Detection of soluble HLA‐G molecules in plasma and amniotic fluid

Abstract

Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P<0.05) lower than in HLA-A11 negative ones (mean: 24.5+/-2.0 SEM ng/ml; n=80). sHLA-G levels in women at delivery (mean: 22.9+/-2.2 SEM ng/ml; n=28) were in the range of controls but were significantly (P<0.001) reduced in the corresponding cord bloods (mean: 13.8+/-1.5 SEM ng/ml; n=28). sHLA-G levels in amniotic fluids (mean: 15.5 + 1.0 SEM ng/ml; n=50) were significantly (P<0.001) lower than in plasma. sHLA-G levels were 5 and 11% of those of sHLA-I antigens in plasmas and amniotic fluids, respectively. Individual sHLA-G levels were not correlated with sHLA-I levels. SDS-PAGE analysis of plasma sHLA-G antigens revealed two molecular variants with a 35 kD and a 27 kD MW corresponding to the sizes of sHLA-G1 and -G2 isoforms. In conclusion, our study has shown that the two-step assay we have developed is reliable in measuring sHLA-G antigen levels. This assay will facilitate the analysis of the biological and clinical significance of sHLA-G antigens in plasma.

A Soluble Isoform of the Rhesus Monkey Nonclassical MHC Class I Molecule Mamu-AG Is Expressed in the Placenta and the Testis

“…The immunostaining pattern obtained with 16G1 would suggest that villous cytotrophoblasts, but not syncytiotrophoblasts or Hofbauer cells, are likely to produce free H chain Mamu-AG. Recently, several groups have sought to establish whether soluble HLA-G is present in the serum of pregnant women (15)(16)(17)(18). ELISA-based assays using various Ab capture and detection approaches have indicated the presence of soluble HLA-G not only in the serum of pregnant women, but also in nonpregnant women and in men as well.…”

“…However, it seems unlikely that testicular soluble HLA-G may be the source of circulating serum HLA-G in men, since the patency of the blood-testis barrier would preclude this route of trafficking of the molecule. In addition, the assays used to detect soluble HLA-G typically detect the class I-␤ 2 -microglobulin complex (15)(16)(17)(18), and immunostaining of human testes with 16G1 did not detect soluble HLA-G protein (41). Thus, another site of expression of human soluble HLA-G seems likely.…”

“…Initial studies with an Ab (16G1) raised against the unique C-terminal extension detected the protein within the placenta (14), but not in peripheral lymphocytes. In addition, several groups have now reported assays for detecting soluble HLA-G in peripheral blood and other biological fluids (15)(16)(17)(18). Unexpectedly, soluble HLA-G was detected not only in pregnancy, but also in nonpregnant women as well as males.…”

Human Amniotic Epithelial Cells Possess Hepatocyte-like Characteristics and Functions

“…The amnion has been applied clinically, e.g., in the treatment of burn lesions (Trelford and Trelford-Sauder, 1979), to cover surgical wounds to avoid collusion (Trelford and Trelford-Sauder, 1979), and in ocular surface reconstitution (Koizumi et al, 2000). In addition, human amniotic epithelial (hAE) cells seem to be relatively resistant to rejection even after allotransplantation because of their expression of immunosuppressive factors, such as CD59 (Rooney and Morgan, 1992) and soluble HLA-G (Rebmann et al, 1999). Alternatively, it was suggested that amnion-derived cells may possess multipotency because human amniotic fluid cells contain a minor cell population expressing Oct-4 (Prusa et al, 2003).…”

Human Leukocyte Antigen-G Expression After Heart Transplantation Is Associated With a Reduced Incidence of Rejection

“…[7][8][9] The soluble isoforms HLA-G5 and HLA-G6 have been detected in amniotic fluid and serum of pregnant women. 10,11 HLA-G expression was initially found to be restricted to placenta. More recently, its expression has been detected in thymic epithelial cells, 12 various malignant cells, [13][14][15] and peripheral blood monocytes activated by IL-10.…”