Soluble HLA-G Isoforms Research Articles

HLA-G as a new tumor biomarker: detection of soluble isoforms of HLA-G in the serum and saliva of patients with colorectal cancer.

Recent medical investigations suggest that HLA-G, due to its tolerogenic properties, can be used as a biomarker in the diagnosis, treatment, and prognosis of different neoplasms. This observational prospective pilot study aims at detecting sHLA-G in the serum and saliva of patients diagnosed with colorectal cancer (CRC). For this purpose, we compared the expression of sHLA-G from patients with a control sample from a healthy population. Using the specific enzyme-linked immunosorbent assay (ELISA) method, the expression of sHLA-G in the serum and saliva samples from patients affected by CRC (n = 20) and in a control sample (n = 10) were analyzed. The data showed that in patients with CRC, salivary sHLA-G values were significantly higher than in the control group (18.84U/ml versus 6.3U/ml, p = 0.036). In addition, higher levels of sHLA-G were observed in the saliva of patients with CRC in more advanced stages, compared with patients in early stages (24.2U/ml vs. 8.1U/ml, p = 0.019). A significant correlation was observed between the concentration of sHLA-G in the serum and saliva of the analyzed samples (Spearman correlation 0.7, p = 0.004). This study demonstrates, for the first time, the possibility of detecting sHLA-G in the saliva of patients with CRC, resulting in a less invasive alternative to venipuncture. Likewise, we propose that sHLA-G could be an attractive molecular target based on its significant high levels in advanced stages.

HLA Class Ib Molecules and Immune Cells in Pregnancy and Preeclampsia.

Despite decades of research, the highly prevalent pregnancy complication preeclampsia, “the disease of theories,” has remained an enigma. Indeed, the etiology of preeclampsia is largely unknown. A compiling amount of studies indicates that the pathological basis involves a complex array of genetic predisposition and immunological maladaptation, and that a contribution from the mother, the father, and the fetus is likely to be important. The Human Leukocyte Antigen (HLA)-G is an increasing focus of research in relation to preeclampsia. The HLA-G molecule is primarily expressed by the extravillous trophoblast cells lining the placenta together with the two other HLA class Ib molecules, HLA-E and HLA-F. Soluble isoforms of HLA-G have been detected in the early endometrium, the matured cumulus–oocyte complex, maternal blood of pregnant women, in umbilical cord blood, and lately, in seminal plasma. HLA-G is believed to be involved in modulating immune responses in the context of vascular remodeling during pregnancy as well as in dampening potential harmful immune attacks raised against the semi-allogeneic fetus. In addition, HLA-G genetic variants are associated with both membrane-bound and soluble forms of HLA-G, and, in some studies, with preeclampsia. In this review, a genetic contribution from the mother, the father, and the fetus, together with the presence and function of various immune cells of relevance in pregnancy are reviewed in relation to HLA-G and preeclampsia.

HLA-G gene expression influenced at allelic level in association with end stage renal disease and acute allograft rejection

BackgroundHuman leukocyte antigen (HLA)-G is a non-classical major-histocompatibility complex class-I molecule associated with immunosuppressive function. We have evaluated the impact of HLA-G allele associated with untranslated-region (UTR)-haplotype in end stage renal disease (ESRD) and acute allograft rejection (AR) cases. The mRNA levels of different HLA-G isoforms were evaluated in ESRD and AR cases. Subsequently, the total HLA-G mRNA levels and protein concentration were evaluated against its UTR-haplotype among ESRD and AR cases. MethodologySequence based typing of the promoter region was carried-out to evaluate the impact of HLA-G haplotype in 350 ESRD cases and 300 controls. HLA-G gene expression was evaluated at the transcriptional level using semi-quantitative and quantitative PCR, whereas protein concentration was determined by ELISA among both cases and control. ResultsIncreased risk was observed for G*01:01:01:03, G*01:01:02, G*01:06 and G*01:05:N haplotypes while G*01:01:01:01 and G*01:04:01 haplotypes showed a protective effect in ESRD and AR cases. Higher level of soluble HLA-G isoforms (G5 and G6) was observed among ESRD cases. Reduced levels of soluble isoform (G5) and increased levels of membrane bound (G1 and G3) isoforms were found among AR cases, revealing risk association. Decreased HLA-G expression was observed at both mRNA and protein level for G*01:01:01:03 and G*01:05:N haplotypes in ESRD and AR cases. ConclusionsThese results suggest that the variation in the expression profile of membrane bound and soluble isoforms may modulate the risk for ESRD and AR. UTR-haplotypes appear to be involved in different HLA-G expression patterns at transcriptional and translational levels.

Human leukocyte antigen-G expression in differentiated human airway epithelial cells: lack of modulation by Th2-associated cytokines

BackgroundHuman leukocyte antigen (HLA)-G is a nonclassical class I antigen with immunomodulatory roles including up-regulation of suppressor T regulatory lymphocytes. HLA-G was recently identified as an asthma susceptibility gene, and expression of a soluble isoform, HLA-G5, has been demonstrated in human airway epithelium. Increased presence of HLA-G5 has been demonstrated in bronchoalveolar lavage fluid recovered from patients with mild asthma; this suggests a role for this isoform in modulating airway inflammation though the mechanisms by which this occurs is unclear. Airway inflammation associated with Th2 cytokines such as IL-4 and IL-13 is a principal feature of asthma, but whether these cytokines elicit expression of HLA-G is not known.MethodsWe examined gene and protein expression of both soluble (G5) and membrane-bound (G1) HLA-G isoforms in primary differentiated human airway epithelial cells collected from normal lungs and grown in air-liquid interface culture. Cells were treated with up to 10 ng/ml of either IL-4, IL-5, or IL-13, or 100 ng/ml of the immunomodulatory cytokine IL-10, or 10,000 U/ml of the Th1-associated cytokine interferon-beta, for 24 hr, after which RNA was isolated for evaluation by quantitative PCR and protein was collected for Western blot analysis.ResultsHLA-G5 but not G1 was present in dAEC as demonstrated by quantitative PCR, western blot and confocal microscopy. Neither G5 nor G1 expression was increased by the Th2-associated cytokines IL-4, IL-5 or IL-13 over 24 hr, nor after treatment with IL-10, but was increased 4.5 ± 1.4 fold after treatment with 10,000 U/ml interferon-beta.ConclusionsThese data demonstrate the constitutive expression of a T lymphocyte regulatory molecule in differentiated human airway epithelial cells that is not modulated by Th2-associated cytokines.

Impact of HLA-G in the outcome of vitiligo in Tunisian patients

Background:The human leukocyte antigen (HLA) system in the skin coordinates the pigmentation and immune response and could be implicated in the pathogenesis of vitiligo. Human leukocyte antigen HLA-G is a nonclassic, major histocompatibility complex class I molecule expressed in the extravillous cytotrophoblast at the feto-maternal interface. It is known to protect the fetus from maternal cellular immunity. Analogically, it could be implicated in the pathogenesis of autoimmune diseases such as vitiligo.Aims:To compare the expression of HLA-G between vitiligo patients and healthy controls.Materials and Methods:In the present study, 22 vitiligo patients and 24 healthy controls were investigated to look for a possible correlation between HLA-G expression and this pathology. Expression of HLA-G in cutaneous biopsy specimens was investigated by immunohistochemical analysis.Results:HLA-G was detected in the biopsy specimens of 3 (13%) out of 22 patients. This number was significantly higher in healthy controls 18 (75%) out of 24 as compared to vitiligo patients (P < 0.001).Conclusion:There is significant negative correlation between HLA-G expression and vitiligo. In our mind, upregulation of HLA-G expression in lesional skin could be local (superficial expression) or systemic (soluble HLA-G isoforms) compensation to restore normal pigmentation in lesions.

Isoforms of human leukocyte antigen-G and their inhibitory receptors in human kidney allograft acceptance

Novel therapeutic strategies such as the modulation of dendritic cell and T-cell function have exhibited great potential in clinical transplantation. Human leukocyte antigen (HLA)-G is a molecule that plays a significant role in establishing complex mechanisms to protect semiallogeneic fetuses from rejection by the maternal immune system. The unique characteristics of both cell-surface and soluble isoforms of HLA-G, the formation of disulfide-bonded dimers with the potential to augment inhibitory receptor signaling, and the function of HLA-G as a preferential ligand for the immunoglobulin-like transcript receptors make HLA-G very important in fundamental approaches for the modulation of immune responses to improve allogeneic graft survival in clinical transplantation. Experimental data from several groups as well as our data from experiments involving HLA-G-mediated human tolerogenic dendritic cells in vitro and receptor transgenic mice in vivo indicate that different isoforms of HLA-G have various immunomodulatory effects through the inhibitory receptors. This knowledge is crucial in understanding mechanisms of prolongation of allograft survival. The analyses of HLA-G isoforms and inhibitory receptors in patients with kidney allograft and the relationship among different isoforms of HLA-G, inhibitory receptors, their mediated immunoregulation, and graft acceptance or failure will be discussed here.

Expression of HLA-G in the skin of patients with pemphigus vulgaris.

Studies on HLA-G, a nonpolymorphic antigen of non-classical HLA class I, is of basic and clinical significance. In the present study, the expression of HLA-G proteins in the human skin tissue sections of normal and autoimmune pemphigus vulgaris (PV) individuals were investigated using monoclonal antibodies. The antibodies recognized both membrane-bound and soluble isoforms of HLA-G. RT-PCR was performed to assess the patterns of HLA-G mRNA transcripts in the epidermal cells of PV and normal subjects. HLA-G expression could only be detected at transcriptional level in normal skin tissues. However cells derived from PV subjects expressed detectable HLA-G molecules at both transcriptional and translational levels. In addition, the RT-PCR patterns of HLA-G amplification revealed a reduction in HLA-G2 and an increase in HLA-G1 transcripts in epidermal cells of PV patients as compared to normal cells. These observations further support suggestions in the literature regarding the role of HLA-G in induction of tolerance in autoimmune individuals.

Human Neuroblastoma Cells Trigger an Immunosuppressive Program in Monocytes by Stimulating Soluble HLA-G Release

HLA-G is overexpressed in different tumors and plays a role in immune escape. Because no information is available on HLA-G in relation to human neuroblastoma, we have investigated the expression of membrane-bound and secretion of soluble isoforms of HLA-G in neuroblastoma and functionally characterized their immunosuppressive activities. At diagnosis, serum soluble HLA-G (sHLA-G) levels were significantly higher in patients than in age-matched healthy subjects. In addition, patients who subsequently relapsed exhibited higher sHLA-G levels than those who remained in remission. Neuroblastoma patient sera selected according to high sHLA-G concentrations inhibited natural killer (NK) cell and CTL-mediated neuroblastoma cell lysis. Such lysis was partially restored by serum depletion of sHLA-G. In 6 of 12 human neuroblastoma cell lines, low HLA-G surface expression was not up-regulated by IFN-gamma. Only the ACN cell line secreted constitutively sHLA-G. IFN-gamma induced de novo sHLA-G secretion by LAN-5 and SHSY5Y cells and enhanced that by ACN cells. Primary tumor lesions from neuroblastoma patients tested negative for HLA-G. Neuroblastoma patients displayed a higher number of sHLA-G-secreting monocytes than healthy controls. Incubation of monocytes from normal donors with IFN-gamma or pooled neuroblastoma cell line supernatants significantly increased the proportion of sHLA-G-secreting cells. In addition, tumor cell supernatants up-regulated monocyte expression of CD68, HLA-DR, CD69, and CD71 and down-regulated IL-12 production. Our conclusions are the following: (a) sHLA-G serum levels are increased in neuroblastoma patients and correlate with relapse, (b) sHLA-G is secreted by monocytes activated by tumor cells rather than by tumor cells themselves, and (c) sHLA-G dampens anti-neuroblastoma immune responses.

Quantifying soluble HLA-G in supernatants of cultured embryos as a marker of implantation potential in an assisted reproduction program

HLA-G plays a tolerogenic function at the maternal-fetal interface. Detection of the soluble isoforms of HLA-G (sHLA-G) in culture medium derived from embryos grown in vitro, although technically complex, has gained interest in assisted reproduction programs because of an apparent relationship with embryo competence. Here, an amplified ELISA was designed to measure sHLA-G at the concentrations expected in embryo supernatants using a limiting-dilution assay of the choriocarcinoma JEG-3 cell line as a surrogate model. With this ELISA approach, 111 single embryo culture supernatants, collected 44-48 hours after intracytoplasmic sperm injection, were retrospectively analysed and levels correlated with pregnancy results. The presence of sHLA-G was demonstrated in 22 (19.8%) of the embryo cultures. There was no relationship between sHLA-G levels and grading of embryo morphology. The reproductive outcome of the morphologically normal embryos that were transferred to the women uterus (2-3 per patient) was as follows: in the group of women in which all transferred embryos were sHLA-G negative, the pregnancy and implantation rates were 29% (4 pregnancies/14 women) and 14% (5 gestational sacs/35 embryos transferred), respectively. In contrast, in the group in which the embryo transfers included at least one sHLA-G positive the pregnancy and implantation rates increased to 60% (3/5) and 29% c(4/14) respectively. In conclusion, sHLA-G levels in preimplantation embryo supernatants can be quantified and results suggest positive association with pregnancy likelihood. sHLA-G detection seems to be useful to complement morphology in selecting good quality embryos for increasing implantation rates and reducing multiple gestations.

Stranger in a strange land

Mammalian mothers and their embryos/fetuses are almost invariably genetically different, which raises the question of how the mother's immune system is diverted so as to permit cohabitation with the 'foreign' body. Several decades of research have shown that multiple cooperative systems sanction uteroplacental immune privilege. These systems include production of several varieties of soluble immunosuppressive molecules in the uterus and the placenta and strict regulation of the molecules expressed on or by placental trophoblast cells. Trophoblast, a unique lineage without counterpart in adult tissues, is in direct contact with maternal blood and tissue. The major graft rejection-promoting molecules, human leukocyte antigens (HLAs), are tightly regulated in these cells, with none of HLA-A, HLA-B, or HLA class II antigens expressed. The HLA class Ib antigens, HLA-E, HLA-F, and HLA-G, are detectable on some subpopulations. Our studies have focused on the expression, regulation, and functions of the soluble isoforms of HLA-G, which circulate in maternal blood and are present at high levels in the pregnant uterus. These isoforms are derived from the single HLA-G gene by alternative splicing and are now known to have immunosuppressive properties. Ours and other studies indicate that soluble HLA-G proteins may comprise a unique tolerogenic system for establishing local immune privilege during pregnancy.

Recombinant HLA-G5 and -G6 drive U937 myelomonocytic cell production of TGF-beta1.

Throughout human pregnancy, activated maternal macrophages producing anti-inflammatory cytokines comprise a stable cell population in the uterus. This organ is also massively infiltrated with semiallogeneic, placenta-derived, invasive cytotrophoblast cells, which produce membrane and soluble isoforms of human leukocyte antigen (HLA)-G. Here, we investigated the possibility that two soluble isoforms of HLA-G, HLA-G5 and -G6, program macrophage production of cytokines. The model system consisted of human U937 myelomonocytic cells treated with phorbol 12-myristate 13-acetate (PMA) and interferon-gamma (IFN-gamma), which induced differentiation and activation but did not affect their viability or decrease their expression of the two inhibitory immunoglobulin-like transcript (ILT) receptors for HLA-G, ILT2 and ILT4. Exposure of the PMA/IFN-gamma-treated U937 cells to increasing concentrations of recombinant HLA-G5 or -G6 (rG5 and rG6) stimulated effects common to the two isoforms. High doses of both significantly decreased interleukin (IL)-10 and dramatically increased transforming growth factor-beta1. Differential effectiveness between the isoforms was demonstrated in dose-response studies, as was differential binding to ILT2 and ILT4 in receptor-blocking studies. No effects on production of IL-4, IL-1 receptor antagonist, IL-15, tumor necrosis factor alpha, IL-1beta, or IL-6 were observed. Collectively, the results are consistent with the postulate that environmental programming of decidual macrophages may be dictated in part by their proximity to soluble HLA-G-producing fetal cytotrophoblast cells.

Immunogenicity of the soluble isoforms of HLA-G.

Soluble class Ib HLA-G glycoproteins synthesized in the placenta are abundant in the pregnant uterus and circulate in maternal blood throughout pregnancy. To establish immunogenicity of these proteins, we tested sera from 64 women with at least one successful pregnancy (multigravid), 21 women who had never been pregnant, and 54 males for antibodies to epitopes present on recombinant sHLA-G isoforms (sHLA-G1, sHLA-G2) derived from HLA 6.0 cDNA (HLA-G*0101 allele). By indirect enzyme-linked immunosorbent assay, antibodies to sHLA-G isoforms were identified in six sera, all from multigravid women; all other sera were negative (P = 0.0083). Immunoblots showed that two of the positive sera reacted exclusively with sHLA-G1 and -G2 whereas four reacted to both sHLA-G and pooled HLA class I antigens. To establish potential relationships between anti-sHLA-G and exposure to foreign paternal alleles (*0101, *0103, *0104, *0106), all multigravid women and their partners were genotyped. No relationship between allelic disparity and antibody production was identified. Taken together, these results indicate that (i) tolerance to HLA-G is the usual condition as antibodies to HLA-G were not detected in 91% (58/64) multigravid women, and (ii) pregnancy stimulates loss of tolerance in 9% (6/64) of multigravid women. All six women delivered healthy babies, demonstrating that maternal antibodies to epitopes on sHLA-G do not abrogate pregnancy.

Production and characterization of monoclonal antibodies with specificity for human HLA-G isoforms.

Production of monoclonal antibodies to HLA-G, a nonpolymorphic antigen of non-classical HLA class I, is of basic and clinical importance. In the present study, monoclonal antibodies were prepared which recognize different membrane bound and soluble isoforms of HLA-G, following immunization of BALB/c mice with a synthetic peptide. Use of this peptide (23 residues), which is present in the alpha1 domain of HLA-G, was previously demonstrated to provide antibodies useful for recognition of HLA-G isoforms in human placenta. Antibody-producing hybridomas were screened by an indirect one-step ELISA method. A clone designated 5E6H7, secreted antibodies useful in immunostaining studies involving both surface HLA-G of placental tissues and soluble forms of this antigen in human sera. In addition, unreactivity of this antibody with human lymphocytes and sections of normal human skin was observed by immunofluorescence microscopy, thus demonstrating its specificity for HLA-G.

Soluble HLA-G circulates in maternal blood during pregnancy

Objective: Soluble isoforms of the HLA class Ib gene HLA-G have been identified at the maternal-fetal interface. Because soluble forms of other HLA class I antigens modulate T-cell reactivity and induce cellactivated apoptosis, our goal was to determine whether soluble HLA-G circulates in maternal or fetal blood and to identify the specific isoform. Study Design: Capture enzyme-linked immunosorbent assays with mouse monoclonal antibodies directed toward an epitope present on all isoforms of soluble HLA-G were constructed to identify soluble HLA-G in 44 serum samples from nonpregnant control subjects, 129 serum samples from pregnant women, and 10 samples of term cord blood. Distinguishing between soluble HLA-G1, which is composed of heavy chains complexed with light chains (β2-microglobulin), and soluble HLA-G2, which consists only of heavy chains, was achieved by substituting a monoclonal antibody that requires β2-microglobulin for binding (W6/32) in the capture phase of the enzyme-linked immunosorbent assay. Results: Capture enzyme-linked immunosorbent assays with mouse anti–soluble HLA-G showed that soluble HLA-G was present at all stages of gestation and that levels of soluble HLA-G were statistically significantly higher in serum samples from pregnant women than in serum samples from nonpregnant women. In contrast, W6/32 failed to detect soluble HLA-G in serum samples from pregnant women. Cord serum samples did not contain detectable soluble HLA-G. Conclusion: Collectively, the data indicate that pregnancy is characterized by the presence of soluble HLA-G circulating in maternal blood and strongly suggest that the major isoform is soluble HLA-G2. (Am J Obstet Gynecol 2000;183:682-8.)

Primary cultured human thymic epithelial cells express both membrane-bound and soluble HLA-G translated products

This report demonstrates that both membrane-bound and soluble HLA-G isoforms are present in primary cultured human thymic epithelial cells (TEC). HLA-G transcriptional isoforms have been detected by RT-PCR, using different sets of HLA-G specific primers. A flow cytometry analysis, using two anti-HLA-G mAbs, namely 87G and BFL.1, revealed the presence of HLA-G translated products at the cell surface of a subpopulation of TEC. Finally, it was shown that HLA-G soluble forms were secreted in TEC culture supernatant, using a sandwich elisa with BFL.1 and W6/32 mAbs. These results confirm and extent those previously described showing that HLA-G expressing cells were detectable by immunohistochemistry in thymic medullary epithelial cells.

Placental HLA-G protein expression in vivo: where and what for?

In contrast to HLA-A and -B class Ia genes that are down-regulated in human trophoblast cells, HLA-G class Ib molecules are expressed in the placenta throughout gestation. In addition to extravillous cytotrophoblast that invade the decidua basalis essentially, HLA-G was also observed in endothelial cells of fetal vessels in the chorionic villi as well as in amnion cells and amniotic fluid. Both membrane-bound and soluble HLA-G isoforms have been detected. In view of the recently published functional data showing that HLA-G: (i) has the capability to bind and present peptides; (ii) is recognized by at least three different killing inhibitory receptors; and (iii) is a regulator of HLA-E expression, we can predict that such functions are likely to be exerted by extravillous cytotrophoblast. Of particular importance will be the anti-viral function of HLA-G at this materno-fetal interface, knowing that HLA-G was shown to be expressed by thymic medullary epithelial cells. In addition to these immunological functions, due to its presence on chorionic fetal endothelial cells, we hypothesize that HLA-G could also be a regulator of chorionic villous angiogenesis. Finally, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy.

Immunotolerant functions of HLA-G.

HLA-G is a nonclassical major histocompatibility complex class I molecule selectively expressed on cytotrophoblasts at the fetal-maternal interface, where it plays a role in materno-fetal tolerance. In contrast to classical HLA-A, -B and -C class I molecules, HLA-G is characterized by (i) a tissue-restricted distribution, (ii) a limited polymorphism and (iii) a transcription of spliced messenger RNAs encoding for at least four membrane-bound and two soluble HLA-G isoforms. Extensive studies over the past few years have identified HLA-G as a molecule involved in immune tolerance. In this review, attempts were made to summarize the current state of knowledge of the effects of HLA-G on both natural killer and T cell functions and their implications in materno-fetal tolerance and tumor immunosurveillance.

The functionality of HLA-G is emerging.

In view of the recently published data, the HLA-G class Ib gene appears to be a functional locus. This is based on the following observations: 1) HLA-G is capable of presenting nonamer peptides and of exerting antigen-presenting functions; 2) HLA-G is a ligand for at least three natural killer (NK) and other cell inhibitory receptors of the immunoglobulin superfamily, namely leukocyte immunoglobulin-like receptor-1/immunoglobulin-like transcript (ILT)-2, ILT-4 and p49; 3) in addition to the extravillous cytotrophoblast cells, HLA-G proteins have been detected in endothelial cells of placental chorionic villi, as well as in amniotic fluid and in some medullary thymic epithelial cells; 4) major histocompatibility complex (MHC) class Ib genes that share the unique characteristics of HLA-G, including a high expression in placenta, have been reported in other mammalian species. In addition to the classical MHC class I roles (antigen presentation and ligation to NK receptors inducing inhibitory and/or activatory signals), HLA-G is likely to exert other, novel functions: first, HLA-G was shown to be involved in the control of HLA-E expression by furnishing the appropriate class I leader sequence nonamer peptide; second, we hypothesize that HLA-G could be a regulator of placental angiogenesis; third, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy. Such functional properties, although incompletely understood, are likely to be important in the outcome of human pregnancies but also in normal adult life.