HLA-G Molecules Research Articles

Circulating Vesicular-bound HLA-G as Noninvasive Predictive Biomarker of CLAD After Lung Transplantation.

Circulating extracellular vesicles (EVs) have shown promising results as noninvasive biomarkers for predicting disease outcomes in solid organ transplantation. Because in situ graft cell expression of the tolerogenic molecule HLA-G is associated with acceptance after lung transplantation (LTx), we hypothesized that plasma EV-bound HLA-G (HLA-GEV) levels could predict chronic lung allograft dysfunction (CLAD) development. We analyzed 78 LTx recipients from the Cohort-for-Lung-Transplantation cohort, all in a stable (STA) state within the first year post-LTx. At 3 y, 41 patients remained STA, and 37 had CLAD (bronchiolitis obliterans syndrome, BOS, [n = 32] or restrictive allograft syndrome [n = 5]). HLA-GEV plasma levels were measured at month 6 (M6) and M12 in 78 patients. CLAD occurrence and graft failure at 3 y post-LTx were assessed according to early HLA-GEV plasma levels. In patients with subsequent BOS, (1) HLA-GEV levels at M12 were significantly lower than those in STA patients (P = 0.013) and (2) also significantly lower than their previous levels at M6 (P = 0.04).A lower incidence of CLAD and BOS and higher graft survival at 3 y were observed in patients with high HLA-GEV plasma levels at M12 (high versus low HLA-GEVs patients [cutoff 21.3 ng/mL]: freedom from CLAD, P = 0.002; freedom from BOS, P < 0.001; and graft survival, P = 0.04, [log-rank]). Furthermore, in multivariate analyses, low HLA-GEV levels at M12 were independently associated with a subsequent risk of CLAD, BOS, and graft failure at 3 y (P = 0.015, P = 0.036, and P = 0.026, respectively [Cox models]). This exploratory study suggests the potential of EV-bound HLA-G plasma levels as a liquid biopsy in predicting CLAD/BOS onset and subsequent graft failure.

Isolation of donor gamma globulin obtained from multiparous women and its effects upon expression of HLA-G and HLA-DR molecules on lymphocytes from mothers of children with septal congenital heart defects

A common pathogenetic mechanism of reproductive losses and congenital heart disease (CHD) is associated with immune inflammation in the “mother-embryo” system which affects differentiation and proliferation of cardiovascular progenitor cells. It is hypothesized that this link may be blocked by regulatory auto- and alloimmune antibodies to HLA-G and HLA-DR molecules. Moreover, these antibodies may be present at sufficient amounts in donor immunoglobulins, especially those obtained from the blood of multiparous women. Based on this suggestion, the aim of our study was to obtain enriched gamma globulin fraction from the blood of multiparous women and evaluate its functional effects towards HLA-DR and HLA-G molecules. Isolation of the gammaglobulin fraction (GGF) from the blood plasma of multiparous women was performed using affinity chromatography in several sessions. Purity grade of the resulting protein was analyzed by immunoelectrophoresis, electrophoretic separation of the protein fraction of blood serum and electrophoresis in 4.12% polyacrylamide gel with the addition of SDS (PAGE electrophoresis). PAAG electrophoresis showed that this GGF did not differ from commercial therapeutic intravenous immunoglobulin (IVIG). Assessment of the functional activity of GGF upon HLA-DR and HLA-G molecules was performed in the main group of women and their children with congenital heart disease (n = 38), and control group of women who gave birth to conditionally healthy children (n = 21). To determine the specificity of GGF with respect to HLA-G, HLA-DR molecules, as well as to compare its effect with autologous and allogeneic sera and IVIG, we developed an immunological testing protocol using flow cytometry. The protocol was arranged on the basis of the methodology of “cross-match” approach and Russian patent “Method for determining antibodies to HLA-G”. It was found that the blocking activity of female serum towards autologous (intrinsic) and allogeneic (embryo/fetus/child) HLA-G and HLA-DR molecules may determine the protective effect on development of congenital heart defects in the next generation. Donor human immunoglobulin showed a similar blocking effects to these molecules, possibly due to the presence of alloimmune antibodies to HLA classes I and II. The gammaglobulin fraction obtained from the donor blood of multiparous women has a more pronounced blocking effect on the HLA-G and HLA-DR expression. Hence, this immunobiological preparation can be considered a prototype of therapeutic and prophylactic agent blocking the genesis of congenital heart defects.

Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity

BackgroundThe human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch.MethodsFirst, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous β-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed.ResultsWe found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo.ConclusionThe HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.

Abstract 1362: IOMX-0675, a LILRB1 and LILRB2 cross-specific antibody, effectively repolarizes immunosuppressive myeloid cells and activates T cells leading to potent tumor cell killing

Abstract Recently, myeloid checkpoints in the tumor microenvironment have gained increased attention in the context of tumor immune evasion. LILRB1 (ILT2) and LILRB2 (ILT4) are immunosuppressive receptors of the leukocyte immunoglobulin-like receptor (LILR) family that recognize both classical and non-classical MHC-I molecules (e.g., HLA-G). While tumor-infiltrating myeloid cells express both LILRB1 and LILRB2, lymphoid cells are restricted to LILRB1 expression. LILRB1 and LILRB2 are frequently co-expressed with immune-activating LILR family members LILRA1 and LILRA3 in several tumor indications and upregulated in patients non-responsive to T cell checkpoint blockade. Furthermore, the non-classical MHC-I molecule HLA-G, a major ligand of LILRB1 and LILRB2 in the tumor environment, is overexpressed and associated with poor prognosis in multiple solid cancer types. IOMX-0675, a fully human, Fc-silenced, monoclonal immunoglobulin G1 (IgG1) antibody with a highly differentiated binding profile, that binds selectively with high affinity to the inhibitory receptors LILRB1 and LILRB2, while binding only weakly to the closely related immune-activating LILR family members LILRA1 and LILRA3, was identified from iOmx’ proprietary phage display library. A high-resolution Fab-based Octet assay was established to monitor the IOMX-0675-mediated blocking of LILRB1 or LILRB2 binding to HLA-G. The LILRB1/2 cross-specific antibody promotes phagocytic and pro-inflammatory activity of various macrophage subtypes, which was monitored by flow cytometry and cytokine profiling. In co-cultures of monocyte-derived macrophages with autologous T cells, IOMX-0675 shows high potential to reprogram dose-dependently immunosuppressive macrophage, to further activate pro-inflammatory macrophage subtypes and thereby rescues the activity of the lymphoid immune compartment. Even in environments dominated by LILRA1/A3, IOMX-0675 shows a strong binding capacity to the immunosuppressive LILRB1/B2 receptors and significantly repolarizes the immune environment. In summary, we discovered IOMX-0675, a cross-specific antibody antagonizing both LILRB1 and LILRB2 with high selectivity, that exhibits potent reprogramming of the immunosuppressive myeloid compartment and restores cytotoxic T cell activity in the tumor microenvironment. Negligible binding to immune-activating LILR family members by IOMX-0675 could unleash full anti-tumor activity. The differential binding profile of IOMX-0675 provides best-in-class potential and may maximize anti-tumor efficacy for the benefit of patients with high-unmet medical need, who are resistant to T cell checkpoint blockade. Citation Format: Christina Hartl, Jonas Zantow, Ilona-Petra Maser, Marisa Stebegg-Wagner, Simone Friedrich, Jonas Schilz, Stefan Kaden, Ronny Milde, Eugenia Korotkova, Michail Maraslis, Bettina Langer, Michal Swiat, Carmen Ginzel, Carmen Ammerhauser, Lucille Albert, Leonie Majunke, Alina Huth, Kritika Sudan, Murray Yule, Christine Rothe, Hannes Loferer, Maximilian Aigner, Alexander N. Marziale, Stefan Bissinger. IOMX-0675, a LILRB1 and LILRB2 cross-specific antibody, effectively repolarizes immunosuppressive myeloid cells and activates T cells leading to potent tumor cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1362.

HLA-G Expression/Secretion and T-Cell Cytotoxicity in Missed Abortion in Comparison to Normal Pregnancy.

The main role of HLA-G is to protect the semi-allogeneic embryo from immune rejection by proper interaction with its cognate receptors on the maternal immune cells. Spontaneous abortion is the most common adverse pregnancy outcome, with an incidence rate between 10% and 15%, with immunologic dysregulation being thought to play a role in some of the cases. In this study, we aimed to detect the membrane and soluble HLA-G molecule at the maternal-fetal interface (MFI) and in the serum of women experiencing missed abortion (asymptomatic early pregnancy loss) in comparison to the women experiencing normal early pregnancy. In addition, the proportion of T cells and their cytotoxic profile was evaluated. We observed no difference in the spatial expression of HLA-G at the MFI and in its serum levels between the women with missed abortions and those with normal early pregnancy. In addition, comparable numbers of peripheral blood and decidual total T and γδT cells were found. In addition, as novel data we showed that missed abortion is not associated with altered extravilous invasion into uterine blood vessels and increased cytotoxicity of γδT cells. A strong signal for HLA-G on non-migrating extravilous trophoblast in the full-term normal placental bed was detected. In conclusion, HLA-G production at the MFI or in the blood of the women could not be used as a marker for normal pregnancy or missed abortions.

Cancer cell immunity-related protein co-expression networks are associated with early-stage solid-predominant lung adenocarcinoma.

Solid-predominant lung adenocarcinoma (SPA), which is one of the high-risk subtypes with poor prognosis and unsatisfactory response to chemotherapy and targeted therapy in lung adenocarcinoma, remains molecular profile unclarified. Weighted correlation network analysis (WGCNA) was used for data mining, especially for studying biological networks based on pairwise correlations between variables. This study aimed to identify disease-related protein co-expression networks associated with early-stage SPA. We assessed cancerous cells laser-microdissected from formalin-fixed paraffin-embedded (FFPE) tissues of a SPA group (n = 5), referencing a low-risk subtype, a lepidic predominant subtype group (LPA) (n = 4), and another high-risk subtype, micropapillary predominant subtype (MPA) group (n = 3) and performed mass spectrometry-based proteomic analysis. Disease-related co-expression networks associated with the SPA subtype were identified by WGCNA and their upstream regulators and causal networks were predicted by Ingenuity Pathway Analysis. Among the forty WGCNA network modules identified, two network modules were found to be associated significantly with the SPA subtype. Canonical enriched pathways were highly associated with cellular growth, proliferation, and immune response. Upregulated HLA class I molecules HLA-G and HLA-B implicated high mutation burden and T cell activation in the SPA subtype. Upstream analysis implicated the involvement of highly activated oncogenic regulators, MYC, MLXIPL, MYCN, the redox master regulator NFE2L2, and the highly inhibited LARP1, leading to oncogenic IRES-dependent translation, and also regulators of the adaptive immune response, including highly activated IFNG, TCRD, CD3-TCR, CD8A, CD8B, CD3, CD80/CD86, and highly inhibited LILRB2. Interestingly, the immune checkpoint molecule HLA-G, which is the counterpart of LILRB2, was highly expressed characteristically in the SPA subtype and might be associated with antitumor immunity. Our findings provide a disease molecular profile based on protein co-expression networks identified for the high-risk solid predominant adenocarcinoma, which will help develop future therapeutic strategies.

Analysis of HLA-G 14 bp Insertion/Deletion Polymorphism and HLA-G, ILT2 and ILT4 Expression in Head and Neck Squamous Cell Carcinoma Patients.

HLA-G is the checkpoint molecule involved in the suppression of the immune response. Increased expression of HLA-G and its ILTs receptors have been correlated with tumor progression in various cancer types. In head and neck squamous cell carcinoma (HNSCC) tumors, the effect of HLA-G, ILT2 and ILT4 expression on cancer development has to be explained. The 34 HNSCC patients and 98 controls were genotyped for the HLA-G 14 bp ins/del polymorphism. In HNSCC lesions, HLA-G, ILT2 and ILT4 mRNA expression was analysed using real-time PCR. The association between HLA-G, ILT2 and ILT4 mRNA expression and clinical variables (age at onset, TNM staging system and p16 positivity) was also evaluated. No genetic association between the HLA-G 14 bp ins/del and HNSCC risk was detected (p > 0.05). However, in the non-metastatic HNSCC group, a significantly higher HLA-G mRNA expression was noted in tumors in the T4 stage compared to those in the T1 and T2 stages (p = 0.0289). ILT2 mRNA expression was significantly increased in non-metastatic vs. metastatic tumors (p = 0.0269). Furthermore, a significantly higher ILT4 mRNA expression was noted in tumors in the T1+T2 stage compared to those in the T3 stage (p = 0.0495). Our results suggest that the HLA-G molecule creates an immunological microenvironment involved in HNSCC development.

B cell immune profiles in dysbiotic vermiform appendixes of pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid tumors and is resistant to immunotherapy. B cells play an essential role in PDAC progression and immune responses, both locally and systemically. Moreover, increasing evidence suggests that microbial compositions inside the tumor, as well as in the oral cavity and the gut, are important factors in shaping the PDAC immune landscape. However, the gut-associated lymphoid tissue (GALT) has not previously been explored in PDAC patients. In this study, we analyzed healthy vermiform appendix (VA) from 20 patients with PDAC and 32 patients with colon diseases by gene expression immune profiling, flow cytometry analysis, and microbiome sequencing. We show that the VA GALT of PDAC patients exhibits markers of increased inflammation and cytotoxic cell activity. In contrast, B cell function is decreased in PDAC VA GALT based on gene expression profiling; B cells express significantly fewer MHC class II surface receptors, whereas plasma cells express the immune checkpoint molecule HLA-G. Additionally, the vermiform appendix microbiome of PDAC patients is enriched with Klebsiella pneumoniae, Bifidobacterium animalis, and Adlercreutzia equolifaciens, while certain commensals are depleted. Our findings may suggest impaired B cell function within the GALT of PDAC patients, which could potentially be linked to microbial dysbiosis. Additional investigations are imperative to validate our observations and explore these potential targets of future therapies.

Hypomethylation-induced regulatory programs in T cells unveiled by transcriptomic analyses.

Regulatory T cells (Tregs) are essential mediators of tolerance mitigating aberrant immune responses. While naturally occurring Treg (nTreg) development and function are directed by epigenetic events, induced Treg (iTreg) identity and mechanisms of action remain elusive. Mirroring the epigenetic circuits of nTregs, we and others have used hypomethylation agents (HAs) to ex vivo convert T cells into iTregs (HA-iTregs) and further showed that the suppressive properties of the HA-iTregs are predominantly confined in an emergent population, which de novo expresses the immunomodulatory molecule HLA-G, consequently providing a surface marker for isolation of the suppressive HA-iTreg compartment (G+ cells). We isolated the HA-induced G+ cells and their G- counterparts and employed high-throughput RNA-sequencing (RNA-seq) analyses to uncover the G+-specific transcriptomic changes guiding T cells toward a regulatory trajectory upon their exposure to HA. We found a distinct transcriptional upregulation of G+ cells accompanied by enrichment of immune-response-related pathways. Although single-cell RNA-seq profiling revealed regulatory G+ cells to have molecular features akin to nTregs, when assessed in conjunction with the comparative transcriptomic analysis and profiling of secreted cytokines against the non-suppressive G- cells, FOXP3 and other T-helper signatures appear to play a minor role in their suppressive phenotype. We found an ectopic expression of IDO-1 and CCL17/22 in G+ cells, denoting that in vitro exposure of T cells to HA may well unlock myeloid suppressor genes. This report provides transcriptional data shaping the molecular identity of a highly purified and potent HA-iTreg population and hints toward ectopic myeloid-specific molecular mechanisms mediating HA-iTreg function.

Expression pattern of HLA-G and HLA-DR molecules on lymphocytes of women and their children with septal congenital heart defects

The aim of our study was to evaluate the features of HLA-G and HLA-DR expression on lymphocytes of women and their children with congenital heart defects (CHD) under the influence of allogeneic and autologous blood sera.38 women and their children with sporadic septal congenital heart defects (main group) were examined. The comparison groups included 21 women and their children without congenital heart disease (comparison group 1), as well as 17 apparently healthy men (comparison group 2). A total of 115 individuals were examined. The cross-match studies were carried out using a CytoFlex flow cytometer (Beckman Coulter, USA). The effects of autologous and allogeneic blood sera on HLA-G and HLA-DR expression on lymphocytes were evaluated. Statistical processing of the obtained results was carried out using Statistica for WINDOWS software packages from StatSoftInc. Version 10.0 and MedCalc 17.5.3. by the rules of variation statistics.The expression of HLA-G and HLA-DR molecules on the lymphocytes did not significantly change under the influence of autologous serum from men and women of children with CHD. At the same time, in women with more than two births of apparently healthy children, autologous serum significantly suppressed expression of HLA-G and HLA-DR on their lymphocytes. In particular, a pronounced and significant suppression was noted with autologous serum for HLA-DR molecules on CD3-positive lymphocytes. One may suggest that inflammation in the mother-embryo system is limited by this mechanism. Other significant differences concerned the effect of autologous and allogeneic (maternal) sera on the expression of HLA-G and HLA-DR molecules on the children’s lymphocytes. We have shown that in the group of children with septal CHD, autologous and allogeneic sera did not suppress the expression of HLA-G and HLA-DR on lymphocytes. At the same time, in the group of apparently healthy children, autologous and allogeneic (maternal) sera suppressed the expression of HLA-G and HLA-DR on lymphocytes. Moreover, the suppressive effect upon expression of both HLA-G and HLA-DR was significantly higher in allogeneic (maternal) sera than in autologous serum (p &lt; 0.01). This effect seems to be determined by the presence of autoand alloimmune antibodies to HLA-G and HLA-DR molecules in blood serum of multiparous women.The suppressor activity of female sera against allogeneic (embryo / fetus / child) and autologous (intrinsic) HLA-G and HLA-DR antigenic molecules may determine a protective effect related to development of septal congenital heart defects in offspring.

Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation.

Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.

Contribution of HLA-G and FOXP3 genes and proteins in the severity of cervical intraepithelial neoplasia during HPV infection

Human Papillomavirus (HPV) persistence leads to the chronification of cervical inflammation, where HLA-G and Foxp3; immunomodulatory molecules, may contribute to the aggravation of the lesion and cancerization. Here, we evaluated the synergic effect of these two molecules in the worsening of the lesion in presence of HPV infection.Hundred and eighty (180) women cervical cells and biopsies were collected for (i) HLAG Sanger sequencing and gene expression, and (ii) HLA-G and Foxp3 molecule expressions by immunohistochemistry.53 women were HPV+ against 127 women HPV−. HPV+ women were more at risk of having cytological changes (p ≤ 0.0123), histological changes (p < 0.0011), and cervical lesion (p = 0.0004). The HLA-G + 3142CC genotype predisposed women to infection (p = 0.0190), while HLA-G + 3142C and +3035 T alleles were associated with HLA-G5 transcript expression. Both sHLA-G (p = 0.030) and Foxp3 (p = 0.0002) proteins were higher in cervical lesion as well as in high-grade lesion. In addition, sHLA-G+ cells were positively correlated to Foxp3+ cells in presence of HPV infection and in cervical grade II/III injuries.In conclusion, HPV may use HLA-G and Foxp3 as a way of host immune escape contributing to the persistence of infection and inflammation, leading to the cervical lesion and the worsening of lesions.

Opposing impacts of HLA-G haplotypes PROMO-G010104-UTR-3 and PROMO-G010101b/c-UTR-4 on risk of recurrent implantation failure

Research questionThe human leukocyte antigen (HLA) class Ib molecules HLA-F and HLA-G are implicated in pregnancy success, but how do HLA-G and HLA-F genetic polymorphisms impact recurrent implantation failure (RIF)? DesignProspective cohort study at a fertility clinic including a cohort of 84 women experiencing RIF and 35 IVF controls to assess the influence of HLA-G haplotypes and diplotypes and HLA-F single nucleotide polymorphisms (SNP) on RIF. ResultsOver-representation trends for HLA-F SNP genotypes rs1362126, rs2523405 and rs2523393, previously linked with a short time-to-pregnancy, were detected in female control groups compared with RIF patients with no identified pathology linked to infertility. The HLA-G promoter haplotype PROMO-G010101b/c linked with the HLA-G 3’-untranslated region (3’UTR) haplotype UTR-4, which previously has been associated with positive IVF outcome and pregnancy success, was less frequent in the RIF group. For RIF patients carrying the UTR-4 haplotype, the odds ratio (OR) was 0.27 (95% CI 0.12–0.66; P = 0.0044, Pc = 0.026). The HLA-G PROMO-G010104-UTR-3 haplotype was associated with an increased risk of RIF. For RIF patients carrying the UTR-3 haplotype, the OR was 5.86 (95% CI 1.52–26.23; P = 0.0115, Pc = 0.069). ConclusionsThese results show that specific HLA-G haplotypes based on the promoter region and the 3’UTR are either associated with an increased risk of reduced fertility, including the manifestation of RIF, and lower chance of achieving pregnancy, or with a reduced risk of experiencing RIF.

Association of the HLA-G rs66554220 variant with non-segmental vitiligo and its clinical features in Northwestern Mexico population.

The HLA-G molecule functions as a critical immunomodulatory checkpoint, its expression is significantly associated with pathological processes that may be responsible in part for autoimmune conditions such as non-segmental vitiligo (NS-V), characterized by chronic skin depigmentation. In this sense, the rs66554220 (14 bp ID) variant located in the 3'UTR, implicated in the regulation of HLA-G production, is associated with autoimmune diseases. To evaluate the role of the HLA-G rs66554220 variant in NS-V and its clinical features in Northwestern Mexicans. We genotyped the rs66554220 variant by SSP-PCR in 197 NS-V patients and 198 age-sex matched non-related healthy individuals (HI). Del allele and genotype Del/Ins were the most prevalent in both study groups (NS-V/HI = 56%/55% and 46.70%/46.46%, respectively). Despite lacking association between the variant and NS-V, we found an association of the Ins allele in different inheritance models with familial clustering, onset of the illness, universal clinical subtype and Koebner's phenomenon. The rs66554220 (14 bp ID) variant is not a risk factor for NS-V in the Mexican population studied. To our knowledge, this is the first report about the topic in the Mexican population and worldwide that includes clinical features related with this HLA-G genetic variant.

Lower HLA-G levels in extravillous trophoblasts of human term placenta in gestational diabetes mellitus than in normal controls

The non-classical human leucocyte antigen (HLA) class I molecule HLA-G is widely known to play a major role in feto-maternal tolerance. We tested the hypothesis that HLA-G expression is altered in placentas of women with gestational diabetes mellitus (GDM) in a specific pattern that depends on fetal sex. HLA-G expression was analysed in a total of 80 placentas (40 placentas from women with GDM and 40 healthy controls) by immunohistochemistry using the semi-quantitative immunoreactive score (IRS). Double immunofluorescence staining identified the cells expressing HLA-G in the decidua and allowed evaluation of the expression pattern. We found a significant (p < 0.001) reduction of HLA-G expression in extravillous cytotrophoblasts (EVTs) in the placentas of women with GDM as compared to the healthy controls and were able to demonstrate that this downregulation was not due to a loss of cell number, but to a loss of expression intensity. A special change in the cell pattern of EVTs was observed, with these cells showing an obvious decrease in HLA-G expression on their cell surface. No significant differences according to fetal sex were found. These data show a possible association between decreased HLA-G expression and presence of GDM and provide new insights into altered placental function in women with GDM.

The Impact of HLA-G and HLA-E Polymorphisms on CMV Reinfection in Liver Transplant Recipients.

Cytomegalovirus (CMV) reinfection in transplant patients has been associated with graft loss and decreased patient survival. In this regard, the HLA-G molecule has the immunomodulatory characteristic and its soluble isoforms have important roles in immunity to viruses. The 14bp insertion/deletion polymorphism impacts HLA-G mRNA stability. Regarding the HLA-E molecule, two nonsynonymous alleles, HLA-E*0101, and HLA-E*0103 are different in their functions including the affinity of the relative peptide. To explore the possible link between HLA-G and HLA-E polymorphisms with CMV reinfection among liver transplant recipients (LTRs). In this study, a total of 140 liver transplantations were performed; of which 70 CMV-reactivated LTRs and 70 CMV non-reactivated ones were recruited. The cut-off value of CMV DNA was determined to be 100 copies/mL. PCR evaluated different genotypes for HLA-G and ARMS-PCR for HLA-E*0101 and *0103. Neither the HLA-G genotypes (-14 bp/-14bp and +14bp/+14 bp homozygous genotypes with the p-values: 0.43, and 0.13, respectively +14 bp⁄-14 bp heterozygous genotype with p-value: 0.49) nor the HLA-E genotypes (HLA-E*0101/0103, HLA-E*0101/0101, and HLA-E*0103/0103 with the p-values: 0.152, 0.249, and 0.391, respectively) had any association with CMV reinfection in the LTRs. No difference was observed in the HLA-E and HLA-G genotype frequencies between our studied groups. Further studies are needed to explore other genetic variations and evaluate soluble HLA-G and HLA-E levels in the transplant population.

The impact of soluble HLA-G in IVF/ICSI embryo culture medium on implantation success.

The HLA-G molecule is widely accepted as an important factor for pregnancy success. Its expression has been detected in the extravillous trophoblasts. Soluble HLA-G (sHLA-G) was found in the genital tract, pre-implanted embryos as well as in seminal fluid. In this study, we investigated the concentration of sHLA-G (sHLA-G1 and sHLA-G5) in media from 344 single cultured embryos following in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). The level of sHLA-G (U/ml) was tested with a sandwich enzyme-linked immunosorbent assay (ELISA) kit. We correlated sHLA-G secretion with ovarian stimulation protocols, the type of embryo transfer (fresh or frozen cycle) and the quality of the embryos. The ovarian stimulation protocol affects the secretion of sHLA-G by the embryo. Embryos obtained from the long agonist protocol secreted more sHLA-G than those originating from the short antagonist protocol (p = 0.0001). Embryos whose transfer resulted in a clinical pregnancy and/or live birth secreted more sHLA-G compared to those whose transfer ended without pregnancy. This was particularly observable in embryos following the long ovarian stimulation protocol and from a frozen embryo cycle. In conclusion, sHLA-G secreted by the embryo has an impact on implantation and live birth and could be a developmental potential marker of the embryo. Its concentration depends on the ovarian stimulation protocol used.

The double-sided of human leukocyte antigen-G molecules in type 1 autoimmune hepatitis.

The immunomodulatory effects of HLA-G expression and its role in cancers, human liver infections and liver transplantation are well documented, but so far, there are only a few reports addressing autoimmune liver diseases, particularly autoimmune hepatitis (AIH).Method and materialsWe analyzed the genetic and phenotypic characteristics of HLA-G in 205 type 1 AIH patients (AIH-1) and a population of 210 healthy controls from Sardinia (Italy).ResultsAnalysis of the HLA-G locus showed no substantial differences in allele frequencies between patients and the healthy control population. The HLA-G UTR-1 haplotype was the most prevalent in both AIH-1 patients and controls (40.24% and 34.29%). Strong linkage was found between the HLA-G UTR-1 haplotype and HLA-DRB1*03:01 in AIH-1 patients but not controls (D’ = 0.92 vs D’ = 0.50 respectively; P = 1.3x10-8). Soluble HLA-G (sHLA-G) levels were significantly lower in AIH-1 patients compared to controls [13.9 (11.6 – 17.4) U/mL vs 21.3 (16.5 – 27.8) U/mL; P = 0.011]. Twenty-four patients with mild or moderate inflammatory involvement, as assessed from liver biopsy, showed much higher sHLA-G levels compared to the 28 patients with severe liver inflammation [33.5 (23.6 – 44.8) U/mL vs 8.8 (6.1 – 14.5) U/mL; P = 0.003]. Finally, immunohistochemistry analysis of 52 liver biopsies from AIH-1 patients did not show expression of HLA-G molecules in the liver parenchyma. However, a percentage of 69.2% (36/52) revealed widespread expression of HLA-G both in the cytoplasm and the membrane of plasma cells labeled with anti-HLA-G monoclonal antibodies.ConclusionThis study highlights the positive immunomodulatory effect of HLA-G molecules on the clinical course of AIH-1 and how this improvement closely correlates with plasma levels of sHLA-G. However, our results open the debate on the ambiguous role of HLA-G molecules expressed by plasma cells, which are pathognomonic features of AIH-1.

Higher risk for chronic graft-versus-host disease (GvHD) in HLA-G mismatched transplants following allogeneic hematopoietic stem cell transplantation: A retrospective study.

Graft-versus-host disease (GvHD) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is highly influenced by the degree of HLA matching between recipient and donor. The HLA-class Ib molecule HLA-G has been shown to promote tolerogenicity through its interaction with inhibitory receptors found on several immunocompetent cells. We hypothesized that in an allo-HSCT setting, HLA-G mismatches may negatively impact the HLA-G-mediated tolerogenicity either due to inefficient interaction with the inhibitory receptors of the transplanted immune cells or due to direct allorecognition of mismatched HLA-G on host cells by the immune cells of the donor. In order to explore this hypothesis, we investigated the impact of HLA-G mismatching in 2.083 10/10 matched high resolution HLA-typed allo-HSCT transplants. We found that the risk of chronic GvHD was significantly higher in HLA-G-mismatched transplant cases as compared with the HLA-G-matched control group (HR: 1.46, 95%CI=1.11-1.91, p= 0.006). Sub-analysis of the mismatch vector revealed that this effect was only detectable in the GvH (HR: 1.89, 95%CI 1.39-2.57, p < 0.001) but not the HvG direction (HR: 1.01, 95%CI=0.63-1.63, p= 0.967). In addition, the negative impact of HLA-G mismatching on chronic GvHD was only significant in younger patients (<30y HR: 3.02, 95%CI=1.25-7.28, p=0.014; >29y HR: 1.28, 95%CI=0.94-1.72, p= 0.113). Our results indicate that HLA-G mismatches may contribute to the onset of chronic GvHD, especially in younger patients and should therefore be avoided when possible.

Non-Classical HLA Determinants of the Clinical Response after Autologous Stem Cell Transplantation for Systemic Sclerosis.

Systemic Sclerosis (SSc) is a chronic autoimmune disease with high morbidity and mortality. Autologous Hematopoietic Stem Cell Transplantation (AHSCT) is the best therapeutic option for rapidly progressive SSc, allowing increased survival with regression of skin and lung fibrosis. The immune determinants of the clinical response after AHSCT have yet to be well characterized. In particular, the pivotal role of the Human Leukocyte Antigen (HLA) system is not well understood, including the role of non-classical immuno-modulatory HLA-E and HLA-G molecules in developing tolerance and the role of Natural Killer cells (NK) in the immunomodulation processes. We retrospectively tested whether the genetic and/or circulating expression of the non-classical HLA-E and HLA-G loci, as well as the imputed classical HLA determinants of HLA-E expression, influence the observed clinical response to AHSCT at 12- and 24-month follow-up. In a phenotypically well-defined sample of 46 SSc patients classified as clinical responders or non-responders, we performed HLA genotyping using next-generation sequencing and circulating levels of HLA-G and quantified HLA-E soluble isoforms by ELISA. The -21HLA-B leader peptide dimorphism and the differential expression level of HLA-A and HLA-C alleles were imputed. We observed a strong trend towards better clinical response in HLA-E*01:03 or HLA-G 14bp Del allele carriers, which are known to be associated with high expression of the corresponding molecules. At 12-month post-AHSCT follow-up, higher circulating levels of soluble HLA-E were associated with higher values of modified Rodnan Skin Score (mRSS) (p = 0.0275), a proxy of disease severity. In the non-responder group, the majority of patients carried a double dose of the HLA-B Threonine leader peptide, suggesting a non-efficient inhibitory effect of the HLA-E molecules. We did not find any correlation between the soluble HLA-G levels and the observed clinical response after AHSCT. High imputed expression levels of HLA-C alleles, reflecting more efficient NK cell inhibition, correlated with low values of the mRSS 3 months after AHSCT (p = 0.0087). This first pilot analysis of HLA-E and HLA-G immuno-modulatory molecules suggests that efficient inhibition of NK cells contributes to clinical response after AHSCT for SSc. Further studies are warranted in larger patient cohorts to confirm our results.