Soluble HLA-G Research Articles

HLA-G expression during hookworm infection in pregnant women

IntroductionHLA-G plays a key role on immune tolerance. Pathogens can induce soluble HLA-G (sHLA-G) production to down-regulate the host immune response, creating a tolerogenic environment favorable for their dissemination. To our knowledge, no study has yet been conducted to assess the relationship between sHLA-G and geohelminth infections. MethodsThe study was conducted in Allada, Southeastern Benin, from 2011−2014. The study population encompassed 400 pregnant women, included before the end of the 28th week of gestation and followed-up until delivery. At two antenatal care visits and at delivery, stool and blood samples were collected. Helminths were diagnosed by means of the Kato-Katz concentration technique. We used quantile regression to analyze the association between helminth infections and sHLA-G levels during pregnancy. ResultssHLA-G levels gradually increased during pregnancy and reached maximal levels at delivery. Prevalence of helminth infections was low, with a majority of hookworm infections. We found significantly more hookworm-infected women above the 80th quantile (Q80) of the distribution of the mean sHLA-G level (p < 0.03, multivariate quantile regression). Considering only women above the Q80 percentile, the mean sHLA-G level was significantly higher in hookworm-infected compared to uninfected women (p = 0.04). ConclusionHigh levels of sHLA-G were associated with hookworm infection in pregnant women. This result is consistent with the potential involvement of sHLA-G in immune tolerance induced by helminths during pregnancy.

Influence of immune-checkpoint inhibitor and HLA-G in patients with Cervical Cancer

Abstract A better understanding of the role of Immune checkpoint inhibitors (ICI) and immunoregulatory genes HLA-G is essential for the development of immunotherapeutic strategies in cancer. This study aims to evaluate the influence of CTLA-4, PD-1 and polymorphisms of HLA-G and soluble HLA-G (sHLA-G) in patients with cervical cancer (CC), different degrees of Cervical Intraepithelial Neoplasia (CIN), and healthy control (HC). One hundred and seventy-four (174) non-pregnant Brazilian women, sexually active, older than 18 years, were evaluated. These patients were stratified according to the results of the anatomopathological examination in: CC group n=24, Low-grade squamous intraepithelial lesions (LSIL) n=66, High-grade squamous intraepithelial lesions (HSIL) n=84. HC group consisted of 47 women without cytological and/or colposcopic abnormalities detected. The peripheral blood of these patients was collected for analysis of CTLA-4, PD-1 and HLA-G polymorphisms using real-time PCR. The analysis indicated a significantly higher 14bpDel allele frequency in CC when compared to HC group (P=0.030, OR=1.71, 95%CI 1.01–2.74). Moreover, the 14bpDel/Del genotype frequency was also significantly higher in patients with CC group (P = 0.035, OR=2.23, 95%CI 1.05–4.97). Qualitative analysis revealed that the production of sHLA-G is detectable in 52.03% (n=77) of all patients groups and 34.29 %(n=12) in HC groups. This analysis revealed a trend of significance (P= 0.063; OR=2.08, 95%CI=0.96–4.48) in CC group. The polymorphisms of CTLA-4 and 14 bp HLA-G are associated with a significantly increased risk of CC. HLA-G 14bp Del/Del polymorphism, and the serum sHLA-G can act as a promising biomarker for might be a genetic risk factor for CC.

A role for allosteric disulfides in regulating the function of human killer cell receptor KIR2DL4

Abstract KIR2DL4 is an unusual member of the killer cell Ig-like receptor (KIR) family expressed in NK cells. It resides mostly in endosomes and activates NK cells in response to soluble agonists, such as soluble HLA-G and a monoclonal antibody. KIR2DL4 signals from endosomes for a unique proinflammatory and proangiogenic response, which has relevance to placental development due to the restricted expression of its ligand, HLA-G, by fetal trophoblast cells during early pregnancy. To understand the nature and regulation of KIR2DL4–HLA-G interactions, we used an unbiased random mutagenesis approach and screened mutants of KIR2DL4 in a cellular assay for their ability to load soluble HLA-G into KIR2DL4+ endosomes. With this approach, amino acid residues in KIR2DL4 that are important for endosomal localization and HLA-G recognition were identified. Specifically, we have identified a putative allosteric cysteine in the extracellular domain of this receptor, which may be involved in a disulfide switching mechanism to control receptor internalization and ligand binding. Protein disulfide isomerase (PDI) acting at the cell surface may be responsible for this switch as inhibition of PDI interfered with internalization of KIR2DL4 and uptake of HLA-G into endosomes. The configuration of the relevant disulfide bond is -RHstaple, which is a hallmark of many allosteric disulfides. Thus, KIR2DL4 localization and function is regulated by allosteric disulfide bonds at the cell surface. Studies are underway to explore the dynamic regulation of receptor structure by this allosteric disulfide switch to control ligand binding, localization and signaling by this receptor.

Natural Killer Cells as Key Players of Tumor Progression and Angiogenesis: Old and Novel Tools to Divert Their Pro-Tumor Activities into Potent Anti-Tumor Effects.

Immune cells, as a consequence of their plasticity, can acquire altered phenotype/functions within the tumor microenvironment (TME). Some of these aberrant functions include attenuation of targeting and killing of tumor cells, tolerogenic/immunosuppressive behavior and acquisition of pro-angiogenic activities. Natural killer (NK) cells are effector lymphocytes involved in tumor immunosurveillance. In solid malignancies, tumor-associated NK cells (TANK cells) in peripheral blood and tumor-infiltrating NK (TINK) cells show altered phenotypes and are characterized by either anergy or reduced cytotoxicity. Here, we aim at discussing how NK cells can support tumor progression and how induction of angiogenesis, due to TME stimuli, can be a relevant part on the NK cell-associated tumor supporting activities. We will review and discuss the contribution of the TME in shaping NK cell response favoring cancer progression. We will focus on TME-derived set of factors such as TGF-β, soluble HLA-G, prostaglandin E2, adenosine, extracellular vesicles, and miRNAs, which can exhibit a dual function. On one hand, these factors can suppress NK cell-mediated activities but, on the other hand, they can induce a pro-angiogenic polarization in NK cells. Also, we will analyze the impact on cancer progression of the interaction of NK cells with several TME-associated cells, including macrophages, neutrophils, mast cells, cancer-associated fibroblasts, and endothelial cells. Then, we will discuss the most relevant therapeutic approaches aimed at potentiating/restoring NK cell activities against tumors. Finally, supported by the literature revision and our new findings on NK cell pro-angiogenic activities, we uphold NK cells to a key host cellular paradigm in controlling tumor progression and angiogenesis; thus, we should bear in mind NK cells like a TME-associated target for anti-tumor therapeutic approaches.

The Role of Soluble HLA-G in the Vertical Transmission of Toxoplasma gondii

The Role of Soluble HLA-G in the Vertical Transmission of Toxoplasma gondii

The histone demethylase PHF8 facilitates alternative splicing of the histocompatibility antigen HLA-G.

Histone3-lysine9 (H3K9) residues not only control gene expression, but also contribute to RNA splicing. Here, the H3K9 histone demethylase PHF8 was investigated in endothelial cells for its involvement in alternative splicing. An angiogenic sprouting assay shows the importance of PHF8 for endothelial cells. Immunoprecipitation reveals that PHF8 interacts with U1 spliceosomal proteins, such as SRPK1 and snRNP70. We identify the histocompatibility antigen HLA-G as a target of PHF8. The inclusion of HLA-G intron 4, with concomitant RNA Polymerase II accumulation at this intron is controlled by PHF8 and H3K9. Soluble HLA-G is generated after PHF8 knockdown, which leads to reduced T-cell proliferation. Collectively, PHF8 knockdown generates the immunosuppressive alternative splice product soluble HLA-G, which is secreted by endothelial cells to elicit a potential inhibitory effect on inflammation.

Immune Modulation in HLA-G Expressing Head and Neck Squamous Cell Carcinoma in Relation to Human Papilloma Virus Positivity: A Study From Northeast India.

Background: Tumor specific ectopic expression of the immunomodulatory molecule, HLA-G is known to mediate immune tolerance and promote carcinogenesis. Viruses too employ strategies to evade immune surveillance. Considering the role of both HLA-G and HPV in tumor growth and progression, it is pertinent to investigate the relationship between HLA-G and HPV in context of immune modulation in HNSCC.Method: A hospital based case–control study was conducted in histopathologically confirmed HNSCC tissues. HLA-G isoform expression and HPV association studies were carried out and mRNA levels of HLA-G, markers of proliferation and differentiation (ki-67, keratin 18, cyclin D1), immune checkpoint molecules (IL-10, PD-1. TGF-β), SOCS (SOCS1 and SOCS3) and pro-inflammatory cytokine IFN-γ were determined.Results: Higher expression of HLA-G was noted in HPV positive tumors (5.14 fold, p = 0.002). HLA-G7 was the most frequent isoform (29/80) found in HNSCC particularly in HPV positive tumors (13/16). In HPV negative tumors, all the checkpoint molecules were upregulated along with pro–inflammatory IFN-γ. In contrast, in HPV positive tumors, IFN-γ expression was higher (2.12 fold) but levels of IL-10, PD-1, TGF-β, SOCS1 and SOCS3 were markedly lower (fold change of IL-10 = 0.37, PD1 = 0.41, TGF-β = 0.17, SOCS1 = 0.055, SOCS3 = 0.027). HPV positive tumors were more proliferative and differentiated with higher expression of ki-67 and keratin18 (6.25 fold, p = 0.079 and 10.62 fold, p = 0.009). Decreased expression of cyclin D1 was noted in HPV positive tumors (6.94 fold, p = 0.006) than HPV negative tumors (17.69 fold). Also, HLA-G7 expressing HPV positive tumors showed lowest expression of cyclin D1. Interestingly, SOCS showed normal expression in HLA-G7 expressing HPV negative tumors (1.2 and 1.4 fold). IFN-γ was downregulated in HPV positive tumors without HLA-G7 (0.31 fold).Conclusion: Our data suggests that SOCS were downregulated irrespective of HLA-G positivity and IFN- γ expression appeared to be mediated by HLA-G. SOCS are reported to have anti-tumor activity and also SOCS and soluble HLA-G are known to interfere with cell cycle progression. Hence, through regulating HLA-G expression, HPV positive tumors could mediate immune suppression by manipulating SOCS, IFN-γ, IL-10 and cyclin D1 pathways which needs further exploration.

High Expression of HLA-G in Ovarian Carcinomatosis: The Role of Interleukin-1β

The present study focuses on the influence of the tumor microenvironment on the expression of HLA-G in ovarian cancer and its impact on immune cells. We used carcinomatosis fluids (n = 16) collected from patients diagnosed with epithelial ovarian cancer, detected by an increase in CA125 levels. Our results indicate that HLA-G is expressed by 1) ascitic cell clusters, 2) stromal cells (hospicells) extracted from cancer cell clusters, and 3) cancer cell lines and tumor cells. The origin of HLA-G was linked to inflammatory cytokines present in the cancer microenvironment. In parallel, the ascitic fluid of patients with ovarian cancer contains soluble HLA-G (sHLA-G). The mesothelial cell layer and submesothelial tissues, as well as the immune cell infiltrate, do not secrete HLA-G. In contrast, sHLA-G is absorbed by peritoneal tissues along with mesothelial layers as well as immune cell infiltrates. We demonstrated that interleukin-1β along with TGF-β can be a major HLA-G–inducing factor that upregulates HLA-G expression through the NF-κB pathway. The level of HLA-G in ascites correlated positively with the expression of T regulatory (T-regs) cells, while it negatively correlated with the expression of natural killer and memory cells in tumor-infiltrating immune cells. In conclusion, the production of HLA-G is associated with the presence of inflammatory cytokines and is strongly correlated with microenvironment tolerant cells such as T-regs and diminution of NK and memory T cells.

HLA-G is upregulated in advanced endometriosis

ObjectiveTo assess whether the HLA-G immunomodulatory protein is potentially involved in the pathophysiology of endometriosis or disease progression. Study designCross-sectional observational study of 227 women who underwent laparoscopy, being 146 for endometriosis excision and 81 for elective tubal ligation (control group). Soluble HLA-G (sHLA-G) levels in the serum and peritoneal fluid (PF), as well as the HLA-G protein expression in matched eutopic and ectopic endometrium of women with and without endometriosis were evaluated by ELISA and immunohistochemistry assays, respectively. Women with endometriosis were separated into groups according to the initial (I/II, n = 60) and advanced (III/IV, n = 86) stages of disease. sHLA-G measurement was performed only in women with matched serum and PF samples in both the control (CTRL; n = 77) and endometriosis (EDT; I–II, n = 60; III–IV, n = 83) groups. HLA-G protein expression was evaluated in 26 women with deep endometriosis (I–II, n = 12; III–IV, n = 14) and 22 controls. ResultsHigher concentrations of sHLA-G (P = 0.013) in the serum but not in the PF were observed in women with advanced endometriosis compared to the control group. In situ expression of HLA-G protein was also higher in ectopic (P = 0.018) but not in eutopic endometrium of women with advanced endometriosis compared to control group. ConclusionOur findings suggest that HLA-G upregulation in advanced stages may contribute to the state of immunosuppression in endometriosis as disease progresses.

Increased HLA-G Expression in Term Placenta of Women with a History of Recurrent Miscarriage Despite Their Genetic Predisposition to Decreased HLA-G Levels.

Human leukocyte antigen (HLA)-G is an immune modulating molecule that is present on fetal extravillous trophoblasts at the fetal-maternal interface. Single nucleotide polymorphisms (SNPs) in the 3 prime untranslated region (3′UTR) of the HLA-G gene can affect the level of HLA-G expression, which may be altered in women with recurrent miscarriages (RM). This case-control study included 23 women with a medical history of three or more consecutive miscarriages who delivered a child after uncomplicated pregnancy, and 46 controls with uncomplicated pregnancy. Genomic DNA was isolated to sequence the 3′UTR of HLA-G. Tissue from term placentas was processed to quantify the HLA-G protein and mRNA levels. The women with a history of RM had a lower frequency of the HLA-G 3′UTR 14-bp del/del genotype as compared to controls (Odds ratio (OR) 0.28; p = 0.039), which has previously been related to higher soluble HLA-G levels. Yet, HLA-G protein (OR 6.67; p = 0.006) and mRNA (OR 6.33; p = 0.010) expression was increased in term placentas of women with a history of RM as compared to controls. In conclusion, during a successful pregnancy, HLA-G expression is elevated in term placentas from women with a history of RM as compared to controls, despite a genetic predisposition that is associated with decreased HLA-G levels. These findings suggest that HLA-G upregulation could be a compensatory mechanism in the occurrence of RM to achieve an ongoing pregnancy.

The Role of Soluble HLA-G in the Vertical Transmission of Toxoplasma gondii

Soluble human leukocyte antigen G (sHLA-G) plays a key role in pregnancy through interaction with decidual natural killer (dNK) cell inhibitory receptors at the maternal-fetal interface. To demonstrate the possible role of sHLA-G during the pregnancy with Toxoplasma gondii infection, we compared the concentration of a murine functional homolog of sHLA-G, Qa-2, in T. gondii infected and non-infected pregnant C57BL/6 mice, and that of sHLA-G in BeWo culture supernatant. In addition, the levels of KIR2DL4 expressed on human dNK cells and NKG2A in pregnant mice were evaluated. We showed that T. gondii infection result in significant increase in the level of Qa-2 and NKG2A in pregnant mice. sHLA-G and KIR2DL4 in human samples were also significantly upregulated under the condition of T. gondii infection. The further treatment with sHLA-G antibody could reduce the expression level of KIR2DL4 which was upregulated by T. gondii infection. In summary, sHLA-G could upregulate the expression level of KIR2DL4 which lead to excessive immunological tolerance, and further contributed to T. gondii immunity escaping and affecting fetus via vertical transmission which may lead to adverse outcomes.

Soluble HLA-G Expression Inversely Correlates With Fetal Microchimerism Levels in Peripheral Blood From Women With Scleroderma.

Women with scleroderma (SSc) maintain significantly higher quantities of persisting fetal microchimerism (FMc) from complete or incomplete pregnancies in their peripheral blood compared to healthy women. The non-classical class-I human leukocyte antigen (HLA) molecule HLA-G plays a pivotal role for the implantation and maintenance of pregnancy and has often been investigated in offspring from women with pregnancy complications. However data show that maternal HLA-G polymorphisms as well as maternal soluble HLA-G (sHLA-G) expression could influence pregnancy outcome. Here, we aimed to investigate the underlying role of maternal sHLA-G expression and HLA-G polymorphisms on the persistence of FMc. We measured sHLA-G levels by enzyme linked immunosorbent assay in plasma samples from 88 healthy women and 74 women with SSc. Male Mc was quantified by DYS14 real-time PCR in blood samples from 58 women who had previously given birth to at least one male child. Furthermore, eight HLA-G 5′URR/3′UTR polymorphisms, previously described as influencing HLA-G expression, were performed on DNA samples from 96 healthy women and 106 women with SSc. Peripheral sHLA-G was at lower concentration in plasma from SSc (76.2 ± 48.3 IU/mL) compared to healthy women (117.5 ± 60.1 IU/mL, p < 0.0001), independently of clinical subtypes, autoantibody profiles, disease duration, or treatments. Moreover, sHLA-G levels were inversely correlated to FMc quantities (Spearman correlation, p < 0.01). Finally, women with SSc had lower sHLA-G independently of the eight HLA-G 5′URR/3′UTR polymorphisms, although they were statistically more often homozygous than heterozygous for HLA-G polymorphism genotypes −716 (G/T), −201 (G/A), 14 bp (ins/del), and +3,142 (G/A) than healthy women. In conclusion, women with SSc display less sHLA-G expression independently of the eight HLA-G polymorphisms tested. This decreased production correlates with higher quantities of persisting FMc commonly observed in blood from SSc women. These results shed some lights on the contribution of the maternal HLA-G protein to long-term persistent fetal Mc and initiate new perspectives in this field.

Tropheryma whipplei Increases Expression of Human Leukocyte Antigen-G on Monocytes to Reduce Tumor Necrosis Factor and Promote Bacterial Replication

Infection with Tropheryma whipplei has a range of effects-some patients can be chronic carriers without developing any symptoms, whereas others can develop systemic Whipple disease, characterized by a lack a protective inflammatory immune response. Alterations in HLA-G function have been associated with several diseases. We investigated the role of HLA-G during T whipplei infection. Sera, total RNA, and genomic DNA were collected from peripheral blood from 22 patients with classic Whipple's disease, 19 patients with localized T whipplei infections, and 21 asymptomatic carriers. Levels of soluble HLA-G in sera were measured by enzyme-linked immuosorbent assay, and expressions of HLA-G and its isoforms were monitored by real-time polymerase chain reaction. HLA-G alleles were identified and compared with a population of voluntary bone marrow donors. Additionally, monocytes from healthy subjects were stimulated with T whipplei, and HLA-G expression was monitored by real-time polymerase chain reaction and flow cytometry. Bacterial replication was assessed by polymerase chain reaction in the presence of HLA-G or inhibitor of tumor necrosis factor (TNF) (etanercept). HLA-G mRNAs and levels of soluble HLA-G were significantly increased in sera from patients with chronic T whipplei infection compared with sera from asymptomatic carriers and control individuals. No specific HLA-G haplotypes were associated with disease or T whipplei infection. However, T whipplei infection of monocytes induced expression of HLA-G, which was associated with reduced secretion of TNF compared with noninfected monocytes. A neutralizing antibody against HLA-G increased TNF secretion by monocytes in response to T whipplei, and a TNF inhibitor promoted bacteria replication. Levels of HLA-G are increased in sera from patients with T whipplei tissue infections, associated with reduced production of TNF by monocytes. This might promote bacteria colonization in patients.

HLA-G: Facts and Fictions

Human leukocyte antigen (HLA)-G is a nonclassical MHC class I molecule with modulatory effects on NK and T cells. Unlike classical HLA class I molecules, HLA-G has seven isoforms, three of which are soluble. Soluble HLA-G molecules are reportedly able to transduce negative signals to immune cells after interacting with their corresponding receptors. The expression of these molecules plays significant roles in maternal tolerance against semi-allogenic fetuses. Overexpression of HLA-G in tumors and increased serum levels of soluble HLA-G have been reported in different malignancies, and these changes may be involved in tumoral immune evasion and cancer progression. To improve immune responses against tumor cells, the downmodulation of HLA-G by siRNA or blocking monoclonal antibodies can be helpful in cancer immunotherapy. Additionally, HLA-G can be considered a potential biomarker for the diagnosis and/or prognosis of certain cancers. Although polymorphism of the HLA-G gene-coding region is more limited than in classical HLA class I, some genetic variations in regulatory regions of the gene control the expression level of this molecule. Furthermore, epigenetic factors such as infections may affect the expression of HLA-G in infection-related cancers.

Elevated soluble human leukocyte antigen G levels in patients after allogeneic stem cell transplantation are associated with less severe acute and chronic graft-versus-host disease.

HLA-G is a non-classical class I molecule which induces tolerance in allogeneic situations by inhibition of cytotoxic NK and CD8 + T cells and by induction of regulatory T cells. Concordantly, in solid organ transplantation HLA-G is associated with a lower risk for acute and chronic rejection, whereas its role in allogeneic stem cell transplantation (allo-SCT) is less established. We here present detailed analyses of HLA-G-levels in patients after allo-SCT showing a correlation of elevated soluble HLA-G (sHLA-G) levels with less severe acute (p = 0.06) and chronic GvHD (p = 0.0025) and with a superior overall survival (p = 0.03). Soluble HLA-G levels are also positively correlated with the frequency of regulatory T cells in vivo. These clinical data are corroborated by in vitro analyses showing that patients-derived sHLA-G inhibit allogeneic immune responses. ATG-treatment of patients dominantly affects the sHLA-G levels post allo-SCT. Thus, this study highlights the association of elevated sHLA-G levels with less severe acute and chronic GvHD and provides additional functional analyses elucidating possible tolerance-inducing mechanisms of sHLA-G in the context of allo-SCT.

HLA-G Haplotypes Are Differentially Associated with Asthmatic Features.

Human leukocyte antigen (HLA)-G, a HLA class Ib molecule, interacts with receptors on lymphocytes such as T cells, B cells, and natural killer cells to influence immune responses. Unlike classical HLA molecules, HLA-G expression is not found on all somatic cells, but restricted to tissue sites, including human bronchial epithelium cells (HBEC). Individual variation in HLA-G expression is linked to its genetic polymorphism and has been associated with many pathological situations such as asthma, which is characterized by epithelium abnormalities and inflammatory cell activation. Studies reported both higher and equivalent soluble HLA-G (sHLA-G) expression in different cohorts of asthmatic patients. In particular, we recently described impaired local expression of HLA-G and abnormal profiles for alternatively spliced isoforms in HBEC from asthmatic patients. sHLA-G dosage is challenging because of its many levels of polymorphism (dimerization, association with β2-microglobulin, and alternative splicing), thus many clinical studies focused on HLA-G single-nucleotide polymorphisms as predictive biomarkers, but few analyzed HLA-G haplotypes. Here, we aimed to characterize HLA-G haplotypes and describe their association with asthmatic clinical features and sHLA-G peripheral expression and to describe variations in transcription factor (TF) binding sites and alternative splicing sites. HLA-G haplotypes were differentially distributed in 330 healthy and 580 asthmatic individuals. Furthermore, HLA-G haplotypes were associated with asthmatic clinical features showed. However, we did not confirm an association between sHLA-G and genetic, biological, or clinical parameters. HLA-G haplotypes were phylogenetically split into distinct groups, with each group displaying particular variations in TF binding or RNA splicing sites that could reflect differential HLA-G qualitative or quantitative expression, with tissue-dependent specificities. Our results, based on a multicenter cohort, thus support the pertinence of HLA-G haplotypes as predictive genetic markers for asthma.

The HLA-G 14 bp insertion/deletion polymorphism and its association with soluble HLA-G levels in women with recurrent miscarriages.

HLA-G, a nonclassical class-Ib gene is mainly expressed on extravillous trophoblasts at the fetal-maternal interface. HLA-G molecule is considered to play an important role in maternal immune suppression during pregnancy. The 14 bp insertion/deletion polymorphism (rs66554220) in exon eight of the HLA-G gene influences HLA-G mRNA stability and isoform splicing patterns. In this study, 202 recurrent miscarriage (RM) women with two or more than two consecutive miscarriages, their 202 partners and 204 fertile control women with at least one live birth and no miscarriages were analyzed for 14 bp insertion/deletion polymorphism. Soluble HLA-G (sHLA-G) levels were also determined and compared between randomly selected 111 RM women and 111 control women using QAYEE-Bio ELISA kits. Student's t test and χ2 test were used to depict the statistical differences. The results showed no significant differences for 14 bp allele and genotype frequencies between the study groups. However, our study showed a significant difference (P = .0107) for sHLA-G levels in RM women and control women. Furthermore, a significant difference (P = .0135) for sHLA-G levels in relation to +/-14 bp heterozygous genotype was seen between the two groups. The 14 bp allele sharing between the partners did not show any significant association with the number of miscarriages in RM couples. The association of 14 bp polymorphism and recurrent miscarriages was not significant in our study.

The role of HLA-G in parasitic diseases.

Little attention has been devoted to the role of HLA-G gene and molecule on parasitic disorders, and the available studies have focused on malaria, African and American trypanosomiasis, leishmaniosis, toxoplasmosis and echinococcosis. After reporting a brief description regarding the role of the cells of innate and adaptive immune system against parasites, we reviewed the major features of the HLA-G gene and molecule and the role of HLA-G on the major cells of immune system. Increased levels of soluble HLA-G (sHLA-G) have been observed in patients presenting toxoplasmosis and in the active phase of echinococcosis. In addition, increased sHLA-G has also been associated with increased susceptibility to malaria and increased susceptibility to develop human African trypanosomiasis (HAT). In contrast, decreased membrane-bound HLA-G has been reported in placenta of patients infected with Plasmodium falciparum and in heart and colon of patients presenting Chagas disease. The 3' untranslated region of the HLA-G gene has been the main focus of studies on malaria, HAT and Chagas disease, exhibiting distinct patterns of associations. Considering that HLA-G is an immune checkpoint molecule, inhibiting the activity of several cells of the immune system, the excessive neoexpression and the increased sHLA-G levels together with the decreased constitutive tissue expression of membrane-bound HLA-G may be detrimental to the host infected with parasite agents.

The Influence of Cytomegalovirus on Expression of HLA-G and its Ligand KIR2DL4 by Human Peripheral Blood Leucocyte Subsets.

HLA-G is a non-classical class I HLA antigen, normally expressed in high levels only on extravillous cytotrophoblast. It has immunosuppressive properties in pregnancy and has also been found to be upregulated on leucocytes in viral infection. In this study, proportions of all leucocyte subsets expressing HLA-G were found to be low in healthy subjects positive or negative for cytomegalovirus (CMV). Significantly greater proportions of CD4+ CD69+ and CD56+ T cells expressed HLA-G compared to other T cells. However, following stimulation with CMV antigens or intact CMV, proportions of CD4+, CD8+, CD69+ and CD56+ T cells, and also B cells expressing HLA-G, were significantly increased in CMV+ subjects. Despite some subjects having alleles of HLA-G associated with high levels of expression, no relationship was found between HLA-G genotype and expression levels. Purified B cells from CMV+ subjects stimulated in mixed culture with CMV antigens showed significantly increased HLA-G mRNA expression by real-time polymerase chain reaction. Serum levels of soluble HLA-G were similar in CMV- and CMV+ subjects but levels in culture supernatants were significantly higher in cells from CMV+ than from CMV- subjects stimulated with CMV antigens. The HLA-G ligand KIR2DL4 was mainly expressed on NK cells and CD56+ T cells with no differences between CMV+ and CMV- subjects. Following stimulation with IL-2, an increase in the proportion of CD56+ T cells positive for KIR2DL4 was found, together with a significant decrease in CD56dimCD16+ NK cells. The results show that CMV influences HLA-G expression in healthy subjects and may contribute to viral immune evasion.

P058 Renal replacement therapy: Variability in HLA-G expression as an outcome indicator

Aim The non-classical HLA class I molecule, HLA-G has unique features like restricted expression, limited polymorphism, and alternative splicing led isoforms. HLA-G plays crucial immunomodulatory role in allogenic situations like pregnancy and allo-transplantation. This molecule creates a tolerogenic microenvironment through interaction with receptors ILT-2/ILT-4/KIR2DL4 on immune cells. The posttranscriptional regulation and mRNA stability is altered due to 3’UTR region polymorphism impacting miRNA interactions. To investigate the clinical relevance of HLA-G in patients undergoing renal transplantation. For this study, 50 healthy controls and 39 recipient-donor pairs were enrolled of which, 11 recipients experienced graft rejection episodes. Methods HLA-G exon8-14bp INDEL polymorphism was evaluated by PCR-SSP and sequencing. The 14bp genotype (ins/del, del/del and ins/ins) distribution was evaluated. Serum soluble HLA-G levels (pre and serial post transplant days) were measured by ELISA. Results For analysis, recipients were segregated into rejection (R) and non-rejection (NR) or well functioning graft (WFG) groups. Higher percentage of NR recipients carried ins/ins genotype as compared to the R group (32.1% vs 20% respectively). In healthy controls, the median sHLA-G level was 31.7 U/ml and group with ins/ins genotype had highest levels. In transplant recipients, sHLA-G levels were significantly higher than controls. Median sHLA-G levels were lower (105.4 U/ml) among rejection group, in comparison to WFG (260.4 U/ml). Throughout the post-transplant period, sHLA-G levels were better maintained in the WFG group. In the rejection group, the sHLA-G levels improved only after anti-rejection therapy, however it remained lower than WFG. Conclusions The HLA-G 3’UTR genotyping and serial sHLA-G level monitoring are likely to predict adverse event like rejection. HLA-G expression on alloactivated recipient cells is underway to better understand functional relevance.