Using four different HLA-G-recognizing monoclonal antibodies (mAb), we investigated whether this nonclassical HLA class I molecule could be expressed in placental cell types other than extravillous cytotrophoblasts (evct) in which HLA-G has already been detected. Immunohistochemical analysis was performed on serial cryosections of first trimester placenta as well as on maternal decidual tissue. In addition to some proliferative evct, the recently described BFL.1 mAb also slightly stained some villous cytotrophoblast (vct) stem cells located near cell columns and cell islands, which until now have been considered as HLA-G negative. The same staining pattern was obtained with the 16G1 mAb raised against the soluble HLA-G isoform, whereas neither 87G nor HCA2 reacted with vct but did strongly label the invasive populations of evct, including interstitial and endovascular trophoblasts. Surprisingly, BFL.1 strongly and reproducibly stained endothelial cells in the fetal capillaries present in the mesenchymal core of the chorionic villi, whereas none of the other surrounding cellular components were stained. The same specific labeling was obtained, although with less intensity, with the three other HLA-G-recognizing mAb. In contrast, maternal endothelial cells present in spiral arteries of the decidua parietalis remained unstained. This unexpected cellular localization suggests that HLA-G may be present as a soluble form during the whole period of fetal vascularization and/or exert a nonimmunological function related to the endothelial cell type, in particular in the angiogenesis process which is highly active, until term, in chorionic villi.

The human MHC class Ib gene HLA-G is transcribed and translated in different placental cell subpopulations during pregnancy. In addition to this restricted tissue distribution, HLA-G proteins were also recently detected in the thymus of HLA-G transgenic mice, as well as in some human thymic epithelial cells (TEC). There was a need to further define the phenotype of the HLA-G-expressing cells in the human thymus as well as the type of translated forms that they produce. Using several HLA-G-specific mAb and immunohistochemistry performed on cryosections of human thymi at different ages, we found that the HLA-G-expressing cells are present on medullary cells exhibiting the epithelial morphological type 6. Co-localization experiments performed by double or triple immunofluorescence staining demonstrate that these HLA-G-expressing cells express various cytokeratins, epithelial cell markers but not the CD83 dendritic cell marker. We further show by ELISA measurements that a subset of primary cultured human TEC also expresses soluble HLA-G. Therefore, HLA-G protein tissue distribution is not restricted solely to placental cells. A subpopulation of medullary TEC also expresses HLA-G both at their cell surface and in secreted form, raising the question of the functional significance of such MHC class Ib molecules. Whether thymic soluble and/or membrane-bound HLA-G contribute to inhibit NK cells or to a negative selection of autoreactive T cells which could be harmful in case of pregnancy and/or to a positive selection of viral peptides/HLA-G-restricted CD8(+) T cells remains to be demonstrated.

In contrast to the highly polymorphic HLA class Ia genes that exhibit a broad somatic tissue distribution, the restricted constitutive expression of HLA-G to trophoblast and a subset of thymic epithelial cells suggests tight transcriptional control of this MHC class Ib gene. Transactivation of MHC class I genes is mediated by three major regulatory modules present in their promoter region namely enhancer A, ISRE, and SXY. The 220-bp promoter sequence of HLA-G comprises modified enhancer A and SXY modules and lacks the ISRE which renders this gene unresponsive to NK-kappaB, IRF1, and class II transactivator DNA-binding factors. A number of other HLA-G upstream regulatory elements have recently been described. Using different transgenic HLA-G mouse models under the control of the HLA-G promoter, several groups have shown by in situ hybridization and/or qualitative or quantitative RT-PCR that constitutive HLA-G transcriptional expression in placental tissue decreased with gestational time. This suggests that once the placenta is fully formed, the functions of HLA-G might not be so crucial.

No abstract

Recent studies of the nonclassical HLA-G class I gene provide insight into its function(s) during pregnancy. The HLA-G gene can be transcribed in different isoforms resulting from alternative splicings and encoding membrane-bound and soluble proteins. These different mRNA species have been found in the various trophoblast cell subpopulations that constitute the maternofetal interface in the human placenta. The raising of antibodies to HLA-G has introduced new tools to determine in which types of trophoblast cells and in which other tissues these transcriptional isoforms are translated in functional proteins. The HLA-G gene exhibits a certain amount of polymorphism, the exon three that encodes the alpha 2 external domain showing the most extensive nucleotide variability. It remains to be determined whether the homozygosity of some HLA-G alleles constitutes a real disadvantage in terms of pregnancy or resistance to specific pathogens. Regarding the potential antigen-presenting function(s) of HLA-G, two isoforms are capable of binding an identical set of nonamer peptides derived from a variety of intracellular proteins. The ligand motif contains three anchor residues and is similar to that of classical HLA class I molecules. Experiments are being performed to identify the recognizing cells and to determine whether HLA-G induces a cytolytic (including anti-viral) T-cell response or in some other way represses natural killer-cell functions.