The deubiquitinase USP2a promotes tumor immunosuppression by stabilizing immune checkpoint B7-H4 in lung adenocarcinoma harboring EGFR-activating mutants.

B7-H4 is an immune checkpoint crucial for inhibiting CD8+ T-cell activity. A clinical trial is underway to investigate B7-H4 as a potential immunotherapeutic agent. However, the regulatory mechanism of B7-H4 degradation via the ubiquitin-proteasome pathway (UPP) remains poorly understood. In this study, we discovered that proteasome inhibitors effectively increased B7-H4 expression, while EGFR-activating mutants promoted B7-H4 expression through the UPP. We screened B7-H4 binding proteins by co-immunoprecipitation and mass spectrometry and found that USP2a acted as a deubiquitinase of B7-H4 by removing K48- and K63-linked ubiquitin chains from B7-H4, leading to a reduction in B7-H4 degradation. EGFR mutants enhanced B7-H4 stability by upregulating USP2a expression. We further investigated the role of USP2a in tumor growth in vivo. Depletion of USP2a in L858R/LLC cells inhibited tumor cell proliferation, consequently suppressing tumor growth in immune-deficient nude mice by destabilizing downstream molecules such as Cyclin D1. In an immune-competent C57BL/6 mouse tumor model, USP2a abrogation facilitated infiltration of CD95+CD8+ effector T cells and hindered infiltration of Tim-3+CD8+ and LAG-3+CD8+ exhausted T cells by destabilizing B7-H4. Clinical lung adenocarcinoma samples showed a significant correlation between B7-H4 abundance and USP2a expression, indicating the contribution of the EGFR/USP2a/B7-H4 axis to tumor immunosuppression. In summary, this study elucidates the dual effects of USP2a in tumor growth by stabilizing Cyclin D1, promoting tumor cell proliferation, and stabilizing B7-H4, contributing to tumor immunosuppression. Therefore, USP2a represents a potential target for tumor therapy.

Dehydroepiandrosterone attenuated the immune escape of oral squamous cell carcinoma through NF-κB p65/miR-15b-5p/B7-H4 axis.

OBJECTIVES: We aimed to explore the effects and mechanisms of action of dehydroepiandrosterone (DHEA) on immune evasion of oral squamous cell carcinoma (OSCC) to provide evidence for enhancing the effect of immunotherapy. MATERIALS AND METHODS: A xenograft mouse model and immunohistochemistry were used to reveal the patterns of tumor-infiltrating lymphocytes (TILs). The CAL27 and SCC VII cell lines were used for the in vitro study. Western blotting, qPCR, immunofluorescence, and flow cytometry were used to evaluate the expression of B7-H4. Recombinant mouse B7-H4 protein (rmB7-H4) and PG490, an inhibitor of NF-κB p65 were used for the "rescue study." Gain- and loss-of-function, luciferase reporter, and chromatin immunoprecipitation assays were performed to verify this mechanism. RESULTS: DHEA inhibited tumor growth in an OSCC xenograft mouse model, increased CD8 + cells, and decreased FOXP3 + cells in TILs. DHEA reduced the expression of B7-H4 in CAL27 and SCC VII cells RmB7-H4 reverses the effect of DHEA on tumor growth and TIL patterns. DHEA increased the expression of miR-15b-5p and activated its transcriptional factor NF-κB p65. Further experiments demonstrated that miR-15b-5p inhibited B7-H4 expression by binding to its 3'-UTR regions, and NF-κB p65 activated miR-15b transcription. PG490 reversed the effects of DHEA on tumor growth, antitumor immunity in the OSCC xenograft model, and the expression/phosphorylation of NF-κB p65, miR-15b-5p, and B7-H4. CONCLUSIONS: This study indicates that DHEA attenuates the immune escape of OSCC cells by inhibiting B7-H4 expression, providing new insights for cancer immunotherapy.

Aberrant expression of B7-H4 and B7-H5 contributes to the development of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (CSCC) is the second most common malignant tumor of the skin. B7 homolog 4 (B7-H4) and B7-H5 (B7 homolog 5) are associated with a variety of tumors. Investigate the potential role of B7-H4 and B7-H5 in regulating the tumorigenesis and progression of CSCC. B7-H4 and B7-H5 transcriptome data were collected from GEO and TCGA databases and subjected to bioinformatical analysis by protein-protein interaction (PPI) network, functional enrichment analysis, immune analysis, and drug-gene interaction prediction analysis. We characterized the expression of B7-H4 and B7-H5 in carcinoma tissues of CSCC patients by immunohistochemistry. Meanwhile, the clinical correlation of B7-H4 and B7-H5 in CSCC was explored by statistical analysis. B7-H4 and B7-H5 genes were under-expressed in CSCC and correlated with tumor staging. According to GO and KEGG Pathway enrichment analysis, B7-H4, and B7-H5 can regulate the proliferation and activation of T cells, lymphocytes, and monocytes, and the expression of cytokines, such as IL-6 and IL-10, in CSCC. B7-H4 and B7-H5 are also jointly involved in the occurrence and development of CSCC via the JAK-STAT and Notch signaling pathways. We found that B7-H4 and B7-H5 proteins were abnormally highly expressed in CSCC tissue and correlated with tumor size and stage. Our findings offer new insights into the pathogenesis of CSCC and suggest that B7-H4 and B7-H5 are novel tissue biomarkers and promising therapeutic targets for CSCC.

B7-H4 reduces the infiltration of CD8+T cells and induces their anti-tumor dysfunction in gliomas.

B7-H4 is a promising immune checkpoint molecule in tumor immunotherapy. Our previous study showed that high B7-H4 expression was strongly correlated with deficiency in tumor infiltrated lymphocytes (TILs) in glioma patients. On this basis, we investigated the impact of B7-H4 on CD8+TILs in gliomas and the associated molecular mechanism here. B7-H4-positive tumor samples (n=129) from our glioma cohort were used to assess B7-H4 expression and CD8+TIL quantification by immunohistochemistry. CD8+TILs from five glioma patients cultured with B7-H4 protein were used to evaluate anti-tumor dysfunction by flow cytometry and ELISpot. An orthotopic murine glioma model was used to investigate the role of B7-H4 in glioma CD8+TILs by immunohisto- chemistry and flow cytometry. CD8+TILs from glioma patients cultured with B7-H4 protein were used to explore the potential molecular mechanism by RNA sequencing and western blot. Our results showed that glioma CD8+TIL density was negatively correlated with B7-H4 expression both in glioma patient cohort (P < 0.05) and orthotopic glioma murine model (P < 0.01). B7-H4 also lowered the expression of CD137 and CD103 (P < 0.05 for both) in glioma CD8+TILs and reduced their secretion of the anti-tumor cytokines IFN-γ and TNF-α (P < 0.01 for both) in a dose-dependent manner. Furthermore, B7-H4 was found to induce early dysfunction of glioma CD8+TILs by downregulating the phosphorylation of AKT and eNOS (P < 0.05 for both). In conclusion, B7-H4 reduced the infiltration of glioma CD8+TILs and induced an anti-tumor dysfunction phenotype. B7-H4 may also impair the anti-tumor function of glioma CD8+TILs via the AKT-eNOS pathway. These results indicated that B7-H4 may serve as a potential target in future glioma immunotherapy.

Prognostic Value of B7H4 Expression in Patients with Solid Cancers: A Systematic Review and Meta-Analysis.

V-set domain-containing T-cell activation inhibitor 1 (aliases VTCN1, B7H4) participates in tumour immune escape by delivering inhibitory signals to T cells. The purpose of this article was to assess the B7H4 prognostic value in solid cancers. Three databases were searched for relevant articles. The main endpoints were overall survival (OS), disease-specific survival (DSS), progression-free survival (PFS), recurrence-free survival (RFS), and disease-free survival (DFS). Appropriate hazard ratios (HRs) were pooled. The R studio software (version 4.0.3) was used for data analysis. Thirty-one studies met the inclusion criteria. High expression of B7H4 was associated with worse OS (HR = 1.52, 95% CI: 1.37-1.68) but not with DSS (HR = 1.14, 95% CI: 0.49-2.63), RFS (HR = 1.77, 95% CI: 0.75-4.18), DFS (HR = 1.29, 95% CI: 0.8-2.09), or PFS (HR = 1.71, 95% CI: 0.91-3.2) in patients with solid cancers. High expression of B7H4 is associated with a poorer prognosis in patients with solid cancers. B7H4 is a promising prognostic biomarker and immunotherapeutic target for various solid cancers because of its activity in cancer immunity and tumourigenesis.

Epithelial Expressed B7-H4 Drives Differential Immunotherapy Response in Murine and Human Breast Cancer.

Combinations of immune checkpoint inhibitors (ICI, including anti-PD-1/PD-L1) and chemotherapy have been FDA approved for metastatic and early-stage triple-negative breast cancer (TNBC), but most patients do not benefit. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with anti-PD-L1 resistance. However, little is known about its regulation and effect on immune cell function in breast cancers. We assessed murine and human breast cancer cells to identify regulation mechanisms of B7-H4 in vitro. We used an immunocompetent anti-PD-L1-sensitive orthotopic mammary cancer model and induced ectopic expression of B7-H4. We assessed therapy response and transcriptional changes at baseline and under treatment with anti-PD-L1. We observed B7-H4 was highly associated with epithelial cell status and transcription factors and found to be regulated by PI3K activity. EMT6 tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. In addition, tumor-infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. Paradoxically, in human breast cancer, B7-H4 expression was associated with survival benefit for patients with metastatic TNBC treated with carboplatin plus anti-PD-L1 and was associated with no change in response or survival for patients with early breast cancer receiving chemotherapy plus anti-PD-1. While B7-H4 induces tumor resistance to anti-PD-L1 in murine models, there are alternative mechanisms of signaling and function in human cancers. In addition, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 independent of PD-L1. SIGNIFICANCE: This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target.

Antibody-Drug Conjugates: Advancing from Magic Bullet to Biological Missile.

Precision drug development is focusing on targeting tumor cell surface proteins for therapeutic delivery, maximizing biomarker identified on-target damage to the tumor while minimizing toxicity. A recent article demonstrated high expression of B7-H4 antigen on resistant ovarian cancer cells and described preclinical activity of B7-H4-directed antibody-drug conjugate. See related article by Gitto et al., p. 1567.

A SOX9-B7x axis safeguards dedifferentiated tumor cells from immune surveillance to drive breast cancer progression.

How dedifferentiated stem-like tumor cells evade immunosurveillance remains poorly understood. We show that the lineage-plasticity regulator SOX9, which is upregulated in dedifferentiated tumor cells, limits the number of infiltrating T lymphocytes in premalignant lesions of mouse basal-like breast cancer. SOX9-mediated immunosuppression is required for the progression of in situ tumors to invasive carcinoma. SOX9 induces the expression of immune checkpoint B7x/B7-H4 through STAT3 activation and direct transcriptional regulation. B7x is upregulated in dedifferentiated tumor cells and protects them from immunosurveillance. B7x also protects mammary gland regeneration in immunocompetent mice. In advanced tumors, B7x targeting inhibits tumor growth and overcomes resistance to anti-PD-L1 immunotherapy. In human breast cancer, SOX9 and B7x expression are correlated and associated with reduced CD8+ T cell infiltration. This study, using mouse models, cell lines, and patient samples, identifies a dedifferentiation-associated immunosuppression mechanism and demonstrates the therapeutic potential of targeting the SOX9-B7x pathway in basal-like breast cancer.

Expression analysis of inhibitory B7 family members in Alzheimer's disease.

Alzheimer's disease (AD) is a global health problem due to its complexity, which frequently makes the development of treatment methods extremely difficult. Therefore, new methodologies are necessary to investigate the pathophysiology of AD and to treat AD. The interaction of immune modulation and neurodegeneration has added new dimensions in current knowledge of AD etiology and offers an attractive opportunity for the discovery of novel biomarkers and therapies. Using quantitative polymerase chain reaction, we compared the expression levels of inhibitory B7 family members (B7-1, B7-2, B7-H1, B7-DC, B7-H3, B7-H4, B7-H5, B7-H7, and ILDR2), as immune regulators, in the peripheral blood of late-onset AD (LOAD) patients (n = 50) and healthy individuals (n = 50). The levels of B7-2, B7-H4, ILDR2, and B7-DC expression were significantly higher in-patient blood samples than in control blood samples. Furthermore, we discovered a substantial positive correlation between all gene expression levels. In addition, the current study indicated that ILDR2, B7-H4, B7-2, and B7-DC might serve as diagnostic biomarkers to identify LOAD patients from healthy persons. The present work provides additional evidence for the significance of inhibitory B7 family members to the etiology of LOAD.

Spatial Immunoprofiling of Adenoid Cystic Carcinoma Reveals B7-H4 Is a Therapeutic Target for Aggressive Tumors.

PURPOSE: Adenoid cystic carcinoma (ACC) is a heterogeneous malignancy, and no effective systemic therapy exists for metastatic disease. We previously described two prognostic ACC molecular subtypes with distinct therapeutic vulnerabilities, ACC-I and ACC-II. In this study, we explored the ACC tumor microenvironment (TME) using RNA-sequencing and spatial biology to identify potential therapeutic targets. EXPERIMENTAL DESIGN: Tumor samples from 62 ACC patients with available RNA-sequencing data that had been collected as part of previous studies were stained with a panel of 28 validated metal-tagged antibodies. Imaging mass cytometry (IMC) was performed using the Fluidigm Helios CyTOF instrument and analyzed with Visiopharm software. The B7-H4 antibody-drug conjugate AZD8205 was tested in ACC patient-derived xenografts (PDX). RESULTS: RNA deconvolution revealed that most ACCs are immunologically "cold," with approximately 30% being "hot." ACC-I tumors with a poor prognosis harbored a higher density of immune cells; however, spatial analysis by IMC revealed that ACC-I immune cells were significantly restricted to the stroma, characterizing an immune-excluded TME. ACC-I tumors overexpressed the immune checkpoint B7-H4, and the degree of immune exclusion was directly correlated with B7-H4 expression levels, an independent predictor of poor survival. Two ACC-I/B7-H4-high PDXs obtained 90% complete responses to a single dose of AZD8205, but none were observed with isotype-conjugated payload or in an ACC-II/B7-H4 low PDX. CONCLUSIONS: Spatial analysis revealed that ACC subtypes have distinct TMEs, with enrichment of ACC-I immune cells that are restricted to the stroma. B7-H4 is highly expressed in poor-prognosis ACC-I subtype and is a potential therapeutic target.

The immune checkpoint molecule, VTCN1/B7-H4, guides differentiation and suppresses proinflammatory responses and MHC class I expression in an embryonic stem cell-derived model of human trophoblast.

The placenta acts as a protective barrier to pathogens and other harmful substances present in the maternal circulation throughout pregnancy. Disruption of placental development can lead to complications of pregnancy such as preeclampsia, intrauterine growth retardation and preterm birth. In previous work, we have shown that expression of the immune checkpoint regulator, B7-H4/VTCN1, is increased upon differentiation of human embryonic stem cells (hESC) to an in vitro model of primitive trophoblast (TB), that VTCN1/B7-H4 is expressed in first trimester but not term human placenta and that primitive trophoblast may be uniquely susceptible to certain pathogens. Here we report on the role of VTCN1 in trophoblast lineage development and anti-viral responses and the effects of changes in these processes on major histocompatibility complex (MHC) class I expression and peripheral NK cell phenotypes.

The stromal tumor-infiltrating lymphocytes, cancer stemness, epithelial-mesenchymal transition, and B7-H4 expression in ovarian serous carcinoma.

BACKGROUND: B7-H4 is expressed in various types of cancers and its expression inversely correlates with the degree of tumor-infiltrating lymphocytes (TILs). Studies have shown the relationship between B7-H4, cancer stem cell (CSC) properties, and epithelial-mesenchymal transition (EMT) in various cancers. However, very few studies have investigated the relationship between B7-H4, TILs, cancer stemness, and EMT in epithelial ovarian cancer (EOC). The present study aimed to elucidate whether B7-H4 is involved in immune evasion and examine whether B7-H4 is associated with cancer stemness or EMT in ovarian serous carcinoma, the most common type of EOC. The clinical significance of B7-H4 was also investigated to evaluate its potential as a therapeutic target. METHODS: A total of 145 patients included in this study. The degree of stromal TILs was evaluated using hematoxylin and eosin (H&E)-stained slides. Immunohistochemical analysis of B7-H4, CSC-related biomarkers (CD24, CD44s, CD133, and ALDH1), and EMT-related biomarkers (E-cadherin, N-cadherin, and vimentin) was performed using tissue microarray. qRT-PCR for VTCN1, CD24, CD44, PROM1, ALDH1, CDH1, CDH2, and VIM genes was performed on 38 frozen tissue samples. The mRNA expression levels were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) online analysis tool. RESULTS: B7-H4 protein expression positively correlated with the degree of stromal TILs. CD24, CD44s, and CD133 expression showed a positive correlation with B7-H4 expression at both the protein and mRNA levels, but ALDH1 correlated only at the protein level. E-cadherin expression was positively correlated with B7-H4 expression at both the protein and mRNA levels. N-cadherin and vimentin expression was inversely related to B7-H4 expression only at the mRNA level. B7-H4 positive patients were associated with higher tumor grade and lower overall survival rate than B7-H4 negative patients, especially in ovarian serous carcinoma with low stromal TILs. CONCLUSIONS: The present study demonstrates that B7-H4 may not be involved in the immune evasion mechanism, but is involved in cancer stemness and mesenchymal-epithelial transition. In addition, B7-H4 may be a therapeutic target for the treatment of ovarian serous carcinoma, especially with low stromal TILs.

Effect of B7-H4 downregulation induced by Toxoplasma gondii infection on dysfunction of decidual macrophages contributes to adverse pregnancy outcomes.

BACKGROUND: Toxoplasma gondii infection during pregnancy can lead to fetal defect(s) or congenital complications. The inhibitory molecule B7-H4 expressed on decidual macrophages (dMφ) plays an important role in maternal-fetal tolerance. However, the effect of B7-H4 on the function of dMφ during T. gondii infection remains unclear. METHODS: Changes in B7-H4 expression on dMφ after T. gondii infection were explored both in vivo and in vitro. B7-H4-/- pregnant mice (pregnant mice with B7-H4 gene knockout) and purified primary human dMφ treated with B7-H4 neutralizing antibody were used to explore the role of B7-H4 signaling on regulating the membrane molecules, synthesis of arginine metabolic enzymes and cytokine production by dMφ with T. gondii infection. Also, adoptive transfer of dMφ from wild-type (WT) pregnant mice or B7-H4-/- pregnant mice to infected B7-H4-/- pregnant mice was used to examine the effect of B7-H4 on adverse pregnancy outcomes induced by T. gondii infection. RESULTS: The results illustrated that B7-H4-/- pregnant mice infected by T. gondii had poorer pregnancy outcomes than their wild-type counterparts. The expression of B7-H4 on dMφ significantly decreased after T. gondii infection, which resulted in the polarization of dMφ from the M2 toward the M1 phenotype by changing the expression of membrane molecules (CD80, CD86, CD163, CD206), synthesis of arginine metabolic enzymes (Arg-1, iNOS) and production of cytokines (IL-10, TNF-α) production. Also, we found that the B7-H4 downregulation after T. gondii infection increased iNOS and TNF-α expression mediated through the JAK2/STAT1 signaling pathway. In addition, adoptive transfer of dMφ from a WT pregnant mouse donor rather than from a B7-H4-/- pregnant mouse donor was able to improve adverse pregnancy outcomes induced by T. gondii infection. CONCLUSIONS: The results demonstrated that the downregulation of B7-H4 induced by T. gondii infection led to the dysfunction of decidual macrophages and contributed to abnormal pregnancy outcomes. Moreover, adoptive transfer of B7-H4+ dMφ could improve adverse pregnancy outcomes induced by T. gondii infection.

[Expression and significance of immune checkpoint B7-homolog 4 in endometrial cancer].

Objective: To investigate the expression of B7 homolog 4 (B7-H4) and its clinical significance in endometrial cancer. Methods: A total of 833 patients with endometrial cancer admitted to Peking Union Medical College Hospital, Chinese Academy of Medical Sciences from 2009 to 2019, were enrolled. The expression of B7-H4, mismatch repair (MMR), p53, programmed cell death ligand 1 (PD-L1) protein, and CD8+ T lymphocyte density in endometrial cancer tissues were detected by the EnVision two-step method of immunohistochemical staining. First-generation sequencing (Sanger method) was used to determine molecular subtyping of endometrial cancer. The χ2 test was used to compare the differences in positive expression rate of B7-H4 protein in endometrial cancer tissues with different clinicopathological features and molecular subtyping, PD-L1 protein expression, and CD8+ T lymphocyte density. Survival analyses [including recurrence-free survival (RFS) and disease-specific survival (DSS)] were performed for 664 patients with follow-up time≥3 months, with a median follow-up time of 31 months (range: 4-121 months), and the Cox proportional hazards regression model was used to analyze the relevant factors affecting the prognosis of patients with endometrial cancer. Results: (1) The median age of 833 patients was 58 years (range: 25-88 years); pathological type: 595 with endometrioid carcinoma, 238 with non-endometrioid carcinoma; surgical-pathological staging: 542 cases at stage Ⅰ, 38 cases at stage Ⅱ, 173 cases at stage Ⅲ, and 45 cases at stage Ⅳ. Molecular subtyping was performed in 590 patients, including 50 with POLE mutation, 163 with mismatch repair defect (MMR-d) type, 246 with nospecific molecular change (NSMP) type, and 131 with p53 mutation subtype. (2) B7-H4 protein was expressed with brownish-yellow stainind in the cell membrane and cytoplasm of endometrial carcinoma, and the positivity rate of B7-H4 protein was 71.5% (596/833). The positivity rates of B7-H4 protein among patients with different age, surgical-pathological stage, tumor grade, pathological type, depth of muscular invasion, presence or absence of lymphovascular space invasion, and molecular subtype were significantly different (all P<0.05). The positivity rates of B7-H4 protein among patients with different PD-L1 protein expression and CD8+ T lymphocyte density were not significantly different (P>0.05). The 5-year RFS (83.9%) and DSS (87.3%) of B7-H4 protein-positive patients had an increasing trend compared with the 5-year RFS (77.2%) and DSS (78.1%) of B7-H4 protein-negative patients, but there were not statistically significant differences (P=0.053, P=0.083). (3) Univariate analysis showed that the 5-year RFS and DSS of patients with different age, tumor grade, surgical-pathological stage, pathological type, depth of muscular invasion, lymphovascular space invasion, and molecular subtype were significantly different (all P<0.01). There were no significant differences in 5-year RFS (P=0.184, P=0.113) and DSS (P=0.549, P=0.247) among patients with different CD8+ T lymphocyte density and PD-L1 protein expression. Further analysis according to molecular subtype, the results of CD8+ T lymphocyte density and PD-L1 protein expression showed that the 5-year RFS and DSS of B7-H4 protein-positive patients were higher than those of B7-H4 protein-negative patients with NSMP subtype, low density of CD8+ T lymphocyte and PD-L1 protein-negative endometrial carcinoma (all P<0.05), however, there was no significant difference in 5-year DSS between B7-H4 protein-positive patients and B7-H4 protein-negative patients with PD-L1 protein-negative endometrial cancer (P=0.060). Multivariate analysis showed that positive expression of B7-H4 protein was an independent factor for 5-year RFS (HR=0.27, 95%CI: 0.09-0.78, P=0.016) and DSS (HR=0.16, 95%CI: 0.05-0.58, P=0.005) in patients with NSMP subtype endometrial carcinoma. In patients with low-density CD8+ T lymphocytes endometrial cancer, positive expression of B7-H4 protein was an independent factor for 5-year RFS (HR=0.45, 95%CI: 0.26-0.80, P=0.006), but it was not an independent factor for 5-year DSS. In patients with PD-L1 protein-negative endometrial cancer, B7-H4 protein was not an independent factor for 5-year RFS. Conclusion: B7-H4 protein expressed highly in endometrial carcinoma tissues, and its high expression is closely related to clinicopathological features, molecular subtype of p53 mutant and NSMP, and the favorable prognosis of patients with low density of CD8+ T lymphocyte immunophenotype endometrial carcinoma.

V-set domain containing T-cell activation inhibitor-1 (VTCN1): A potential target for the treatment of autoimmune diseases.

Autoimmunity eventuates when the immune system attacks self-molecules as a result of the breakdown in immune tolerance. Targeting autoimmune diseases via immunomodulation has become an essential strategy in today's era. A B7 superfamily member immune checkpoint, the V-set domain containing T-cell activation inhibitor-1 (VTCN1), also known as B7-H4, B7S1, and B7x, is involved in negatively regulating T-cell activation. VTCN1 transcript has been reported in various lymphoid and non-lymphoid tissues, but its protein expression is restricted, indicating its translational regulation. Dysregulation of VTCN1 has resulted in the exacerbation of various autoimmune diseases. Moreover, increased soluble form of VTCN1 in the patient's sera positively correlates with the disease progression and severity. The current review summarizes all the reports till date, unfolding the role of VTCN1 in various autoimmune diseases and its therapeutic potential.

The immunological role of B7-H4 in pregnant women with Sars-Cov2 infection.

PROBLEM: T-cells are key players in fighting the coronavirus disease 2019 (COVID-19). The checkpoint molecule B7-H4, a member of the B7 family, can inhibit T-cell activation and proliferation by inhibiting NF-kb expression. We aimed to elucidate the immunological role of soluble B7-H4 (sB7-H4) and B7-H4 in pregnant women suffered from an acute Sars-Cov2 infection. METHODS: Expression levels of sB7-H4 and cytokines were detected by enzyme linked immunosorbent assay. B7-H4 and cytokines mRNA expression was analyzed by qPCR, and B7-H4 and NF-κb (p65) protein levels were investigated by western blot and immunofluorescence staining in placenta chorionic villous and decidual basalis tissues of COVID-19 affected women and healthy controls. RESULTS: Fibrinoid necrosis in the periphery of placental villi was increased in the COVID-19-affected patients. sB7-H4 protein in maternal and cord blood serum and IL-6/IL-10 were increased while leukocytes were decreased during SARS-CoV-2 infection. Serum sB7-H4 level was increased according to the severity of SARS-Cov-2 infection. Cytokines (IL-6, IL-18, IL-1ß, TNF-α), B7-H4 mRNA and protein in the decidual basalis tissues of COVID-19-infected pregnant women were significantly increased compared to healthy controls. IL-18 and IL-1ß were significantly increased in the placenta chorionic villous samples of COVID-19 affected patients, while NF-κb (p65) expression was decreased. CONCLUSIONS: The expression of the immunological marker sB7-H4 correlated with the severity of COVID-19 disease in pregnant women. sB7-H4 and B7-H4 can be used to monitor the progression of COVID-19 infection during pregnancy, and for evaluating of the maternal immune status.

Exosomal B7-H4 from irradiated glioblastoma cells contributes to increase FoxP3 expression of differentiating Th1 cells and promotes tumor growth.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor. Although numerous postoperative therapeutic strategies have already been developed, including radiotherapy, tumors inevitably recur after several years of treatment. The coinhibitory molecule B7-H4 negatively regulates T cell immune responses and promotes immune escape. Exosomes mediate intercellular communication and initiate immune evasion in the tumor microenvironment (TME). OBJECTIVE: This study aimed to determine whether B7-H4 is upregulated by radiation and loaded into exosomes, thus contributing to immunosuppression and enhancing tumor growth. METHODS: Iodixanol density-gradient centrifugation and flow cytometry were used to verify exosomal B7-H4. Naïve T cells were differentiated into Th1 cells, with or without exosomes. T cell-secreted cytokines and markers of T cell subsets were measured. Mechanistically, the roles of B7-H4, and ALIX in GBM were analyzed using databases and tissue samples. Co-immunoprecipitation, and pull-down assays were used to tested the direct interactions between ATM and ALIX or STAT3. In vitro ATM kinase assays, western blotting, and site-directed mutation were used to assess ATM-mediated STAT3 phosphorylation. Finally, the contribution of exosomal B7-H4 to immunosuppression and tumor growth was investigated in vivo. RESULTS: Exosomes from irradiated GBM cells decreased the anti-tumor immune response of T cell in vitro and in vivo via delivered B7-H4. Mechanistically, irradiation promoted exosome biogenesis by increasing the ATM-ALIX interaction. Furthermore, the ATM-phosphorylated STAT3 was found to directly binds to the B7-H4 promoter to increase its expression. Finally, the radiation-induced increase in exosomal B7-H4 induced FoxP3 expression during Th1 cell differentiation via the activated STAT1 pathway. In vivo, exosomal B7-H4 decreased the radiation sensitivity of GBM cells, and reduced the survival of GBM mice model. CONCLUSION: This study showed that radiation-enhanced exosomal B7-H4 promoted immunosuppression and tumor growth, hence defining a direct link between irradiation and anti-tumor immune responses. Our results suggest that co-administration of radiotherapy with anti-B7-H4 therapy could improve local tumor control and identify exosomal B7-H4 as a potential tumor biomarker.

Altered Levels of Negative Costimulatory Molecule V-Set Domain-Containing T-Cell Activation Inhibitor-1 (VTCN1) and Metalloprotease Nardilysin (NRD1) are Associated with Generalized Active Vitiligo.

BACKGROUND: Vitiligo is characterized by depigmented macules on the skin caused due to autoimmune destruction of melanocytes. V-set domain-containing T-cell activation inhibitor-1 (VTCN1) is a negative costimulatory molecule that plays a vital role in suppressing autoimmunity and tuning immune response. Nardilysin (NRD1), a metalloproteinase, cleaves membrane-tethered VTCN1 resulting in the shedding of soluble-VTCN1 (sVTCN1). However, the role of VTCN1 and NRD1 in vitiligo pathogenesis is unexplored. OBJECTIVES AND METHODS: This study was aimed to (i) Investigate the association of VTCN1 intronic polymorphisms (rs10923223 T/C and rs12046117 C/T) with vitiligo susceptibility in Gujarat population by using Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) (ii) Estimate VTCN1 & NRD1 transcript levels from peripheral blood mononuclear cells (PBMCs) and skin samples of vitiligo patients by real-time PCR, (iii) Estimate sVTCN1 and NRD1 protein levels from plasma by ELISA and (iv) Estimate VTCN1 protein levels in the skin samples of vitiligo patients by immunofluorescence. RESULTS: The analysis revealed increased VTCN1 and NRD1 transcript levels in the skin (p = .039, p = .021 respectively), increased sVTCN1 and NRD1 levels (p = .026, p = .015 respectively) in the plasma, and decreased VTCN1 protein levels (p = .0002) in the skin of vitiligo patients as compared to healthy controls. The genetic analysis revealed no significant association of VTCN1 intronic polymorphisms rs10923223 T/C and rs12046117 C/T with vitiligo susceptibility in Gujarat population (p = .359, p = .937, respectively). CONCLUSIONS: The present study revealed altered VTCN1 and NRD1 expressions in the blood and skin of vitiligo patients, suggesting their potential role in the development and progression of Vitiligo.

Aryl Hydrocarbon Receptor Directly Regulates VTCN1 Gene Expression in MCF-7 Cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.