PGRMC2 and HLA-G regulate immune homeostasis in a microphysiological model of human maternal-fetal membrane interface.

Chorion trophoblasts (CTCs) and immune cell-enriched decidua (DECs) comprise the maternal-fetal membrane interface called the chorio-decidual interface (CDi) which constantly gets exposed to maternal stressors without leading to labor activation. This study explored how CTCs act as a barrier at CDi. The roles of human leukocyte antigen (HLA)-G and progesterone receptor membrane component 2 (PGRMC2) in mediating immune homeostasis were also investigated. The CDi was recreated in a two-chamber microfluidic device (CDi-on-chip) with an outer chamber of primary DECs and immune cell line-derived innate immune cells and an inner chamber of wild-type or PGRMC2 or HLA-G knockout immortalized CTCs. To mimic maternal insults, DECs were treated with lipopolysaccharide, poly(I:C), or oxidative stress inducer cigarette smoke extract. Expression levels of inflammation and immunity genes via targeted RNA sequencing, production of soluble mediators, and immune cell migration into CTCs were determined. In CDi-on-chip, decidua and immune cells became inflammatory in response to insults while CTCs were refractory, highlighting their barrier function. HLA-G and PGRMC2 are found to be vital to immune homeostasis at the CDi, with PGRMC2 serving as an upstream regulator of inflammation, HLA-G expression, and mesenchymal-epithelial transition, and HLA-G serving as a frontline immunomodulatory molecule, thus preventing fetal membrane compromise.

The novel non-classical HLA-G*01:55 identified in a Chinese individual.

The non-classical HLA-G*01:55 allele differs from G*01:01:12 at one position in exon 4.

The full-length genomic sequence of the HLA-G*01:19 allele.

Full genomic sequence shows HLA-G*01:19 differs from HLA-G*01:04:01:01 only at position 99 in exon 2.

The novel non-classical HLA-G*01:46 identified in a Brazilian individual.

Non-classical HLA-G*01:46 differs from G*01:01:03:03 at one position in exon 3.

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

BACKGROUND: The 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. METHODS: First, 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 ß-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. RESULTS: We 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. CONCLUSION: The 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.

LILRB1-HLA-G axis defines a checkpoint driving natural killer cell exhaustion in tuberculosis.

Chronic infections, including Mycobacterium tuberculosis (Mtb)-caused tuberculosis (TB), can induce host immune exhaustion. However, the key checkpoint molecules involved in this process and the underlying regulatory mechanisms remain largely undefined, which impede the application of checkpoint-based immunotherapy in infectious diseases. Here, through adopting time-of-flight mass cytometry and transcriptional profiling to systematically analyze natural killer (NK) cell surface receptors, we identify leukocyte immunoglobulin like receptor B1 (LILRB1) as a critical checkpoint receptor that defines a TB-associated cell subset (LILRB1+ NK cells) and drives NK cell exhaustion in TB. Mechanistically, Mtb-infected macrophages display high expression of human leukocyte antigen-G (HLA-G), which upregulates and activates LILRB1 on NK cells to impair their functions by inhibiting mitogen-activated protein kinase (MAPK) signaling via tyrosine phosphatases SHP1/2. Furthermore, LILRB1 blockade restores NK cell-dependent anti-Mtb immunity in immuno-humanized mice. Thus, LILRB1-HLA-G axis constitutes a NK cell immune checkpoint in TB and serves as a promising immunotherapy target.

HLA-G*01:01:01:34 allele discovered in two individuals from a tribal community of Western Maharashtra, India.

HLA-G*01:01:01:34 differs from HLA-G*01:01:01:01 by one nucleotide in intron 3 at position 1432 (G to A).

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.

HLA-G isoforms, HLA-C allotype and their expressions differ between early abortus and placenta in relation to spontaneous abortions.

INTRODUCTION: Spontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. METHODOLOGY: The study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10-12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12-15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. RESULTS: Membrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%). Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. CONCLUSION: Our data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA-C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.

Role of HLA-G in tumors and upon COVID-19 infection.

HLA-G expression of tumors and upon viral infections is involved in their immune escape leading to the evasion from both T and NK cell recognition. The underlying mechanisms of HLA-G expression in both pathophysiologic conditions are broad and range from genetic abnormalities to epigenetic, transcriptional and posttranscriptional regulation. This review summarizes the current knowledge of the frequency, regulation and clinical relevance of HLA-G expression upon neoplastic and viral transformation, its interaction with components of the microenvironment as well as its potential as diagnostic marker and/or therapeutic target. In addition, it discusses urgent topics, which have to be addressed in HLA-G research.

Polymorphism of HLA and Susceptibility of Breast Cancer.

Breast cancer (BC) is the second most common malignancy in the world. Numerous studies have demonstrated the association between human leukocyte antigen (HLA) and cancer. The occurrence and development of BC are closely linked to genetic factors. Human leukocyte antigens G and E (HLA-G and HLA-E) are non-classical major histocompatibility complex (MHC) class I molecules. These molecules play an important role in immune surveillance by inhibiting the cytotoxic and natural killer T cells responsible for immune escape. The expression of HLA-G and HLA-E has been associated with several diseases, including tumors. The HLA system plays a key role in the escape of tumor cells from immune surveillance. This review aims to determine the correlation between BC susceptibility and HLA markers specific HLA alleles such as HLA-B07, HLA-DRB111, HLA-DRB113, and HLA-DRB115 are associated with an increased risk of developing BC. Furthermore, HLA-G mutations have been attributed to an elevated likelihood of metastasis in BC patients. Understanding the complex associations between the HLA system and BC development is critical for developing novel cancer prevention, detection, and treatment strategies. This review emphasizes the importance of analyzing HLA polymorphisms in the management of BC patients, as well as the urgent need for further research in this area.

[Research progress on HLA-G in non-small cell lung cancer].

Human leukocyte antigen G (HLA-G) belongs to the non-classic major histocompatibility complex class Ib (MHC Ib) molecule, including membrane and soluble isoforms. HLA-G regulates the function of various immune cells through corresponding receptors. It is one of the important immunological mechanisms of maternal-fetal tolerance and tumor immune escape. Non-small cell lung cancer (NSCLC) patients account for the highest proportion of lung cancer patients and have poor prognoses. Studies have shown that the gene polymorphism and expression level of HLA-G is closely related to the occurrence and development of NSCLC. It is suggested that HLA-G can be used as a potential biomarker for the early diagnosis, subtype differentiation, treatment, and prognosis of NSCLC patients. HLA-G has clinical value as an auxiliary diagnostic basis. An in-depth study of its mechanism can provide new strategies for the diagnosis and treatment of NSCLC patients.

The correlation between soluble human leukocyte antigen (sHLA-G) levels and +3010 polymorphism.

Human leukocyte antigen-G (HLA-G) is classified as non-classical HLA, located in the short arm of chromosome 6 and composed of seven introns and eight exons. The HLA-G gene has a lower frequency polymorphism in the coding area and higher variability at the regulatory 5'- and 3'-untranslated regions linked to HLA-G microRNA regulation. HLA-G molecule is known to have an immunomodulatory and tolerogenic features role. In 199 Saudi individuals, we examined the association between plasma soluble HLA-G (sHLA-G) levels and eight polymorphic different sites, including 14 bp ins/del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G single nucleotide polymorphisms (SNPs) in exon 8 in the HLA-G gene. Our results revealed higher frequency for rs17179101C (97%), rs1707T (92%) and rs9380142A (73%) alleles. Greater frequencies for the tested genotypes were observed in 3027C/C (rs17179101) (93%), 14 bp (rs1704) ins/del (92%), +3003T/T (rs1707) (85%) and +3035C/T (rs17179108) (79%) SNP genotypes. Moreover, we observed a significant association of sHLA-G with +3010G/C (rs1710) SNP. In conclusion, we showed a significant association between 3010G/C (rs1710) SNP and the sHLA-G level among our sample for Saudi populations. Our findings demonstrated that specific SNP within the HLA-G gene is linked to sHLA-G molecule secretion, suggesting sHLA-G levels may be regulated genetically.

Prognostic significance of HLA-G in patients with colorectal cancer: a meta-analysis and bioinformatics analysis.

PURPOSE: Human leukocyte antigen-G (HLA-G) has been reported to be aberrantly expressed in colorectal cancer (CRC); however, its prognostic value remains controversial. Hence, our meta-analysis aims to assess the prognostic value of HLA-G in CRC patients based on published literature and The Cancer Genome Atlas (TCGA) datasets. METHODS: A systematic search was conducted on relevant studies retrieved from four electronic databases including PubMed, Embase, Web of Science and Cochrane Library. Hazard ratios (HRs) with 95% confidence intervals (CIs) were recorded to be applied as effective values. Fixed-effects models or random-effects models were applied on the basis of the value of heterogeneity (I 2). Publication bias was analyzed by Begg's and Egger's tests. In addition, the results were validated by using TCGA datasets. RESULTS: Thirteen studies comprising 3896 patients were incorporated into this meta-analysis. The pooled results showed that HLA-G expression was significantly associated with poor overall survival (OS) in both the univariate analysis (HR = 1.44, 95% CI: 1.14-1.83, P = 0.002) and the multivariate analysis (HR = 1.55, 95% CI: 1.23-1.95, P < 0.001). Nevertheless, the expression of HLA-G is not related to age, sex, tumor type, tumor differentiation, TNM stage, or distant metastasis but lymph node metastasis. Notably, the prognosis of colorectal cancer was not consistent with the analysis result from TCGA data. CONCLUSION: HLA-G expression was significantly related to poor OS in CRC according to the results of our meta-analysis. However, we found that the prognostic significance was inconsistent with our results according to the TCGA data in CRC. Hence, more research is still needed to further illustrate the prognostic role of HLA-G in CRC.

Dynamics of Soluble Forms of the Immune Checkpoint Components PD-1/PD-L1/B7-H3, CD314/ULBP1, and HLA-G in Peripheral Blood of Melanoma Patients Receiving Blockers of Programmed Cell Death Protein PD-1.

The content of the soluble forms of immune checkpoint components sPD-1, sPD-L1 in blood serum, and sB7-H3, sCD314, sULBP1, sHLA-G in blood plasma of 30 melanoma patients receiving immunotherapy with anti-PD-1 antibodies (nivolumab, pembrolisumab) was measured before and in 4 and 8 weeks after the start of immunotherapy. The control group comprised 70 practically healthy donors. Standard immunoassay kits were used. In melanoma patients, the levels of sPD-L1 and sB7-H3 were significantly higher than in the control group (p<0001), sPD-1 level did not differ from the control, while sCD314 and sHLA-G levels were insignificantly decreased. During therapy, opposite changes in the levels of markers in individual patients were observed, and frequently after the initial increase (or decrease) after the first 4 weeks normalization did occur in the further 4 weeks. No statistically significant associations between the initial levels of markers and direction of their changes during treatment were found, but some trends indicating to the potential benefits from assessment of soluble forms of immune checkpoint proteins for evaluation and monitoring of the efficiency of the therapy with immune checkpoint blockers were revealed: significant decrease of sB7-H3 and sPD-1 levels in the course of treatment, higher initial sPD-1 level in patients with future progression than in those with stabilization or partial effect, and lower progression frequency in patients with increasing sPD-1 and sPD-L1 levels than in those with decreasing markers levels.

HLA-G 3'UTR haplotype analyses in HCV infection and HCV-derived cirrhosis, hepatocellular carcinoma and fibrosis.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Chronic HCV infection is also an important cause of hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV has the capacity to evade immune surveillance by altering the host immune response. Moreover, variations in immune-related genes can lead to differential susceptibility to HCV infection as well as interfere on the susceptibility to the development of hepatic fibrosis, cirrhosis and HCC. The human leucocyte antigen G (HLA-G) gene codes for an immunomodulatory protein known to be expressed in the maternal-foetal interface and in immune-privileged tissues. The HLA-G 3' untranslated region (3'UTR) is important for mRNA stability, and variants in this region are known to impact gene expression. Studies, mainly focusing in a 14 bp insertion/deletion polymorphism, have correlated HLA-G 3'UTR with susceptibility to viral infections, but other polymorphic variants in the HLA-G 3'UTR might also affect HCV infection as they are inherited as haplotypes. The present study evaluated HLA-G 3'UTR polymorphisms and performed linkage disequilibrium test and haplotype assembly in 286 HCV infected patients who have developed fibrosis, cirrhosis or HCC, as well as in 129 healthy control subjects. Haplotypes UTR-1, UTR-2 and UTR-3 were the most observed in HCV+ patients, in the frequencies of 0.276, 0.255 and 0.121, respectively. No statistically significant difference was observed between HCV+ and control subjects, even when patients were grouped according to outcome (HCC, cirrhosis or fibrosis). Despite that, some trends in the results were observed, and therefore, we cannot rule out the possibility that variants associated to high HLA-G expression can be involved in HCV infection susceptibility.

BRAF, TERT and HLA-G Status in the Papillary Thyroid Carcinoma: A Clinicopathological Association Study.

As BRAF, TERT, HLA-G, and microRNAs have been individually associated with papillary thyroid carcinoma (PTC), we aimed to evaluate the individual and collaborative role of these markers in PTC in the same patient cohort. HLA-G and BRAF tumor expression was evaluated by immunohistochemistry. Using molecular methods, BRAFV600E and TERT promoter mutations were evaluated in thyroid fine needle aspirates. MicroRNA tumor profiling was investigated using massively parallel sequencing. We observed strong HLA-G (67.96%) while BRAF (62.43%) staining was observed in PTC specimens. BRAF overexpression was associated with poor response to therapy. The BRAFV600E (52.9%) and TERTC228T (13%) mutations were associated with extrathyroidal extension, advanced-age, and advanced-stage cancer. The TERT rs2853669 CC+TC genotypes (38%) were overrepresented in metastatic tumors. Nine modulated microRNAs targeting the BRAF, TERT, and/or HLA-G genes were observed in PTC and involved with cancer-related signaling pathways. The markers were individually associated with PTC features, emphasizing the synergistic effect of BRAFV600E and TERTC228T; however, their collaborative role on PTC outcome was not fully demonstrated. The differentially expressed miRNAs targeting the BRAF and/or HLA-G genes may explain their increased expression in the tumor milieu.

HLA-G Gene Variability Is Associated with Papillary Thyroid Carcinoma Morbidity and the HLA-G Protein Profile.

Human leukocyte antigen (HLA)-G is an immune checkpoint molecule that is highly expressed in papillary thyroid carcinoma (PTC). The HLA-G gene presents several functional polymorphisms distributed across the coding and regulatory regions (5'URR: 5' upstream regulatory region and 3'UTR: 3' untranslated region) and some of them may impact HLA-G expression and human malignancy. To understand the contribution of the HLA-G genetic background in PTC, we studied the HLA-G gene variability in PTC patients in association with tumor morbidity, HLA-G tissue expression, and plasma soluble (sHLA-G) levels. We evaluated 185 PTC patients and 154 healthy controls. Polymorphic sites defining coding, regulatory and extended haplotypes were characterized by sequencing analyses. HLA-G tissue expression and plasma soluble HLA-G levels were evaluated by immunohistochemistry and ELISA, respectively. Compared to the controls, the G0104a(5'URR)G*01:04:04(coding)UTR-03(3'UTR) extended haplotype was underrepresented in the PTC patients, while G0104a(5'URR)G*01:04:01(coding)UTR-03(3'UTR) was less frequent in patients with metastatic and multifocal tumors. Decreased HLA-G tissue expression and undetectable plasma sHLA-G were associated with the G010102a(5'URR)G*01:01:02:01(coding)UTR-02(3'UTR) extended haplotype. We concluded that the HLA-G variability was associated with PTC development and morbidity, as well as the magnitude of the encoded protein expression at local and systemic levels.

Human leukocyte antigen-G 14 bp insertion/deletion polymorphism contributes to preeclampsia risk in Asian population: A systematic review and meta­analysis.

Preeclampsia remains enigmatic and responsible for vast maternal and fetal morbidity and mortality worldwide. Our objective was to assess the strength of the effect of the 14 bp deletion/insertion polymorphism in exon 8 of the 3'UTR region of the human leukocyte antigen-G (HLA-G) gene on preeclampsia risk across different populations. A systematic review by a meta-analysis was performed to summarize the scattered epidemiologic evidence, which remains inconclusive and controversial. A systematic literature search according to the PRISMA guidelines was conducted to screen relevant publications. Odds ratio and corresponding 95% confidence interval were estimated to measure the magnitude of the association between this polymorphism and preeclampsia onset. Thirty studies comprising 9402 subjects were eligible. Pooled estimates suggested that both fetal and paternal insertion variants were significantly associated with increased odds of this disease. Nevertheless, the presence of the 14 bp insertion sequence in mothers does not seem to increase the risk of preeclampsia. Moreover, the results of subgroup analysis suggested that the fetal, maternal, and paternal polymorphism has a significant deleterious impact on the preeclampsia risk in the Asian population. In addition, the significant association between the paternal polymorphism and preeclampsia in primigravida was observed in the pooled estimation with a small sample size. By summarizing the amount of significant evidence, our study nominated this polymorphism as a potential biomarker for early risk stratification for Asians. Further large-scale validation is needed to establish fully solid and conclusive evidence for the impact of the insertion polymorphism on preeclampsia risk.

HLA-G alleles and their impacts on placental HSV-1 infection in women from southern Brazil.

The Human Leukocyte Antigen G (HLA-G) is an immunoregulatory molecule with a critical role in pregnancy success. HLA-G alleles are associated with differential susceptibility to multiple conditions, including gestational problems, infectious diseases, and viral persistence. Of note, both herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) can impair HLA-G expression, interfering with HLA-G-associated immunoregulation. On the other hand, the impacts of HLA-G alleles on susceptibility to Herpesviridae infection is a neglected issue. Therefore, this study evaluated HLA-G allele frequencies and their associations with placental Herpesviridae infection in women from southern Brazil. Placenta samples were collected soon after delivery, and detection of viral DNA of HSV-1, HSV-2 and human cytomegalovirus (HCMV) was performed by polymerase chain reaction (PCR). A fragment of HLA-G (exons 2-4) was amplified by PCR, sequenced, and analyzed to allele determination. One hundred and seventy women had their alleles determined. Overall, 25 HLA-G alleles were found, distributed into 56 different genotypes. The most frequent alleles were G* 01:01:01 and G* 01:01:02, found in 37.9 % and 16.5 % of samples, respectively. Among the 170 women, 89 (52.4 %) tested positive for Herpesviridae DNA in the placenta, 55 (32.3 %) tested negative, 3 (1.8 %) were negative for HSV-1 and HSV-2 (with absent HCMV data), and 23 (13.5 %) were undetermined. The G* 01:01:01 allele was significantly associated with an increased risk of placental HSV-1 infection (p = 0.0151; OR=1.837; IC=1.108-3.045). This study describes new information concerning placental HLA-G alleles in women from southern Brazil and helps explain how genetic background can modify susceptibility to placental infections.