Soluble HLA-G isoforms: technical deficiencies lead to misinterpretations.

The recent report authored by Blaschitz et al. (in press) would lead the reader to believe that human trophoblast cells cannot synthesize or secrete any of the soluble isoforms derived from splice variants of the HLA-G message. They propose that all of the soluble HLA-G presumed to be produced in trophoblast cells is, in fact, cleaved membrane HLA-G1. An interesting idea. Yet several published works have presented solid, well documented evidence that argues against this position. The original article describing the molecular structure of soluble HLA-G (Fujii et al., 1994) showed that soluble protein encoded by the intron 4-containing mRNA could be clearly distinguished from membrane HLA-G1 by isoelectric focusing (IEF). Further, a class I protein (reactive with W6/32) with an IEF pattern completely overlapping with soluble HLA-G from transfected cells was demonstrated in JEG-3 choriocarcinoma cells. Also in that study, PCR primers specific for soluble HLA-G-encoding mRNA were clearly described and used to confirm the protein work. In 2002, Solier and colleagues showed that soluble, intron-4 retaining HLA-G1 (now known as HLA-G5) mRNA is present in villous cytotrophoblast cells and that the HLA-G5 protein is secreted by the cells (Solier et al., 2002). In 2003, a team working in the Geraghty laboratory reported that membrane HLA-G1 is essentially absent in placentas, whereas HLA-G5 is abundant in both villous cytotrophoblast cells and syncytiotrophoblast (Ishitani et al., 2003). HLA-G1 was identified only in migrating extravillous cytotrophoblast cells using the highly specific o1G monoclonal antibody. Also in that study, the soluble HLA-G1 specific antibody 16G1 was used to isolate protein directly from term placentas. Bound peptide was isolated from this complex and shown to have a nearly identical profile to peptide isolated from membrane HLA-G1, which was also isolated from placentas. These peptides collectively matched very precisely the peptide binding motif previously described for HLA-G (some peptides were identical), and that work left no doubt as to the source of that peptide by N-terminal sequencing of the heavy chain (Lee et al., 1995). Taken together, these facts leave no doubt that 16G1 does bind HLA-G produced in vivo, and can only be reconciled with the conclusions made by Blaschitz study if one posits that 16G1 instead binds membrane HLA-G1. That conclusion in turn contradicts several other studies including some of the results presented in the Blaschitz et al. paper (Blaschitz et al., in press). At the next level of study alternative HLA-G protein forms were investigated. Newly generated, fully characterized, isoform-specific monoclonal antibodies developed in the Hunt laboratory demonstrated that villous cytotrophoblast cells contain HLA-G5 and HLA-G6 mRNA but express only HLA-G5 protein (Morales et al., 2003). By contrast, expression of HLA-G2/G6 protein was identified exclusively in extravillous cytotrophoblast cells. Despite this strong and consistent evidence put forth by three independent laboratories, it is always important to critically examine new evidence contradicting previously held conclusions. Taking the major features of the Blaschitz paper one at a time, let us look at the evidence they present to support their conclusions.