modulating CD4(+) T cell proliferation. Soluble HLA-G1 may also contribute to the control of implantation.

Surface expression of HLA-C antigen by human extravillous trophoblast.

In this paper definitive evidence that the classical class I product, HLA-C, is expressed on the surface of normal trophoblast cells is provided. HLA-C transcripts were sequenced from cDNA isolated from first trimester trophoblast cells obtained by flow cytometric sorting. Both paternal and maternal alleles were transcribed. HLA-C proteins were demonstrated by biochemical analysis and found on the cell surface in association with beta(2)-microglobulin. Upregulation of cell surface HLA-C but not HLA-G expression after interferon (IFN)-gamma treatment was demonstrated by flow cytometric analysis. Immunohistology has confirmed HLA-C is expressed by all extravillous subpopulations in vivo. The question of whether trophoblast HLA-C molecules interact with decidual NK cells expressing killer Ig-like receptors (KIR) has also been addressed. Our results demonstrate that extravillous trophoblast expresses at least two HLA class I molecules, HLA-G and HLA-C on the cell surface.

Primary cultured human thymic epithelial cells express both membrane-bound and soluble HLA-G translated products.

This report demonstrates that both membrane-bound and soluble HLA-G isoforms are present in primary cultured human thymic epithelial cells (TEC). HLA-G transcriptional isoforms have been detected by RT-PCR, using different sets of HLA-G specific primers. A flow cytometry analysis, using two anti-HLA-G mAbs, namely 87G and BFL.1, revealed the presence of HLA-G translated products at the cell surface of a subpopulation of TEC. Finally, it was shown that HLA-G soluble forms were secreted in TEC culture supernatant, using a sandwich ELISA with BFL.1 and W6/32 mAbs. These results confirm and extent those previously described showing that HLA-G expressing cells were detectable by immunohistochemistry in thymic medullary epithelial cells.

Regulation of B cell development in mouse bone marrow.

Cultured bone marrow cells, after in vitro treatment with hydroxyurea (HU) - a DNA synthesis inhibitor which kills cells in the S phase of the cell cycle - generated 40 to 70% more B cells than untreated control cells. This was shown by fluorescent-activated cell sorter analysis of labelled cells using FITC-F(ab')2 rabbit anti-mouse IgM and functional tests with LPS. The maximum increase was reached after 24 hr of incubation with HU while 6 or 2 hr of exposure had less effect. The effect of HU was dose dependent with a maximum at 4 mM. The same increase of B cells was observed with foetal liver cells but not with spleen or lymph node cells after 24 hr of in vitro HU treatment. Dialysed supernatants from HU treated bone marrow, spleen or foetal liver cells were themselves able to augment the B cell maturation in bone marrow cultures (test cells) as compared with supernatants from untreated cells, showing that soluble factors were involved. Preliminary data showed that inhibitory factors for B cell maturation were produced by normal bone marrow, spleen and thymus cells in vitro and their formation was prevented by HU pretreatment or irradiation (2500 R) whereas stimulatory factors were produced by lymph node cells. Cell separation experiments suggested that T cells and/or adherent cells may be involved in the production of these soluble factors. These data suggest that early B cell development may be under homeostatic control.

[HLA-G and local placental immunity]. / HLA-G et immunité placentaire locale.

HLA-G is a non-classical major histocompatibility complex class I gene, exhibiting unique structure and whose tissue distribution is mainly restricted to the placenta. Its function contributes to modulate local placental immunity during pregnancy. Major structural characteristics are as follows: a unique promoter region, distinct from the other MHC class I promoters described to date, mRNA alternatively spliced into a variety of membrane-bound isoforms and two major soluble forms. HLA-G expression in the placenta is found mainly in extravillous cytotrophoblast that invade decidual tissue and maternal spiral arteries as well as villous cytotrophoblast (soluble form). Soluble HLA-G1 isoforms might play an important role in the embryonic implantation. HLA-G was also found to induce apoptosis of activated CD8(+) T cells. HLA-G also modulates cytokine secretion of NK cells upon interaction with specific receptors.