Natural Killer Cells Promote Fetal Development through the Secretion of Growth-Promoting Factors.

Natural killer (NK) cells are present in large populations at the maternal-fetal interface during early pregnancy. However, the role of NK cells in fetal growth is unclear. Here, we have identified a CD49a+Eomes+ subset of NK cells that secreted growth-promoting factors (GPFs), including pleiotrophin and osteoglycin, in both humans and mice. The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset. Decreases in this GPF-secreting NK cell subset impaired fetal development, resulting in fetal growth restriction. The transcription factor Nfil3, but not T-bet, affected the function and the number of this decidual NK cell subset. Adoptive transfer of induced CD49a+Eomes+ NK cells reversed impaired fetal growth and rebuilt an appropriate local microenvironment. These findings reveal properties of NK cells in promoting fetal growth. In addition, this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy.

HLA-G neo-expression modifies genetic programs governing tumor cell lines.

The development of immunotherapies has proved to be clinically encouraging to re-establish the immune function modified by the expression of immune inhibitory molecules in tumors. However, there are still patients with poor survival rates following treatment. The elucidation of molecular mechanisms triggered by the neo-expression of particular IC in tumors would constitute a major step toward better understanding tumor evolution and would help to design future clinical protocols. To this end, we investigate the modifications triggered by the neo-expression of the immune checkpoints HLA-G in ccRCC tumor cells. We demonstrate, for the first time, that HLA-G modifies key genes implicated mainly in tumor development, angiogenesis, calcium flow and mitochondria dynamics. The involvement of HLA-G on the expression of genes belonging to these pathways such as ADAM-12, NCAM1 and NRP1 was confirmed by the CRISPR/Cas9-mediated edition of HLA-G. The data reveal multifaceted roles of HLA-G in tumor cells which are far beyond the well-known function of HLA-G in the immune anti-tumor response. This warrants further investigation of HLA-G and these new partners in tumors of different origin so as to propose future new treatments to improve health patient's outcome.

Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000  g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000  g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000  g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.

Three types of HLA-G+ extravillous trophoblasts that have distinct immune regulatory properties.

During pregnancy, invading HLA-G+ extravillous trophoblasts (EVT) play a key role in placental development, uterine spiral artery remodeling, and prevention of detrimental maternal immune responses to placental and fetal antigens. Failures of these processes are suggested to play a role in the development of pregnancy complications, but very little is known about the underlying mechanisms. Here we present validated methods to purify and culture primary HLA-G+ EVT from the placental disk and chorionic membrane from healthy term pregnancy. Characterization of HLA-G+ EVT from term pregnancy compared to first trimester revealed their unique phenotypes, gene expression profiles, and differing capacities to increase regulatory T cells (Treg) during coculture assays, features that cannot be captured by using surrogate cell lines or animal models. Furthermore, clinical variables including gestational age and fetal sex significantly influenced EVT biology and function. These methods and approaches form a solid basis for further investigation of the role of HLA-G+ EVT in the development of detrimental placental inflammatory responses associated with pregnancy complications, including spontaneous preterm delivery and preeclampsia.

Mutant B2M-HLA-E and B2M-HLA-G fusion proteins protects universal chimeric antigen receptor-modified T cells from allogeneic NK cell-mediated lysis.

Recent studies have indicated the antitumor activity and reduced allogeneic response of universal chimeric antigen receptor-modified T (UCAR T) cells lacking endogenous T cell receptors and beta-2 microglobulin (B2M) generated using gene-editing technologies. However, these cells are vulnerable to lysis by allogeneic natural killer (NK) cells due to their lack of human leukocyte antigen (HLA) class I molecule expression. Here, constitutive expression of mutant B2M-HLA-E (mBE) and B2M-HLA-G (mBG) fusion proteins in anti-CD19 UCAR T (UCAR T-19) cells was conducted to protect against allogeneic NK cell-mediated lysis. The ability of cells expressing mBE or mBG to resist NK cell-mediated lysis was observed in gene-edited Jurkat CAR19 cells. UCAR T-19 cells constitutively expressing the mBE and mBG fusion proteins were manufactured and showed effective and specific anti-tumor activity. Constitutive expression of the mBE and mBG fusion proteins in UCAR T-19 cells prevented allogeneic NK cell-mediated lysis. In addition, these cells were not recognizable by allogeneic T cells. Additional experiments, including those in animal models and clinical trials, are required to evaluate the safety and efficacy of UCAR T-19 cells that constitutively express mBE and mBG.

NKG2C+ NK Cells for Immunotherapy of Glioblastoma Multiforme.

In glioblastoma, non-classical human leucocyte antigen E (HLA-E) and HLA-G are frequently overexpressed. HLA-E loaded with peptides derived from HLA class I and from HLA-G contributes to inhibition of natural killer (NK) cells with expression of the inhibitory receptor CD94/NKG2A. We investigated whether NK cells expressing the activating CD94/NKG2C receptor counterpart were able to exert anti-glioma effects. NKG2C+ subsets were preferentially expanded by a feeder cell line engineered to express an artificial disulfide-stabilized trimeric HLA-E ligand (HLA-E*spG). NK cells expanded by a feeder cell line, which facilitates outgrowth of conventional NKG2A+, and fresh NK cells, were included for comparison. Expansion via the HLA-E*spG feeder cells selectively increased the fraction of NKG2C+ NK cells, which displayed a higher frequency of KIR2DL2/L3/S2 and CD16 when compared to expanded NKG2A+ NK cells. NKG2C+ NK cells exhibited increased cytotoxicity against K562 and KIR:HLA-matched and -mismatched primary glioblastoma multiforme (GBM) cells when compared to NKG2A+ NK cells and corresponding fresh NK cells. Cytotoxic responses of NKG2C+ NK cells were even more pronounced when utilizing target cells engineered with HLA-E*spG. These findings support the notion that NKG2C+ NK cells have potential therapeutic value for treating gliomas.

Immunomodulating functions of human leukocyte antigen-G and its role in graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapeutic strategy to treat several hematological malignancies and non-hematological malignancies. However, graft-versus-host disease (GVHD) is a frequent and serious transplant-related complication which dramatically restrains the curative effect of allo-HSCT and a significant cause of morbidity and mortality in allogeneic HCT recipients. Effective prevention of GVHD mainly depends on the induction of peripheral immune tolerance. Human leukocyte antigen-G (HLA-G) is a non-classical MHC class I molecule with a strong immunosuppressive function, which plays a prominent role in immune tolerance. HLA-G triggers different reactions depending on the activation state of the immune cells and system. It also exerts a long-term immune tolerance mechanism by inducing regulatory cells. In this present review, we demonstrate the immunomodulatory properties of human leukocyte antigen-G and highlight the role of HLA-G as an immune regulator of GVHD. Furthermore, HLA-G could also serve as a good predictor of GVHD and represent a new therapeutic target for GVHD.

Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.

Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the ß2-microglobulin (ß2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized ß2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with ß2m, thus accounting for ß2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.

Extended loci histocompatibility matching in HSCT-Going beyond classical HLA.

Unrelated haematopoietic stem cell transplantation (HSCT) has evolved from an experimental protocol to a potentially curative first-line treatment in a variety of haematologic malignancies. The continuous refinement of treatment protocols and supportive care paired with ongoing achievements in the technological field of histocompatibility testing enabled this transformation. Without a doubt, HLA matching is still the foremost criterion for donor selection in unrelated HSCT. However, HSCT-related treatment complications still occur frequently, often resulting in patients suffering severely or even dying as a consequence of such complications. Current literature indicates that other immune system modulating factors may play a role in the setting of HSCT. In this review, we discuss the current clinical evidence of a possible influence of nonclassical HLA antigens HLA-E, HLA-F, and HLA-G as well as the HLA-like molecules MICA and MICB, in HSCT.

HLA-G: Function, polymorphisms and pathology.

HLA-G immune modulatory genes and molecules are presently being studied by a widespread number of research groups. In the present study, we do not aim to be exhaustive since the number of manuscripts published every year is overwhelming. Instead, our aim is pointing out facts about HLA-G function, polymorphism and pathology that have been confirmed by several different researchers, together with exposing aspects that may have been overlooked or not sufficiently remarked in this productive field of study. On the other hand, we question whether performing mainly studies on HLA-G and disease associations is going to give a clear answer in the future, since 40 years of study of classical HLA molecules association with disease has still given no definite answer on this issue.

The HLA-G Immune Checkpoint Plays a Pivotal Role in the Regulation of Immune Response in Autoimmune Diseases.

The human G-leukocyte antigen (HLA-G) molecule is a non-classical major histocompatibility complex (MHC) class I molecule. The pertinence of HLA-G has been investigated in numerous studies which have sought to elucidate the relevance of HLA-G in pathologic conditions, such as autoimmune diseases, cancers, and hematologic malignancies. One of the main goals of the current research on HLA-G is to use this molecule in clinical practice, either in diagnostics or as a therapeutic target. Since HLA-G antigens are currently considered as immunomodulatory molecules that are involved in reducing inflammatory and immune responses, in this review, we decided to focus on this group of antigens as potential determinants of progression in autoimmune diseases. This article highlights what we consider as recent pivotal findings on the immunomodulatory function of HLA-G, not only to establish the role of HLA-G in the human body, but also to explain how these proteins mediate the immune response.

Expression of classical human leukocyte antigen class I antigens, HLA-E and HLA-G, is adversely prognostic in pancreatic cancer patients.

The expression of classical human leukocyte antigen class I antigens (HLA-I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA-I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA-I and non-classical HLA-I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA-I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune-related genes to characterize PDAC tumor microenvironments. Lower expression of HLA-I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA-E and HLA-G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA-I antigens was significantly correlated with shorter survival. Higher HLA-I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD-L1 and PD-L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA-I in three PDAC cell lines. It also upregulated surface expression of HLA-E, HLA-G and immune checkpoint molecules, including PD-L1 and PD-L2. These results suggest that the higher expression of HLA-I, HLA-E and HLA-G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA-I expression on PDAC cells.

Comprehensive landscape of immune-checkpoints uncovered in clear cell renal cell carcinoma reveals new and emerging therapeutic targets.

Clear cell renal cell carcinoma (ccRCC) constitutes the most common renal cell carcinoma subtype and has long been recognized as an immunogenic cancer. As such, significant attention has been directed toward optimizing immune-checkpoints (IC)-based therapies. Despite proven benefits, a substantial number of patients remain unresponsive to treatment, suggesting that yet unreported, immunosuppressive mechanisms coexist within tumors and their microenvironment. Here, we comprehensively analyzed and ranked forty-four immune-checkpoints expressed in ccRCC on the basis of in-depth analysis of RNAseq data collected from the TCGA database and advanced statistical methods designed to obtain the group of checkpoints that best discriminates tumor from healthy tissues. Immunohistochemistry and flow cytometry confirmed and enlarged the bioinformatics results. In particular, by using the recursive feature elimination method, we show that HLA-G, B7H3, PDL-1 and ILT2 are the most relevant genes that characterize ccRCC. Notably, ILT2 expression was detected for the first time on tumor cells. The levels of other ligand-receptor pairs such as CD70:CD27; 4-1BB:4-1BBL; CD40:CD40L; CD86:CTLA4; MHC-II:Lag3; CD200:CD200R; CD244:CD48 were also found highly expressed in tumors compared to adjacent non-tumor tissues. Collectively, our approach provides a comprehensible classification of forty-four IC expressed in ccRCC, some of which were never reported before to be co-expressed in ccRCC. In addition, the algorithms used allowed identifying the most relevant group that best discriminates tumor from healthy tissues. The data can potentially assist on the choice of valuable immune-therapy targets which hold potential for the development of more effective anti-tumor treatments.

Cytokine stimulation of the choriocarcinoma cell line JEG-3 leads to alterations in the HLA-G expression profile.

The checkpoint molecule human leukocyte antigen (HLA)-G has restricted tissue expression, and plays a role in the establishment of maternal tolerance to the semi-allogenic fetus during pregnancy by expression on the trophoblast cells in the placenta. HLA-G exists in at least seven well-described mRNA isoforms, of which four are membrane-bound and three soluble. Regulation of the tissue expression of HLA-G and its isoforms is relatively unknown. Therefore, it is important to understand the regulation of HLA-G, and the HLA-G+ choriocarcinoma cell line JEG-3 is a widely used cellular model. We hypothesized that cytokines present in the microenvironment can regulate the HLA-G expression profile. In the present study, we systematically stimulated JEG-3 cells with various concentrations of IL-2, IL-4 IL-6, IL-10, IL-12, IL-15, IL-17A, TGF-ß1, TNF-α and IFN-γ1b. The results suggest that IFN-γ plays a role in maintenance of HLA-G expression, while IL-10 might be involved in regulation of the isoform profile.

HLA-G: At the Interface of Maternal-Fetal Tolerance.

During pregnancy, semiallogeneic fetal extravillous trophoblasts (EVT) invade the uterine mucosa without being rejected by the maternal immune system. Several mechanisms were initially proposed by Peter Medawar half a century ago to explain this apparent violation of the laws of transplantation. Then, three decades ago, an unusual human leukocyte antigen (HLA) molecule was identified: HLA-G. Uniquely expressed in EVT, HLA-G has since become the center of the present understanding of fetus-induced immune tolerance. Despite slow progress in the field, the last few years have seen an explosion in our knowledge of HLA-G biology. Here, we critically review new insights into the mechanisms controlling the expression and function of HLA-G at the maternal-fetal interface, and discuss their relevance for fetal tolerance.

HLA-G14bp ins/del polymorphism and post-transplant weight gain in kidney transplantation: potential implications beyond tolerance.

BACKGROUND: Human leukocyte antigen (HLA)-G is a non-classical HLA molecule with immunomodulant and immunosuppressive functions, involved in transplantation tolerance. HLA-G14bp ins/del polymorphism in exon 8 has been associated with allograft rejection and kidney transplant outcome, with controversial results. We investigated associations of HLA-G14bp ins/del polymorphism on onset of some of the main post-transplant risk factors, like excess body weight, lipid abnormalities, increased fasting plasma glucose. Polymorphisms of cytokines with both immunosuppressive and metabolic effects were also assessed for comparisons and associated analysis. METHODS: The present study involved kidney transplant recipients (n = 173) in which body mass index, cholesterol, triglycerides, fasting plasma glucose were registered in the first years after transplantation and analyzed in association with genotypes. Presence of hypertension and smoking habits, demographic, transplant-related and therapeutic data of patients were also recorded. Polymerase chain reaction, sequence-specific primer amplification and Taqman allelic discrimination techniques were used for genotyping of HLA-G14bp ins/del, interleukin (IL)-10(-1082G > A,-819 T > C,-592A > C), transforming growth factor-ß(+ 869 T > C,+915C > G), IL-6(-174G > C), tumor necrosis factor-α(-308G > A) and IL-18(-137G > C,-607C > A). Effects of genotypes on clinical markers at each time point (pre-transplant and 1 to 5 years after transplant) were analyzed using a repeated-measures general linear model analysis; adjustment for potential confounders was performed. RESULTS: Results showed that HLA-G14bp ins/ins was significantly associated with obesity, in particular after transplantation (3 years, p = 0.002, OR = 4.48, 95% CI:1.76-11.41). Post-transplant body mass index was significantly increased in HLA-G14bp ins/ins carriers (3 and 4 years, p = 0.033 and p = 0.044); effects of HLA-G14bp genotypes on post-transplant BMI were confirmed by using repeated-measures analysis and after controlling for confounding variables. Cytokine genotypes did not associate with the examined factors. CONCLUSIONS: The study of transplanted patients allowed to evidence a potential relationship between post-transplant weight gain and HLA-G14bp ins/del polymorphism, previously involved in rejection for its immunosuppressive/tolerogenic activity. This novel association could widen the knowledge of the role and functions of HLA-G molecules in diseases and transplantation.

The Molecular and Functional Characteristics of HLA-G and the Interaction with Its Receptors: Where to Intervene for Cancer Immunotherapy?

Human leukocyte antigen G (HLA-G) mediates maternal-fetal immune tolerance. It is also considered an immune checkpoint in cancer since it may mediate immune evasion and thus promote tumor growth. HLA-G is, therefore, a potential target for immunotherapy. However, existing monoclonal antibodies directed against HLA-G lack sufficient specificity and are not suitable for immune checkpoint inhibition in a clinical setting. For this reason, it is essential that alternative approaches are explored to block the interaction between HLA-G and its receptors. In this review, we discuss the structure and peptide presentation of HLA-G, and its interaction with the receptors Ig-like transcript (ILT) 2, ILT4, and Killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4). Based on our findings, we propose three alternative strategies to block the interaction between HLA-G and its receptors in cancer immunotherapy: (1) prevention of HLA-G dimerization, (2) targeting the peptide-binding groove of HLA-G, and (3) targeting the HLA-G receptors. These strategies should be an important focus of future studies that aim to develop immune checkpoint inhibitors to block the interaction between HLA-G and its receptors for the treatment of cancer.

HLA-G: A New Immune Checkpoint in Cancer?

Human leukocyte antigen G (HLA-G), known as a central protein in providing immune tolerance to the fetus in pregnant women, is also studied for a possible role in tumor development. Many studies have claimed HLA-G as a new immune checkpoint in cancer. Therefore, HLA-G and its receptors might be targets for immune checkpoint blockade in cancer immunotherapy. In order to substantiate that HLA-G is indeed an immune checkpoint in cancer, two important questions need to be answered: (1) To what extent is HLA-G expressed in the tumor by cancer cells? and (2) What is the function of HLA-G in cancer immune evasion? In this review, we discuss these questions. We agree that HLA-G is a potentially new immune checkpoint in cancer, but additional evidence is required to show the extent of intra-tumor and inter-tumor expression. These studies should focus on tumor expression patterns of the seven different HLA-G isoforms and of the receptors for HLA-G. Furthermore, specific roles for the different HLA-G isoforms should be established.

Characterization of HLA-G Regulation and HLA Expression in Breast Cancer and Malignant Melanoma Cell Lines upon IFN-γ Stimulation and Inhibition of DNA Methylation.

The potential role of human leukocyte antigen (HLA)-G as a target for new cancer immunotherapy drugs has increased the interest in the analysis of mechanisms by which HLA-G expression is regulated, and how the expression can be manipulated. We characterized HLA expression in breast cancer and malignant melanoma cell lines and investigated the induction of HLA-G expression by two distinct mechanisms: stimulation with interferon (IFN)-γ or inhibition of methylation by treatment with 5-aza-2'-deoxycytidine (5-aza-dC). The effect of IFN-γ and 5-aza-dC on HLA expression was dependent on the cancer cell lines studied. However, in general, surface expression of HLA class Ia was induced on all cell lines. Surface expression of HLA-G was inconclusive but induction of HLA-G mRNA was prevalent upon treatment with 5-aza-dC and a combination of IFN-γ and 5-aza-dC. IFN-γ alone failed to induce HLA-G expression in the HLA-G-negative cell lines. The results support that HLA-G expression is regulated partly by DNA methylation. Furthermore, IFN-γ may play a role in the maintenance of HLA-G expression rather than inducing expression. The study demonstrates the feasibility of manipulating HLA expression and contributes to the exploration of mechanisms that can be potential targets for immunotherapy in breast cancer and malignant melanoma.

The Autoimmune Regulator (Aire) transactivates HLA-G gene expression in thymic epithelial cells.

The Autoimmune Regulator (Aire) protein coordinates the negative selection of developing thymocytes by inducing the expression of hundreds of tissue-specific antigens within the thymic medulla, which is also a primary site of the expression of the immune checkpoint HLA-G molecule. Considering the immunomodulatory properties of Aire and HLA-G, and considering that the role of the constitutive thymus expression of HLA-G has not been elucidated, we studied the effect of AIRE cDNA transfection on HLA-G expression in 4D6 thymic cells and in the HLA-G-positive JEG-3 choriocarcinoma cells. Aire promoted the transactivation of HLA-G gene by increasing the overall transcription, inducing the transcription of at least G1 and G2/G4 isoforms, and incrementing the occurrence and distribution of intracellular HLA-G protein solely in 4D6 thymic cells. Luciferase-based assays and chromatin immunoprecipitation experiments performed in 4D6 cells revealed that Aire targeted at least two regions within the 5'-untranslated regulatory region (5'-URR) extending 1·4 kb from the first ATG initiation codon. The interaction occurs independently of three putative Aire-binding sites. These results indicate that the Aire-induced upregulation of HLA-G in thymic cells is likely to act through the interaction of Aire with specific HLA-G 5'-URR DNA-binding factors. Such a multimeric transcriptional complex might operate in the thymus during the process of promiscuous gene expression.