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

107

101

The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with β2-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8α expression on PBMC without changing CD3δ expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.

Section: Discussioncontrasting

confidence: 99%

Section: Generation and Biochemical Characterization Of Eukaryotic Rsmentioning

confidence: 98%

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

Morales¹,

Pace

Platt³

et al. 2003

107

101

“…There is considerable debate about the function of the truncated isoforms and in particular whether they are expressed on the cell surface. Riteau et al (26) have reported that HLA-G2, -G3, and -G4 can be expressed on the surface of transfected cells, thereby protecting these cells from lysis by both NK and CTL effector cells, and in this way may contribute to fetal survival (27) However, we (28) and others (25,29) have found that although these truncated forms may be expressed inside the cell, there is no evidence for them reaching the cell surface. Because no mAbs specific for these isoforms are currently available, it is not possible to determine whether they are expressed on the surface of human embryos and have a functional role.…”

Section: Discussionmentioning

confidence: 65%

Differential Expression of Alternatively Spliced Transcripts of HLA-G in Human Preimplantation Embryos and Inner Cell Masses

Yao

Barlow

Sargent

2005

It has been reported that preimplantation human embryos secrete HLA-G, and the levels may be predictive of their ability to implant. However, it is not known which of the membrane-bound (HLA-G 1–4) and soluble (HLA-G 5–6) alternatively spliced forms are present, nor the developmental stage at which they appear. Therefore, we have investigated HLA-G mRNA isoform expression on single embryos at the two-, four-, six-, and eight-cell, morula, and blastocyst stages. The percentage of embryos expressing each HLA-G isoform mRNA increased with developmental stage, but contrary to expectation, HLA-G5 mRNA was not detected in single two- to eight-cell embryos and was only expressed by 20% of morulae and blastocysts. Similarly, soluble HLA-G6 mRNA was not detected until the blastocyst stage and then in only one-third of embryos. In contrast, labeling with MEM G/9 Ab (specific for HLA-G1 and -G5) was observed in 15 of 20 two- to eight-cell embryos and 5 of 5 blastocysts. This disparity between mRNA and protein may be due to HLA-G protein remaining from maternal oocyte stores produced before embryonic genome activation and brings into question the measurement of soluble HLA-G for clinical evaluation of embryo quality. Although HLA-G is expressed in the preimplantation embryo, later it is primarily expressed in the invasive trophoblast of the placenta rather than the fetus. Therefore, we have investigated whether down-regulation of HLA-G first occurs in the inner cell mass (precursor fetal cells) of the blastocyst and, in support of this concept, have shown the absence HLA-G1 and -G5 protein and mRNA.

“…The transfectants were donated by P. Le Bouteiller (Institut National de Science et Recherche Scientifique, Unité 395, Purpan Hospital, Toulouse, France): JAR-HLA-G1 (JAR-G) was produced by transfection of the full length HLA-G1 cDNA under the control of human CMV promoter as described by Mallet et al (27). JAR-HLA-G1m (JAR-G1m) was produced by transfection of pCDNA3/HLA-G1m plasmid, a gift of Dr. M. LopezBotet (Department of Immunology, University Hospital la Princesa, Madrid, Spain), in which HLA-G leader sequence was modified as follows: the methionyl residue at position 2 was mutated in threonine; therefore, it could not provide a good signal peptide for the expression of HLA-E (28).…”

Section: Cell Lines and Transfectantsmentioning

confidence: 99%

Recognition of Nonclassical HLA Class I Antigens by γδ T Cells During Pregnancy

Barakonyi

Kovács

Mikó

et al. 2002

The healthy trophoblast does not express classical HLA-A and HLA-B products; therefore, an MHC-restricted recognition of trophoblast-presented Ags is unlikely. In the decidua and also in peripheral blood of healthy pregnant women, γδ T cells significantly increase in number. We investigated the possible role of γδ T cells in recognition of trophoblast-presented Ags. PBL and isolated γδ T cells from healthy pregnant women as well as from those at risk for premature pregnancy termination were conjugated to choriocarcinoma cells (JAR) transfected with nonclassical HLA Ags (HLA-E, HLA-G). To investigate the involvement of killer-inhibitory/killer-activatory receptors in trophoblast recognition, we tested the effect of CD94 block on cytotoxic activity of Vδ2+ enriched γδ T cells to HLA-E- and/or HLA-G-transfected targets. Lymphocytes from healthy pregnant women preferentially recognized HLA− choriocarcinoma cells, whereas those from pathologically pregnant patients did not discriminate between HLA+ and HLA− cells. Normal pregnancy Vδ2+ T cells conjugated at a significantly increased rate to HLA-E transfectants, whereas Vδ2+ lymphocytes from pathologically pregnant women did not show a difference between those and HLA− cells. Blocking of the CD94 molecule of Vδ2+ lymphocytes from healthy pregnant women resulted in an increased cytotoxic activity to HLA-E-transfected target cells. These data indicate that Vδ2+ lymphocytes of healthy pregnant women recognize HLA-E on the trophoblast, whereas Vδ1 cells react with other than HLA Ags. In contrast to Vδ2+ lymphocytes from healthy pregnant women, those from women with pathological pregnancies do not recognize HLA-E via their killer-inhibitory receptors and this might account for their high cytotoxic activity.