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Expression of HLA class I molecules in trophoblast cells from various locations in normal human first trimester and term placenta was investigated by immunohistochemistry with a panel of monoclonal antibodies against the heavy chains or complete HLA class I molecules complexed with beta2-microglobulin. These reagents were also employed to distinguish between the products of different HLA class I loci. In addition to previously characterized reagents, a novel monoclonal antibody against HLA-A molecules (TU155) was used. Various choriocarcinoma and transfected cell lines served as controls for the specificities of the monoclonal antibodies. Cells in close contact with maternal cells, such as invading trophoblast cells and cells of the basal plate, expressed beta2-m micro globulin in association with HLA-G and HLA-C heavy chains. These class I heavy chains may also have been present as isolated molecules, although not in each of the cells. In contrast, cells of the chorion laeve exclusively expressed HLA-G, and not HLA-A, -B, or -C antigens. Our data support the often discussed immune protective function and the regulatory function of the HLA-G molecule, during invasion. In addition, by using monoclonal antibodies HCA2 (anti-HLA-A and -G), HC10 (anti-HLA-B and -C), TU149 (anti-HLA-B, -C, and some -A alleles), SFR8-B6 (anti-HLA-Bw6 and some -C), LA45 (some HLA-A and -B), TU48 (anti-HLA-Bw4 and some -A), and TU155 (anti-HLA-A), we show the presence of HLA-C molecules in all extravillous trophoblast cells of the cell columns and in the basal plate; the trophoblast cells of the chorion laeve lack this antigen. The function of this molecule is not clear, although a protective function against natural killer cell activity in the endometrium is postulated.

These results show that the amnion epithelium frequently expresses classical HLA class I molecules as well as HLA-G. The expression of HLA-G antigens on amnion epithelial cells and their presence in the amnion fluid, which is continually ingested by the fetus, may be particularly relevant for the induction of peripheral tolerance.

In the context of implantation and pregnancy, several immunomodulating functions have been attributed to the different HLA-G isoforms. Increasing attention is now being addressed to the actively secreted soluble forms, because they might have a systemic function or could be useful as diagnostic tools. However, the cellular source of secretion, even during pregnancy, where HLA-G expression level is known to be highest, is still under debate. To elucidate the conflicting results, we investigated the isoform distribution in human first trimester and term placentas in situ and in vitro. Results obtained by applying immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR show that (1) all of the alpha1 domain-containing HLA-G isoforms are restrictedly expressed in the extravillous cytotrophoblasts (EVCTs) and very few first-trimester syncytiotrophoblasts, which directly cover cell columns, whereas mesenchymal cells of the villous chorion do not express HLA-G; (2) as demonstrated in western blots, trophoblasts express only the HLA-G1 isoform; (3) HLA-G5 and -G6 transcripts could be detected in human term placenta and isolated first-trimester trophoblasts but levels are extremely low; and (4) conditioned media of primary first-trimester trophoblasts, and the chorion laeve-derived trophoblastic cell line AC1-M59 do contain HLA-G1 fragments shed from the cell surface. Our data provide substantial evidence that none of the intron 4-containing isoforms, the so-called actively secreted, soluble HLA-G5 or -G6, are produced by human trophoblasts in situ or in vitro.

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