Decidual γδT cells of early human pregnancy produce angiogenic and immunomodulatory proteins while also possessing cytotoxic potential.

During pregnancy, the maternal immune system must allow and support the growth of the developing placenta while maintaining the integrity of the mother's body. The trophoblast's unique HLA signature is a key factor in this physiological process. This study focuses on decidual γδT cell populations and examines their expression of receptors that bind to non-classical HLA molecules, HLA-E and HLA-G. We demonstrate that decidual γδT cell subsets, including Vδ1, Vδ2, and double-negative (DN) Vδ1-/Vδ2- cells express HLA-specific regulatory receptors, such as NKG2C, NKG2A, ILT2, and KIR2DL4, each with varying dominance. Furthermore, decidual γδT cells produce cytokines (G-CSF, FGF2) and cytotoxic mediators (Granulysin, IFN-γ), suggesting functions in placental growth and pathogen defense. However, these processes seem to be controlled by factors other than trophoblast-derived non-classical HLA molecules. These findings indicate that decidual γδT cells have the potential to actively contribute to the maintenance of healthy human pregnancy.

Prognostic impact of the bone marrow tumor microenvironment, HLA-I and HLA-Ib expression in MDS and CMML progression to sAML.

Genetic aberrations and immune escape are fundamental in MDS and CMML initiation and progression to sAML. Therefore, quantitative and spatial immune cell organization, expression of immune checkpoints (ICP), classical human leukocyte antigen class I (HLA-I) and the non-classical HLA-Ib antigens were analyzed in 274 neoplastic and 50 non-neoplastic bone marrow (BM) biopsies using conventional and multiplex immunohistochemistry and correlated to publicly available dataset. Higher numbers of tissue infiltrating lymphocytes (TILs) were found in MDS/CMML (8.8%) compared to sAML (7.5%) and non-neoplastic BM (5.3%). Higher T cell abundance, including the CD8+ T cell subset, inversely correlated with the number of pathogenic mutations and was associated with blast BM counts, ICP expression, spatial T cell distribution and improved patients' survival in MDS and CMML. In MDS/CMML, higher PD-1/PD-L1/PD-L2 and HLA-I, but lower HLA-G expression correlated with a significantly better patients' outcome. Moreover, a closer spatial proximity of T cell subpopulations and their proximity to myeloid blasts showed a stronger prognostic impact when compared to TIL numbers. In sAML - the continuum of MDS and CMML - the number of TILs had no impact on prognosis, but higher CD28 and HLA-I expression correlated with a better outcome of sAML patients. This study underlines the independent prognostic value of the tumor microenvironment in MDS/CMML progression to sAML, which shows the most pronounced immune escape. Moreover, new prognostic markers, like HLA-G expression and spatial T cell distribution, were described for the first time, which might also serve as therapeutic targets.

EGFR, HLA-G, CD70, c-MET, and NY-ESO1 as potential biomarkers in high grade epithelial ovarian carcinoma.

High grade epithelial ovarian carcinoma is an aggressive tumor. Treatment includes platinum therapy, however it recurs in most patients due to therapy resistance. In this project, we study the immunohistochemical (IHC) expression of five potential biomarkers/prognostic markers in high grade epithelial ovarian carcinoma: EGFR, HLA-G, CD70, c-MET, and NY-ESO1. A cohort of 274 patients is used. We compare the IHC expression with age, stage, ascites status, family history of cancer, disease free survival (DFS) and overall survival (OS). EGFR expression is significantly correlated with family history and worse OS. HLA-G is associated with worse OS. To confirm the results of EGFR and HLA-G, a second separated cohort of 248 patients is used. Positive EGFR expression again shows worse OS, while HLA-G expression has worse prognostic trend. CD70 has a worse OS trend. C-MET and NY-ESO1 do not have any clinical correlations. EGFR can potentially serve as target in future clinical immune therapy trials.

IDO-1 impairs antitumor immunity of natural killer cells in triple-negative breast cancer via up-regulation of HLA-G.

BACKGROUND: Triple-negative breast cancers (TNBC) are highly aggressive malignancies with poor prognosis. As an essential enzyme in the tryptophan-kynurenine metabolic pathway, indoleamine 2,3 dioxygenase-1 (IDO-1) has been reported to facilitate immune escape of various tumors. However, the mechanism underlying the immunosuppressive role of IDO-1 in TNBC remains largely uncharacterized. METHODS: We examined the IDO-1 expression in 93 clinical TNBC tissues and paired adjacent normal tissues, and analyzed the regulation role of environmental cytokines like IFN-γ in IDO-1 expression. The effect of IDO-1 expression in TNBC cells on the function of NK cells were then evaluated and the underlying mechanisms were exploited. RESULTS: IDO-1 expressed in 50 of 93 (54.1%) TNBC patients. TNBC patients with high IDO-1 expression tended to have more infiltrated immune cells including NK cells, which are less active than patients with low IDO-1 expression. NK cells could produce IFN-γ, which induced IDO-1 expression in TNBC cells, whereas IDO-1 impaired the cytotoxicity of co-cultured NK cells by upregulation of HLA-G. Blockade of HLA-G improved the antitumor activity of NK cells to TNBC in vivo. CONCLUSION: TNBC cells induce dysfunction of NK cells through an IFN-γ/IDO-1/HLA-G pathway, which provide novel insights into the mechanisms of TNBC progression and demonstrate the applicability of IDO-1 and HLA-G targeting in the treatment of TNBC.

HLA-E and Its Soluble Form as Indicators of a Sex-Specific Immune Response in Patients with Oral Squamous Cell Carcinoma.

The human leukocyte antigene E (HLA-E) is associated with tumorigenesis in various cancers. Immunoncology along with sex-specific aspects in cancer therapy are now in scientific focus. Therefore, immunohistochemical HLA-E expression was retrospectively analysed in a cohort of oral squamous cell carcinomas (OSCC) after surgical therapy. Then, serum concentration of HLA-E (sHLA-E) was quantified in a prospective cohort by enzyme-linked immunosorbent assay. High HLA-E expression was associated with advanced UICC stage (Spearman's correlation: p = 0.002) and worse survival (Cox-regression: progression-free survival: hazard ratio (HR) 3.129, confidence range (CI) 1.443-6.787, p = 0.004; overall survival: HR 2.328, CI 1.071-5.060, p = 0.033). The sHLA-E concentration was significantly higher in the control group than in tumor group (Mann-Whitney U-test (MW-U): p = 0.021). Within the tumor group, women showed significantly higher sHLA-E levels than men (MW-U: p = 0.049). A closer look at the tumor group and the control group showed that gender-specific differences exist: while no differences in sHLA-E concentration were detectable between female subjects of tumor group and control group (MW-U: p = 0.916), male subjects of tumor group had a significantly lower sHLA-E concentration compared to those of control group (MW-U: p = 0.001). In summary, our results provide evidence for sex-specific differences in immune responses in OSCC. This fact should be considered regarding future immunotherapy regimens.

HLA-Ehigh /HLA-Ghigh /HLA-IIlow Human iPSC-Derived Cardiomyocytes Exhibit Low Immunogenicity for Heart Regeneration.

Although human pluripotent stem cells (hPSCs)-derived cardiomyocytes (hPSC-CMs) can remuscularize infarcted hearts and restore post-infarct cardiac function, post-transplant rejection resulting from human leukocyte antigen (HLA) mismatching is an enormous obstacle. It is crucial to identify hypoimmunogenic hPSCs for allogeneic cell therapy. This study is conducted to demonstrate the immune privilege of HLA-Ehigh /HLA-Ghigh /HLA-IIlow human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs). Ischemia-reperfusion surgery is done to create transmural myocardial infarction in rats. At post-infarct 4 days, hPSC-CMs (1.0×107 cells per kg), including human embryonic stem cell-derived cardiomyocytes (hESC-CMs), HLA-Elow/HLA-Glow/HLA-IIhigh hiPSC-CMs, and HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs, are injected into the infarcted myocardium. Under the treatment of very low dose cyclosporine A (CsA), only HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs survive in vivo and improved post-infarct cardiac function with infarct size reduction. HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs activate the SHP-1 signaling pathway of natural killer (NK) cells and cytotoxic T cells to evade attack by NK cells and cytotoxic T cells. Herein, it is demonstrated that using a clinically relevant CsA dose, HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs repair the infarcted myocardium and restore the post-infarct heart function. HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSCs are less immunogenic and may serve as platforms for regeneration medicine.

The Upregulation of HLA-G1 and miRNA-34a in Lens Epithelial Cells of Diabetic Retinopathy Patients.

Background: Retinopathy of diabetes is a chronic diabetes mellitus complication affecting retinal vessels, and some ocular complications' molecular mechanisms remain obscure. Objective: To evaluate the expression of HLA-G1, HLA-G5, miRNA-181a, and miRNA-34a in the lens epithelial cells of patients with retinopathy of diabetes. Methods: In a case-control study, 30 diabetic patients with retinopathy, 30 diabetic patients without retinopathy, and 30 cataract patients without diabetes mellitus as the control group were enrolled after a full description with details about the study methods and objectives. The expression of HLA G1, HLA G5, miRNA-181a, and miRNA-34a in lens epithelial cells was assessed by quantitative RT PCR. Moreover, the levels of HLA-G protein in aqueous humor were evaluated by the ELISA method. Results: HLA-G1 expression was significantly upregulated in the retinopathy group (P=0.003). The aqueous humor of diabetic retinopathy patients contained significantly higher levels of HLA-G protein compared with the non-diabetic patients (P=0.001). miRNA-181a was significantly downregulated in the diabetic retinopathy group compared with the patients without diabetes (P=0.001). In addition, miRNA-34a was upregulated in the retinopathy group (P=0.009). Conclusion: Taken together, the present results showed that HLA-G1 and miRNA-34a can be valuable markers for diabetic retinopathy. Our data offers new perspectives for improving the control of inflammation in the lens epithelial cells by considering HLA-G and miRNA.

Application of a JEG-3 organoid model to study HLA-G function in the trophoblast.

The human placenta is a unique temporary organ with a mysterious immune tolerance. The formation of trophoblast organoids has advanced the study of placental development. HLA-G is uniquely expressed in the extravillous trophoblast (EVT) and has been linked to placental disorders. With older experimental methodologies, the role of HLA-G in trophoblast function beyond immunomodulation is still contested, as is its role during trophoblast differentiation. Organoid models incorporating CRISPR/Cas9 technology were used to examine the role of HLA-G in trophoblast function and differentiation. JEG-3 trophoblast organoids (JEG-3-ORGs) were established that highly expressed trophoblast representative markers and had the capacity to differentiate into EVT. CRISPR/Cas9 based on HLA-G knockout (KO) significantly altered the trophoblast immunomodulatory effect on the cytotoxicity of natural killer cells, as well as the trophoblast regulatory effect on HUVEC angiogenesis, but had no effect on the proliferation and invasion of JEG-3 cells and the formation of TB-ORGs. RNA-sequencing analysis further demonstrated that JEG-3 KO cells followed similar biological pathways as their wild-type counterparts during the formation of TB-ORGs. In addition, neither HLA-G KO nor the exogenous addition of HLA-G protein during EVT differentiation from JEG-3-ORGs altered the temporal expression of the known EVT marker genes. Based on the JEG-3 KO (disruption of exons 2 and 3) cell line and the TB-ORGs model, it was determined that HLA-G has a negligible effect on trophoblast invasion and differentiation. Despite this, JEG-3-ORG remains a valuable model for studying trophoblast differentiation.

BiTE-Secreting CAR-γδT as a Dual Targeting Strategy for the Treatment of Solid Tumors.

HLA-G is considered as an immune checkpoint protein and a tumor-associated antigen. In the previous work, it is reported that CAR-NK targeting of HLA-G can be used to treat certain solid tumors. However, the frequent co-expression of PD-L1 and HLA-G) and up-regulation of PD-L1 after adoptive immunotherapy may decrease the effectiveness of HLA-G-CAR. Therefore, simultaneous targeting of HLA-G and PD-L1 by multi-specific CAR could represent an appropriate solution. Furthermore, gamma-delta T (γδT) cells exhibit MHC-independent cytotoxicity against tumor cells and possess allogeneic potential. The utilization of nanobodies offers flexibility for CAR engineering and the ability to recognize novel epitopes. In this study, Vδ2 γδT cells are used as effector cells and electroporated with an mRNA-driven, nanobody-based HLA-G-CAR with a secreted PD-L1/CD3ε Bispecific T-cell engager (BiTE) construct (Nb-CAR.BiTE). Both in vivo and in vitro experiments reveal that the Nb-CAR.BiTE-γδT cells could effectively eliminate PD-L1 and/or HLA-G-positive solid tumors. The secreted PD-L1/CD3ε Nb-BiTE can not only redirect Nb-CAR-γδT but also recruit un-transduced bystander T cells against tumor cells expressing PD-L1, thereby enhancing the activity of Nb-CAR-γδT therapy. Furthermore, evidence is provided that Nb-CAR.BiTE redirectes γδT into tumor-implanted tissues and that the secreted Nb-BiTE is restricted to the tumor site without apparent toxicity.

Peripheral HLA-G/ILT-2 immune checkpoint axis in acute and convalescent COVID-19 patients.

The immunosuppressive non-classical human leukocyte antigen-G (HLA-G) can elicits pro-viral activities by down-modulating immune responses. We analysed soluble forms of HLA-G, IL-6 and IL-10 as well as on immune effector cell expression of HLA-G and its cognate ILT-2 receptor in peripheral blood obtained from hospitalised and convalescent COVID-19 patients. Compared with convalescents (N = 202), circulating soluble HLA-G levels (total and vesicular-bound molecules) were significantly increased in hospitalised patients (N = 93) irrespective of the disease severity. During COVID-19, IL-6 and IL-10 levels were also elevated. Regarding the immune checkpoint expression of HLA-G/ILT-2 on peripheral immune effector cells, the frequencies of membrane-bound HLA-G on CD3+ and CD14+ cells were almost identical in patients during and post COVID-19, while the frequency of ILT-2 receptor on CD3+ and CD14+ cells was increased during acute infection. A multi-parametric correlation analysis of soluble HLA-G forms with IL-6, IL-10, activation markers CD25 and CD154, HLA-G, and ILT-2 expression on immune cells revealed a strong positive correlation of soluble HLA-G forms with membrane-bound HLA-G molecules on CD3+/CD14+ cells only in convalescents. During COVID-19, only vesicular-bound HLA-G were positively correlated with the activation marker CD25 on T cells. Thus, our data suggest that the elevated levels of soluble HLA-G in COVID-19 are due to increased expression in organ tissues other than circulating immune effector cells. The concomitant increased expression of soluble HLA-G and ILT-2 receptor frequencies supports the concept that the immune checkpoint HLA-G/ILT-2 plays a role in the immune-pathogenesis of COVID-19.

A Microphysiological Device to Model the Choriodecidual Interface Immune Status during Pregnancy.

During human pregnancy the chorion (fetal) lines decidua (maternal) creating the feto-maternal interface. Despite their proximity, resident decidual immune cells remain quiescent during gestation and do not invade the chorion. Infection and infiltration of activated immune cells toward the chorion are often associated with preterm birth. However, the mechanisms that maintain choriodecidual immune homeostasis or compromise immune barrier functions remain unclear. To understand these processes, a two-chamber microphysiological system (MPS) was created to model the human choriodecidual immune interface under normal and infectious conditions in vitro. This MPS has outer (fetal chorion trophoblast cells) and inner chambers (maternal decidual + CD45+ cells [70:30 ratio]) connected by microchannels. Decidual cells were treated with LPS to mimic maternal infection, followed by immunostaining for HLA-DR and HLA-G, immune panel screening by imaging cytometry by time of flight, and immune regulatory factors IL-8 and IL-10, soluble HLA-G, and progesterone (ELISA). LPS induced a proinflammatory phenotype in the decidua characterized by a decrease in HLA-DR and an increase in IL-8 compared with controls. LPS treatment increased the influx of immune cells into the chorion, indicative of chorionitis. Cytometry by time of flight characterized immune cells in both chambers as active NK cells and neutrophils, with a decrease in the abundance of nonproinflammatory cytokine-producing NK cells and T cells. Conversely, chorion cells increased progesterone and soluble HLA-G production while maintaining HLA-G expression. These results highlight the utility of MPS to model choriodecidual immune cell infiltration and determine the complex maternal-fetal crosstalk to regulate immune balance during infection.

Cadmium inhibits differentiation of human trophoblast stem cells into extravillous trophoblasts and disrupts epigenetic changes within the promoter region of the HLA-G gene.

Cadmium (Cd) is a toxic heavy metal widely distributed in the environment. Maternal whole-blood Cd levels during pregnancy are positively associated with the risk of early preterm birth. We hypothesized that Cd inhibits trophoblast differentiation, resulting in the development of hypertensive disorders of pregnancy and a high risk of early preterm birth. Using the CT27 human trophoblast stem cell line, we found that exposing these cells to 0.1-0.4 µM Cd inhibited their differentiation into extravillous cytotrophoblasts (EVTs). Supporting this finding, we found that expression of the metal-binding protein metallothionein, which suppresses the toxicity of Cd, is low in EVTs. We also found that Cd exposure changes the methylation status of the promoter region of the HLA-G gene, which is specifically expressed in EVTs. Together, these results suggest that Cd inhibits placental formation by suppressing trophoblast differentiation into EVTs. This suppression may underlie the increased risk of gestational hypertension in women with high whole-blood Cd levels.

INPP5A/HLA-G1/IL-10/MMP-21 Axis in Progression of Esophageal Squamous Cell Carcinoma

Background: Background: Type I inositol polyphosphate-5-phosphatase A (INPP5A) is involved in different cellular events, including cell proliferation. Since INPP5A, HLAG1, IL-10, and matrix metalloproteinases (MMP)-21 genes play fundamental roles in esophageal squamous cell carcinoma (ESCC) tumorigenesis, we aimed in this study to clarify the possible interplay of these genes and explore the potential of these chemistries as a predictor marker for diagnosis in ESCC disease. Methods: Methods: Gene expression analysis of INPP5A, HLAG-1, IL-10, and MMP-21 was performed using relative comparative real-time PCR in 56 ESCCs compared to their margin normal tissues. Immunohistochemical staining was accomplished for INPP5A in ESCCs. Analysis of ROC curves and the AUC were applied to evaluate the diagnostic capability of the candidate genes. Results: Results: High levels of HLA-G1, MMP-21, and IL-10 were detected in nearly 23.2%, 62.5%, and 53.5% of ESCCs compared to the normal tissues, respectively, whereas INPP5A underexpression was detected in 19.6% of ESCCs, which all tested genes indicated significant correlations with each other. The protein expression level of INPP5A in ESCC tissues was significantly lower than that of the non-tumor esophageal tissues (p = 0.001). Interestingly, the concomitant expression of the INPP5A/HLA-G1, INPP5A/MMP-21, INPP5A/IL-10, HLA-G1/MMP-21, HLA-G1/IL-10, and MMP-21/IL-10 was significantly correlated with several clinicopathological variables. INPP5A, HLA-G1, MMP-21, and IL-10 showed to be the most appropriate candidates to discriminate tumor/non-tumor groups due to the total AUCs of all combinations (>60%). Conclusion: Conclusion: Our results represent a new regulatory axis containing INPP5A/HLAG-1/IL-10/MMP-21 markers in ESCC development and may provide novel insight into the mechanism of immune evasion mediated by the INPP5A/HLAG-1/IL-10/MMP-21 regulatory network in the disease.

[HADHA Inhibits the Migration and Invasion of HTR-8/SVneo Cells by Regulating PI3K/AKT Signaling Pathway].

Objective: To explore the effects of hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) on the migration and invasion of HTR-8/SVneo cells, a human trophoblast cell line, and its potential mechanism of action. Methods: Immunofluorescence staining was done to evaluate the expression levels of HADHA in samples of normal villi and recurrent spontaneous abortion (RSA) villi at 6-8 weeks. Lentiviral infection system was used to construct stable HTR-8/SVneo cell lines with HADHA overexpression and knockdown. Western blot, qRT-PCR, Wound-healing assay, and Transwell assay were used to determine the effect of HADHA on the migration and invasion of HTR-8/SVneo cells and the expression of relevant genes. Transcriptome sequencing and bioinformatics analysis were done to screen for the potential target genes and signaling pathways regulated by HADHA. The specific molecular mechanism of how HADHA regulates the migration and invasion of HTR-8/SVneo cells was examined by adding the inhibitor of protein kinase B (PKB/AKT). Results: HADHA was highly expressed in extravillous trophoblasts (EVT) of RSA villus samples as compared with samples from the normal control group. In HTR-8/SVneo cells overexpressing HADHA, the expression levels of migration and invasion-related genes, including HLA-G, MMP2, MMP9, and NCAD, were decreased (P<0.01,P<0.05), and the migration and invasion abilities of HTR-8/SVneo cells were weakened (P<0.05). HADHA knockdown increased the expression levels of HLA-G, MMP2, MMP9, and NCAD (P<0.01, P<0.05), and promoted the migration and invasion of HTR-8/SVneo cells (P<0.05). In addition, HADHA overexpression decreased the phosphorylation levels of PI3K and AKT (P<0.05) and inhibited the PI3K/AKT signaling pathway. HADHA knockdown activated the PI3K/AKT signaling pathway. When MK-2206, an AKT inhibitor, was added to stable HTR-8/SVneo cell lines with HADHA knockdown, the migration and invasion of the cells were significantly reduced. Conclusion: HADHA inhibits the migration and invasion of HTR-8/SVneo cells by inhibiting the PI3K/AKT signaling pathway.

BND-22, a first-in-class humanized ILT2-blocking antibody, promotes antitumor immunity and tumor regression.

BACKGROUND: Cancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages. METHODS: We describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems. RESULTS: Collectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies. CONCLUSIONS: BND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer. TRIAL REGISTRATION NUMBER: NCT04717375.

Epigenetic/genetic variations in CG-rich elements of immune-related genes contribute to food allergy development during childhood.

BACKGROUND: Genetic areas of FOXP3 TSDR, human leukocyte antigen-G (HLA-G) upstream of CpG island 96, CpG41 and CpG73 islands of the HLA-DRB1 and HLA-DQB1 genes respectively, previously documented to display immune-modulatory properties, were subjected to epigenetic/genetic analysis to assess their influence in IgE-mediated food allergy (FA) development in children. METHODS: Sixty-four orally challenged and IgE-tested food allergic subjects together with 44 controls were recruited. Targeted pyrosequencing analysis to detect DNA methylation status and genetic variations was utilized and experimental results obtained were analyzed by a statistical software platform and correlated to clinical data. Also, transcription factor (TF) binding sites in study areas were unmasked by the JASPAR prediction database. RESULTS: Parents' smoking was significantly correlated with aberrant methylation patterns, regardless of food allergic or control status. HLA-G promoter region showed a trend for hypomethylation in food allergic subjects, with one of the CG sites displaying significantly decreased methylation values. Rs1233333, residing within the HLA-G promoter region preserved a protective role toward DNA methylation. Variable methylation patterns were recorded for CpG41 of the HLA-DRB1 gene and hypermethylation of the region was significantly correlated with the presence of single nucleotide polymorphisms (SNPs). TFs' recognition sites, located in studied genetic areas and exerting pivotal regulatory biological roles, are potentially affected by divergent DNA methylation status. CONCLUSIONS: We propose that HLA-G expression is triggered by food-derived allergens, providing a TregFoxP3-/HLA-G+ subpopulation generation to promote direct immune tolerance. Furthermore, clear evidence is provided for the underlying co-operation of genetic polymorphisms with epigenetic events, mainly at the CpG41 island of the HLA-DRB1 gene, which needs an extended investigation and elucidation.

Endometrial HLA-F expression is influenced by genotypes and correlates differently with immune cell infiltration in IVF and recurrent implantation failure patients.

STUDY QUESTION: Is human leukocyte antigen (HLA)-F protein expressed in mid-secretory endometrium, and are its expression levels influenced by HLA-F gene polymorphisms and correlated with the abundance of uterine natural killer (uNK) cells and anti-inflammatory M2 macrophages? SUMMARY ANSWER: HLA-F protein is expressed in mid-secretory endometrium, and levels are correlated with immune cell infiltration, plasma progesterone concentrations and HLA-F single-nucleotide polymorphisms (SNPs), however, women experiencing recurrent implantation failure (RIF) show differences when compared to women attending their first IVF treatment. WHAT IS KNOWN ALREADY: The immunomodulatory HLA class Ib molecules HLA-G and HLA-F are expressed on the extravillous trophoblast cells and interact with receptors on maternal immune cells. Little is known regarding HLA-F expression in endometrial stroma and HLA-F function; furthermore, HLA-F and HLA-G SNP genotypes and haplotypes have been correlated with differences in time-to-pregnancy. STUDY DESIGN, SIZE, DURATION: Primary endometrial stromal cell (ESC) cultures (n = 5) were established from endometrial biopsies from women attending IVF treatment at a fertility clinic. Basic HLA-F and HLA-G protein expression by the ESCs were investigated. A prospective controlled cohort study was performed including 85 women with a history of RIF and 36 control women beginning their first fertility treatment and with no history of RIF. In some analyses, the RIF group was divided into unknown cause, male infertility, female infertility, and both female and male infertility. Endometrial biopsies and blood samples were obtained the day equivalent to embryo transfer in a hormone-substituted cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: HLA protein expression by ESCs was characterized using flow cytometry and western blot. In the cohort study, the specific immune markers HLA-F and HLA-G, CD56 and CD16 (NK cells), CD163 (M2 macrophages), FOXP3 (regulatory T cells) and CD138 (plasma cells) were analysed by immunohistochemistry and a digital image analysis system in endometrial biopsies. Endometrial receptivity was assessed by an endometrial receptivity array test (the ERA® test). Endometrial biopsies were examined according to modified Noyes' criteria. SNPs at the HLA-F gene and HLA-G haplotypes were determined. MAIN RESULTS AND THE ROLE OF CHANCE: HLA-F protein is expressed in the endometrium at the time of implantation. Furthermore, the HLA-F protein levels were different according to the womens HLA-F SNP genotypes and diplotypes, which have previously been correlated with differences in time-to-pregnancy. Endometrial HLA-F was positively correlated with anti-inflammatory CD163+ M2 macrophage infiltration and CD56+ uNK cell abundance for the entire cohort. However, this was not the case for CD56+ in the female infertility RIF subgroup. HLA-F levels in the endometrial stroma were negatively correlated with plasma progesterone concentrations in the RIF subgroup with known female infertility. Conversely, HLA-F and progesterone were positively correlated in the RIF subgroup with infertility of the male partner and no infertility diagnosis of the woman indicating interconnections between progesterone, HLA-F and immune cell infiltration. Glandular sHLA-G expression was also positively correlated with uNK cell abundance in the RIF subgroup with no female infertility but negatively correlated in the RIF subgroup with a female infertility diagnosis. LARGE SCALE DATA: Immunohistochemistry analyses of endometrial biopsies and DNA sequencing of HLA genes. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The control group of women attending their first IVF treatment had an anticipated good prognosis but was not proven fertile. A significant age difference between the RIF group and the IVF group reflects the longer treatment period for women with a history of RIF. The standardization of hormonal endometrial preparation, which allowed consistent timing of endometrial and blood sampling, might be a strength because a more uniform hormonal background may more clearly show an influence on the immune marker profile and HLA class Ib levels in the endometrium by other factors, for example genetic polymorphisms. However, the immune marker profile might be different during a normal cycle. WIDER IMPLICATIONS OF THE FINDINGS: The findings further highlight the importance of HLA-F and HLA-G at the implantation site and in early pregnancy for pregnancy success. Diagnostic measures and modulation of the complex interactions between HLA class Ib molecules, maternal immune cells and hormonal factors may have potential to improve fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Region Zealand Health Sciences Research Foundation and the Zealand University Hospital through the ReproHealth Research Consortium ZUH. The authors declared there are no conflicts of interest.

HLAncPred: a method for predicting promiscuous non-classical HLA binding sites.

Human leukocyte antigens (HLA) regulate various innate and adaptive immune responses and play a crucial immunomodulatory role. Recent studies revealed that non-classical HLA-(HLA-E & HLA-G) based immunotherapies have many advantages over traditional HLA-based immunotherapy, particularly against cancer and COVID-19 infection. In the last two decades, several methods have been developed to predict the binders of classical HLA alleles. In contrast, limited attempts have been made to develop methods for predicting non-classical HLA binding peptides, due to the scarcity of sufficient experimental data. Of note, in order to facilitate the scientific community, we have developed an artificial intelligence-based method for predicting binders of class-Ib HLA alleles. All the models were trained and tested on experimentally validated data obtained from the recent release of IEDB. The machine learning models achieved more than 0.98 AUC for HLA-G alleles on validation dataset. Similarly, our models achieved the highest AUC of 0.96 and 0.94 on the validation dataset for HLA-E*01:01 and HLA-E*01:03, respectively. We have summarized the models developed in the past for non-classical HLA and validated the performance with the models developed in this study. Moreover, to facilitate the community, we have utilized our tool for predicting the potential non-classical HLA binding peptides in the spike protein of different variants of virus causing COVID-19, including Omicron (B.1.1.529). One of the major challenges in the field of immunotherapy is to identify the promiscuous binders or antigenic regions that can bind to a large number of HLA alleles. To predict the promiscuous binders for the non-classical HLA alleles, we developed a web server HLAncPred (https://webs.iiitd.edu.in/raghava/hlancpred) and standalone package.

Primary Trophoblast Cultures: Characterization of HLA Profiles and Immune Cell Interactions.

Introduction: Trophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells. Methods: After enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT. Results: The trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-α and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR. Conclusion: We verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions.

HLA-G and CD152 Expression Levels Encourage the Use of Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells as an Alternative for Immunosuppressive Therapy.

Mesenchymal stromal cells (MSCs) have been used in immunosuppressive therapy due to their therapeutic effects, with the HLA-G molecule seeming to play a fundamental role. This work evaluated alternative MSC sources to bone marrow (BM), namely, umbilical cord tissue (UC), adipose tissue (AD) and dental pulp tissue (DP), and the influence of interferon-γ (IFN-γ) and hypoxia on the cultivation of these cells for use in immunosuppression therapies. Expression of costimulatory markers CD40, CD80 and CD86 and immunosuppressive molecules CD152 and HLA-G was analyzed. Lymphocyte inhibition assays were also performed. Sequencing of the HLA-G gene from exons 1 to 5 was performed using next-generation sequencing to determine the presence of alleles. UC-derived MSCs (UCMSCs) expressed higher CD152 and HLA-G1 under standard cultivation. UCMSCs and DP-derived MSCs (DPSCs) secreted similar levels of HLA-G5. All MSC sources inhibited the proliferation of peripheral blood mononuclear cells (PBMCs); growth under regular versus hypoxic conditions resulted in similar levels of inhibition. When IFN-γ was added, PBMC growth was inhibited to a lesser extent by UCMSCs. The HLA-G*01:04:01:01 allele appears to generate a more efficient MSC response in inhibiting lymphocyte proliferation. However, the strength of this conclusion was limited by the small sample size. UCMSCs are an excellent alternative to BM in immunosuppressive therapy: they express high concentrations of inhibitory molecules and can be cultivated without stimuli, which minimizes cost.