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.

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.

Placental HLA-G protein expression in vivo: where and what for?

In contrast to HLA-A and -B class Ia genes that are down-regulated in human trophoblast cells, HLA-G class Ib molecules are expressed in the placenta throughout gestation. In addition to extravillous cytotrophoblast that invade the decidua basalis essentially, HLA-G was also observed in endothelial cells of fetal vessels in the chorionic villi as well as in amnion cells and amniotic fluid. Both membrane-bound and soluble HLA-G isoforms have been detected. In view of the recently published functional data showing that HLA-G: (i) has the capability to bind and present peptides; (ii) is recognized by at least three different killing inhibitory receptors; and (iii) is a regulator of HLA-E expression, we can predict that such functions are likely to be exerted by extravillous cytotrophoblast. Of particular importance will be the anti-viral function of HLA-G at this materno-fetal interface, knowing that HLA-G was shown to be expressed by thymic medullary epithelial cells. In addition to these immunological functions, due to its presence on chorionic fetal endothelial cells, we hypothesize that HLA-G could also be a regulator of chorionic villous angiogenesis. Finally, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy.

Cutting edge: soluble HLA-G1 triggers CD95/CD95 ligand-mediated apoptosis in activated CD8+ cells by interacting with CD8.

The nonpolymorphic soluble HLA-G1 (sHLA-G1) isoform has been reported to be secreted by trophoblast cells at the materno-fetal interface, suggesting that it may act as immunomodulator during pregnancy. In this paper, we report that affinity-purified beta2-microglobulin-associated sHLA-G1 triggered apoptosis in activated, but not resting CD8+ peripheral blood cells. We demonstrate by Western blotting that sHLA-G1 enhanced CD95 ligand expression in activated CD8+ cells. Cytotoxicity was inhibited by preincubation of the cells with a CD95 antagonist mAb (ZB4) or a soluble recombinant CD95-Fc, indicating that apoptosis is mediated through the CD95/CD95 ligand pathway. Finally, we show that such sHLA-G1-induced apoptosis depends on the interaction with CD8 molecules, with cell death being blocked by various CD8 mAbs.

Absence of imprinting of HLA class Ia genes leads to co-expression of biparental alleles on term human trophoblast cells upon IFN-gamma induction.

Human trophoblast cells have developed various efficient regulatory mechanisms to prevent cell surface expression of the classical HLA-A, -B, and (but not always) -C class I molecules. This allows them to escape maternal alloimmune attack during pregnancy. However, recent results have demonstrated that such a lack of expression could be reversed in villous cytotrophoblast cells purified from term placenta by in vitro IFN-gamma treatment. In this context, we investigated whether both maternal and paternal HLA class Ia antigens were co-dominantly expressed in such trophoblast cells. Using polymerase chain reaction sequence-specific primers for HLA-A and HLA-C alleles, we detected transcripts of both paternal and maternal origins, showing that these genes were not affected by genomic imprinting, at least in term placenta. After in vitro IFN-gamma treatment, the polymorphic HLA-A and HLA-B antigens of both parental origins become detectable at the cell surface, as assessed by flow cytometry and/or complement-dependent microtoxicity test. Appearance of paternal antigens on trophoblast cells upon IFN-gamma induction raises the question of the in vivo biological consequences of this phenomena, in term of materno-fetal tolerance and in particular of a potential allogeneic cytotoxic immune response.

Soluble HLA-G: purification from eukaryotic transfected cells and detection by a specific ELISA.

PROBLEM: The detection of soluble forms of human leukocyte antigen-G molecule (sHLA-G) at the maternal-fetal interface suggest that sHLA-G may play a role during pregnancy. To study the potential functions of sHLA-G, we developed a procedure to detect and produce such HLA-G isoforms. METHOD OF STUDY: Transfected cell lines expressing either sHLA-G1s cDNA (JAR-G1s) or an sHLA-G monochain DNA (Fox-G-mono) containing extracellular domains of HLA-G linked to the human beta 2-microglobulin were used. Specific sHLA-G enzyme-linked immunosorbent assay (ELISA), using anti-HLA-G monoclonal antibodies (mAbs) (87G and BFL.1) as coating antibodies and the biotinylated HLA class I mAb, W6/32, to reveal the bound molecules, was then developed. RESULTS: To assess the specificity of the ELISA, we tested cell culture supernatants from the trophoblast-derived JEG-3 cell line and the HLA-G1s-transfected JAR cells, and we detected sHLA-G in both supernatants. sHLA-G monochain was also detected by ELISA in transfected cell supernatants using the conformational mAb, W6/32, showing that the conformation of sHLA-G monochain was proper. Using the same ELISA, sHLA-G was detected in various samples of amniotic fluid. To test the potential role of sHLA-G, sHLA-G has been purified by immunoaffinity columns, using W6/32 mAb, from culture supernatants of HLA-G1s or sHLA-G monochain-transfected cells. CONCLUSION: These important tools will be useful both for the detection of sHLA-G in various biological fluids and in functional tests.

Comparative reactivity of different HLA-G monoclonal antibodies to soluble HLA-G molecules.

Different HLA-G monoclonal antibodies (mAbs) were first evaluated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified beta2-microglobulin (beta2m)-associated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture supernatants from transfected cells. By comparison, the anti-HLA class I mAb 90 did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs, sHLA-G was identified in amniotic fluids as well as in culture supernatants of first trimester and term placental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intron 4-specific mAb (16G1). Reactivity of these different HLA-G mAbs was then compared to determine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and flow cytometry analysis, we showed that they did not compete with each other, which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the alpha1 domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, therefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.