[The Roles of N6-Methyladenosine Modification and Its Regulators in Male Reproduction]. / N6-ç”²åŸºè ºå˜Œå‘¤ä¿®é¥°åŠå ¶è°ƒæŽ§å› å­åœ¨ç”·æ€§ç”Ÿæ®–ä¸­çš„ä½œç”¨.

Infertility affects an estimated 10 to 15 percent of couples worldwide, with approximately half of the cases attributed to male-related issues. Most men diagnosed with infertility exhibit symptoms such as oligospermia, asthenospermia, azoospermia, and compromised sperm quality. Spermatogenesis is a complex and tightly coordinated process of germ cell differentiation, precisely regulated at transcriptional, posttranscriptional, and translational levels to ensure stage-specific gene expression during the development of spermatogenic cells and normal spermiogenesis. N6-methyladenosine (m6A) stands out as the most prevalent modification on eukaryotic mRNA, playing pivotal roles in various biological processes, including mRNA splicing, transportation, and translation. RNA methylation modification is a dynamic and reversible process primarily mediated by "writers", removed by "erasers", and recognized by "readers". In mammals, the aberrant methylation modification of m6A on mRNA is associated with a variety of diseases, including male infertility. However, the precise involvement of disrupted m6A modification in the pathogenesis of human male infertility remains unresolved. Intriguingly, a significant correlation has been found between the expression levels of m6A regulators in the testis and the severity of sperm concentration, motility, and morphology. Aberrant expression patterns of m6A regulatory proteins have been detected in anomalous human semen samples, including those of oligospermia, asthenozoospermia, and azoospermia. Furthermore, the examination of both sperm samples and testicular tissues revealed abnormal mRNA m6A modification, leading to reduced sperm motility and concentration in infertile men. Consequently, it is hypothesized that dysregulation of m6A modification might serve as an integral link in the mechanism of male infertility. This paper presents a comprehensive review of the recent discoveries regarding the spatial and temporal expression dynamics of m6A regulators in testicular tissues and the correlation between deregulated m6A regulators and human male infertility. Previous studies predominantly utilized constitutive or conditional knockout animal models for testicular phenotypic investigations. However, gene suppression in additional tissues could potentially influence the testis in constitutive knockout models. Furthermore, considering the compromised spermatogenesis observed in constitutive animals, distinguishing between the indirect effects of gene depletion on testicular development and its direct impact on the spermatogenic process is challenging, due to their intricate relationship. Such confounding factors might compromise the validity of the findings. To address this challenge, an inducible and conditional gene knockout model may serve as a superior approach. To date, nearly all reported studies have concentrated solely on the level changes of m6A and its regulators in germs cells, while the understanding of the function of m6A modification in testicular somatic cells remains limited. Testicular somatic cells, including peritubular myoid cells, Sertoli cells, and Leydig cells, play indispensable roles during spermatogenesis. Hence, comprehensive exploration of m6A modification within these cells as an additional crucial regulatory mechanism is warranted. In addition, exploration into the presence of unique methylation mechanisms or m6A regulatory factors within the testes is warranted. To elucidate the role of m6A modification in germ cells and testicular somatic cells, detailed experimental strategies need to be implemented. Among them, manipulation of the levels of key enzymes involved in m6A methylation and demethylation might be the most effective approach. Moreover, comprehensive analysis of the gene expression profiles involved in various signaling pathways, such as Wnt/ß-catenin, Ras/MAPK, and Hippo, in m6A-modified germ cells and testicular somatic cells can provide more insight into its regulatory role in the spermatogenesis process. Further research in this area could provide valuable insights for developing innovative strategies to treat male infertility. Finally, considering the mitigation impact of m6A imbalance regulation on disease, investigation concerning whether restoring the equilibrium of m6A modification regulation can restore normal spermatogenesis function is essential, potentially elucidating the pivotal clinical significance of m6A modulation in male infertility.

HSF5 Deficiency Causes Male Infertility Involving Spermatogenic Arrest at Meiotic Prophase I in Humans and Mice.

Meiosis is a specialized cell division process that generates gametes for sexual reproduction. However, the factors and underlying mechanisms involving meiotic progression remain largely unknown, especially in humans. Here, it is first showed that HSF5 is associated with human spermatogenesis. Patients with a pathogenic variant of HSF5 are completely infertile. Testicular histologic findings in the patients reveal rare postmeiotic germ cells resulting from meiotic prophase I arrest. Hsf5 knockout (KO) mice confirms that the loss of HSF5 causes defects in meiotic recombination, crossover formation, sex chromosome synapsis, and sex chromosome inactivation (MSCI), which may contribute to spermatocyte arrest at the late pachytene stage. Importantly, spermatogenic arrest can be rescued by compensatory HSF5 adeno-associated virus injection into KO mouse testes. Mechanistically, integrated analysis of RNA sequencing and chromatin immunoprecipitation sequencing data revealed that HSF5 predominantly binds to promoters of key genes involved in crossover formation (e.g., HFM1, MSH5 and MLH3), synapsis (e.g., SYCP1, SYCP2 and SYCE3), recombination (TEX15), and MSCI (MDC1) and further regulates their transcription during meiotic progression. Taken together, the study demonstrates that HSF5 modulates the transcriptome to ensure meiotic progression in humans and mice. These findings will aid in genetic diagnosis of and potential treatments for male infertility.

The engineered agonistic anti-CD40 antibody potentiates the antitumor effects of ß-glucan by resetting TAMs.

Anti-CD40 antibodies (Abs) have been shown to induce antitumor T-cell responses. We reported that the engineered agonistic anti-CD40 Ab (5C11, IgG4 isotype) recognized human CD40 antigen expressed on a human B lymphoblastoid cell line as well as on splenic cells isolated from humanized CD40 mice. Of note, a single high dosage of 5C11 was able to prohibit tumor growth in parallel with an increase in the population of infiltrated CD8+ T cells. Furthermore, the antitumor effects of 5C11 were enhanced in the presence of ß-glucan along with an increase in the population of infiltrated CD8+ T cells. In addition, the numbers of CD86+ TAMs and neutrophils were elevated in the combination of 5C11 and ß-glucan compared with either 5C11 or ß-glucan alone. Furthermore, the abundance of Faecalibaculum, one of the probiotics critical for tumor suppression, was obviously increased in the combination of 5C11 and ß-glucan-treated mice. These data reveal a novel mechanism of tumor suppression upon the combination treatment of 5C11 and ß-glucan and propose that the combination treatment of agonistic anti-human CD40 antibody 5C11 and ß-glucan could be a promising therapeutic strategy for cancer patients.

Klinefelter syndrome: Etiology and clinical considerations in male infertility.

Klinefelter syndrome (KS) is the most prevalent chromosomal disorder occurring in males. It is defined by an additional X chromosome, 47,XXY, resulting from errors in chromosomal segregation during parental gametogenesis. A major phenotype is impaired reproductive function, in the form of low testosterone and infertility. This review comprehensively examines the genetic and physiological factors contributing to infertility in KS, in addition to emergent assisted reproductive technologies, and the unique ethical challenges KS patients face when seeking infertility treatment. The pathology underlying KS is increased susceptibility for meiotic errors during spermatogenesis, resulting in aneuploid or even polyploid gametes. Specific genetic elements potentiating this susceptibility include polymorphisms in checkpoint genes regulating chromosomal synapsis and segregation. Physiologically, the additional sex chromosome also alters testicular endocrinology and metabolism by dysregulating interstitial and Sertoli cell function, collectively impairing normal sperm development. Additionally, epigenetic modifications like aberrant DNA methylation are being increasingly implicated in these disruptions. We also discuss assisted reproductive approaches leveraged in infertility management for KS patients. Application of assisted reproductive approaches, along with deep comprehension of the meiotic and endocrine disturbances precipitated by supernumerary X chromosomes, shows promise in enabling biological parenthood for KS individuals. This will require continued multidisciplinary collaboration between experts with background of genetics, physiology, ethics and clinical reproductive medicine.

Expression of lymphocyte activation gene-3 on CD4+T cells is regulated by cytokine interleukin-18 in myasthenia gravis.

Myasthenia gravis (MG) is a T cell-dependent, B cell-mediated, and complement-dependent autoimmune disease. Lymphocyte activation gene-3 (LAG-3; CD223) is an immune checkpoint protein that plays an important role in maintaining autoimmune tolerance and homeostasis. To investigate the cytokine-regulated expression pattern of LAG-3, CD4+T cells were sorted from the peripheral blood of healthy volunteers by density gradient centrifugation and stimulated with various cytokines in vitro. The expression of membrane LAG-3 (mLAG-3), membrane a disintegrin and metallopeptidase domain10 (mADAM10) and membrane ADAM17 (mADAM17) on CD4+T cells was detected by flow cytometry; the concentration of soluble LAG-3 (sLAG-3) was detected by ELISA; and the relative expression of genes at the transcriptional level was detected by fluorescence quantitative RT-PCR (qRT-PCR). sLAG-3 levels were significantly increased in the peripheral plasma of AChR Ab-positive patients with MG compared to healthy volunteers, while the percentage of mLAG-3 expression on CD4+T lymphocytes in the peripheral blood of patients with MG was significantly reduced. IL-18 inhibited mLAG-3 levels on CD4+T cells in a concentration-dependent manner. Additionally, the concentration of sLAG-3 in the supernatant increased. After PHA and IL-18 stimulation, ADAM10 and ADAM17 also increased compared to those in the PHA-active group. Moreover, there were significant differences in the expression of mADAM10 and mADAM17 in CD4+T lymphocytes between patients with MG and healthy volunteers. These results suggest that IL-18 may regulate the expression pattern of mLAG-3 in CD4+T cells and sLAG-3 via ADAM10- and ADAM17-mediated pathways, thus affecting the immune effects of CD4+T cells. This study provides a preliminary exploration of the upstream regulatory molecules of the LAG-3 and IL-18/LAG-3 signalling pathways for potential targeted therapy of autoimmune diseases in the future.

High expression of B7-H3 on monocyte/macrophages in tumor microenvironment promotes lung cancer progression by inhibiting apoptosis.

Monocyte/macrophages constitute a significant population of tumor-infiltrating immune cells and play a crucial role in tumor growth, invasion, and metastasis. B7-H3, has immune regulatory functions, however, it is unclear whether B7-H3 expressed on monocyte/macrophages plays a significance role in tumor progression. We found B7-H3 was high-expressed on monocyte/macrophages in tumor microenvironment compared with adjacent tissues in lung cancer, and its expression level was positively correlated with the number of monocyte/macrophages. Furthermore, the expression of B7-H3 was related to clinical stage and lymph node metastasis. Moreover, miR-29a-3p negatively regulated B7-H3, and the expression of B7-H3 on THP-1-derived macrophages was regulated by secreting exosomes containing miR-29a-3p. In addition, knockdown of B7-H3 promoted macrophage apoptosis under hypoxia. Mechanistically, B7-H3 enhanced the antiapoptotic ability of macrophage by up-regulating HIF-1ɑ via activating NF-κB. Taken together, these results imply that B7-H3 as a therapeutic target could hold promise for enhancing anti-tumor immune responses in individuals diagnosed with lung cancer.

A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals.

BACKGROUND: The ubiquitin ligase HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 is essential for the establishment and maintenance of spermatogonia. However, the role of HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 in regulating germ cell differentiation remains unclear, and clinical evidence linking HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 to male infertility pathogenesis is lacking. OBJECTIVE: This study aims to investigate the role of HUWE1 in germ cell differentiation and the mechanism by which a HUWE1 single nucleotide polymorphism increases male infertility risk. MATERIALS AND METHODS: We analyzed HUWE1 single nucleotide polymorphisms in 190 non-obstructive azoospermia patients of Han Chinese descent. We evaluated HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulation by retinoic acid receptor alpha using chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and siRNA-mediated RARα knockdown. Using C18-4 spermatogonial cells, we determined whether HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participated in retinoic acid-mediated retinoic acid receptor alpha signaling. We performed luciferase assays, cell counting kit-8 assays, immunofluorescence, quantitative real-time polymerase chain reaction, and western blotting. We quantified HUWE1 and retinoic acid receptor alpha in testicular biopsies from non-obstructive azoospermia and obstructive azoospermia patients using quantitative real-time polymerase chain reaction and immunofluorescence. RESULTS: Three HUWE1 single nucleotide polymorphisms were significantly associated with spermatogenic failure in 190 non-obstructive azoospermia patients; one (rs34492591) was in the HUWE1 promoter. Retinoic acid receptor alpha regulates HUWE1 gene expression by binding to its promoter. HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participates in retinoic acid/retinoic acid receptor alpha signaling pathway and regulates the expression of germ cell differentiation genes STRA8 and SCP3 to inhibit cell proliferation and reduce γH2AX accumulation. Notably, significantly lower levels of HUWE1 and RARα were detected in testicular biopsy samples from non-obstructive azoospermia patients. CONCLUSIONS: An HUWE1 promoter single nucleotide polymorphism significantly downregulates its expression in non-obstructive azoospermia patients. Mechanistically, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulates germ cell differentiation during meiotic prophase through its participation in retinoic acid/retinoic acid receptor alpha signaling and subsequent modulation of γH2AX. Taken together, these results strongly suggest that the genetic polymorphisms of HUWE1 are closely related to spermatogenesis and non-obstructive azoospermia pathogenesis.

Clinical Significance of Soluble LAG-3 (sLAG-3) in Patients With Cervical Cancer Determined via Enzyme-Linked Immunosorbent Assay With Monoclonal Antibodies.

Background: The tumor microenvironment and tumor immunity have become the focus of research on tumor diagnosis and treatment. Lymphocyte activation gene-3 (LAG-3, CD223) is a newly discovered immunosuppressive receptor that is abnormally expressed in various tumor microenvironments and plays an important role as an immune checkpoint in the tumor immune response. Objective: We developed a novel enzyme-linked immunosorbent assay kit, examined the levels of soluble LAG-3 (sLAG-3) in the serum of patients with cervical cancer, and identified new biomarkers for cervical cancer development. Methods: To investigate the potential biological function of sLAG-3, we generated and characterized 2 novel anti-LAG-3 monoclonal antibodies, namely 4F4 and 4E12. We performed western blotting, immunofluorescence, and immunohistochemistry using hybridoma technology and an enzyme-linked immunosorbent assay kit for detecting human sLAG-3 based on an improved double-antibody sandwich enzyme-linked immunosorbent assay method. The stability and sensitivity of these kits were also assessed. Results: We screened and characterized 2 novel monoclonal antibodies against human LAG-3. The enzyme-linked immunosorbent assay kit also includes a wide range of tests. Using this enzyme-linked immunosorbent assay system, we found that the expression level of sLAG-3 in the peripheral blood of patients with cervical cancer significantly decreased as the disease progressed (P < .0001). Multivariate logistic regression analysis revealed that low sLAG-3 expression was an independent predictor of cervical cancer and related diseases (P < .05). Furthermore, receiver operating characteristic curve analysis showed that sLAG-3 had diagnostic value for cervical cancer metastasis (P < .0001). Conclusion: These data suggest that sLAG-3 is a potential biomarker for cervical cancer development. Therefore, this kit has a certain application value in the diagnosis of cervical cancer.

Hemodynamic Influences of Remimazolam Versus Propofol During the Induction Period of General Anesthesia: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

BACKGROUND: Remimazolam is a novel ultrashort-effect benzodiazepine. In 2020, the US Food and Drug Administration approved it for procedural sedation. Remimazolam is beneficial for consistent sedation and quick recovery in painless gastrointestinal endoscopy. Propofol is one of the most commonly used intravenous anesthetics in clinical practice. Recently, only a few studies have compared propofol with remimazolam for general anesthesia induction. OBJECTIVES: The purpose of our systematic review and meta-analysis was to compare the hemodynamic effects of remimazolam and propofol during the induction of general anesthesia. STUDY DESIGN: Systematic review and meta-analysis of randomized, controlled trials. METHODS: The authors retrieved the PubMed, Embase, Cochrane Library, and Web of Science databases for studies published through September 30, 2022, which reported relevant prospective randomized controlled trials (RCTs) comparing remimazolam with propofol for general anesthesia. The primary outcome was hemodynamic changes, including the absolute value of fluctuation of mean arterial pressure (delta MAP) and heart rate delta HR). The secondary outcomes were the following 2 indicators: the occurrence of total adverse events and the quality of recovery from general anesthesia at 24 hours postsurgery. RevMan 5.4.1 (The Nordic Cochrane Centre for The Cochrane Collaboration) and trial sequential analysis were used to execute the statistical analyses. The different domains of bias were judged by the Cochrane risk of the bias assessment tool. RESULTS: The authors identified 189 papers in PubMed, Embase, Cochrane Library, and Web of Science. Eight articles with 964 patients were selected. The included studies had moderate quality. For primary outcomes, the lower delta HR (mean difference [MD] = -4.99; 95% CI, -7.97 to -2.00; I² = 41.6%; P = 0.001] and delta MAP (MD = -5.91; 95% CI. -8.57 to -3.24; I² = 0%; P < 0.0001) represent more stable hemodynamic characteristics in the remimazolam group. Regarding secondary outcomes, a considerably lower incidence of total adverse events was noted in the remimazolam group than that for the propofol group (odds ratio [OR] = 0.40; 95% CI, 0.28 to 0.58; I² = 63%; P < 0.00001). In comparison to the propofol group, remimazolam achieved an advantage score of quality of recovery -15 in 24 hours postsurgery (MD = 5.31, 95% CI, 1.51 to 9.12; I² = 87%; P = 0.006). LIMITATION: Firstly, there are only a handful of published RCTs on the administration of remimazolam in general anesthesia. In addition, due to patient privacy, we could not extract individual patient data, therefore we could not combine and assess any variations in patient characteristics. CONCLUSION: Evidence suggests that remimazolam has a lower hemodynamic effect during general anesthesia and fewer perioperative adverse effects after general anesthesia than propofol; however, which agent is superior regarding quality benefit in postoperative recovery based on the studies included here remains inconclusive. Additional RCTs with updated meta-analyses to enlarge the sample size and properly analyze the benefit-to-risk ratio to patients are needed to determine the evidence for such a relatively new medicine.

Loss of histone reader Phf7 leads to immune pathways activation via endogenous retroviruses during spermiogenesis.

Genetic studies have elucidated the critical roles of Phf7 in germline development in animals; however, the exact etiology of Phf7 mutations leading to male infertility and the possibility of mechanism-based therapy are still unclear and warrant further investigation. Using the Phf7 knockout mouse model, we verified that genetic defects were responsible for male infertility by preventing histone-to-protamine exchange, as previously reported. The deficiency of spermatogenesis caused by Phf7 deletion through the endogenous retrovirus-mediated activation of the immune pathway is a common mechanism of infertility. Furthermore, we identified PPARα as a promising target of immunity and inflammation in the testis, where endogenous retroviruses are suppressed, and Phf7 as a crucial regulator of endogenous retrovirus-mediated immune regulation and revealed its role as an epigenetic reader. The loss of Phf7 activates immune pathways, which can be rescued by the PPARα agonist astaxanthin. These results showed that astaxanthin is a potential therapeutic agent for treating male infertility. The findings in our study provide insights into the molecular mechanisms underlying male infertility and suggest potential targets for future research and therapeutic development.

Deficiency of N-linked glycosylation impairs immune function of B7-H6.

B7-H6 is a novel immune checkpoint molecule that triggers NK cell cytotoxicity, but the role of N-glycosylation in B7-H6 is poorly understood. We here identified the existence of N-glycosylation of B7-H6 in different cell lines and exogenous expression cells by PNGase F digestion and tunicamycin blockage. Subsequently, we demonstrated that B7-H6 contains 6 functional N-linked glycosylation sites by single site mutation and electrophoresis. Phylogenetical and structural analysis revealed that N43 and N208 glycan are conserved in jawed vertebrates and may thus contribute more to the biological functions. We further demonstrated that N43 and N208 glycosylation are essential for B7-H6 to trigger NK cell activation. Mechanistically, we found that N43 and N208 glycan contributed to the stability and membrane expression of B7-H6 protein. Lack of N208 glycosylation led to membrane B7-H6 shedding, while N43 mutation resulted in impaired B7-H6/NKp30 binding affinity. Together, our findings highlight the significance of N-linked glycosylation in B7-H6 biological functions and suggest potential targets for modulating NK cell-mediated immunity.

Radioimmunotherapy Targeting B7-H3 in situ glioma models enhanced antitumor efficacy by Reconstructing the tumor microenvironment.

Radionuclide drug conjugates (RDCs) with antibodies serve as a novel approach for the treatment of malignant tumors including glioblastoma. However, RDCs require optimal antibodies to work efficiently. Hu4G4, a novel B7-H3-targeting humanized monoclonal IgG1 antibody, is highly specific for the human B7-H3 protein (a marker of tumor cells, including glioblastoma cells). Herein, we established 131I-labeled hu4G4 (131I-hu4G4) and showed that it specifically bound to B7-H3 with high affinity (Kd = 0.99 ± 0.07 nM) and inhibited the growth of U87 cells in vitro. 131I-hu4G4 displayed potent in situ antitumor activity in a mouse model of glioma based on GL261 Red-Fluc-B7-H3 cells. More importantly, 131I-hu4G4 remodeled the tumor microenvironment and promoted the transformation of glioma from "cold" to "hot" tumors by promoting CD4+ and CD8+ T cell infiltration and the polarization of M2 to M1. Therefore, the antitumor activity observed with 131I-hu4G4, together with its ability to enhance antitumor immune responses, makes it a novel candidate for radioimmunotherapy of glioblastoma.

B7-H3 and CD47 co-expression in gastric cancer is a predictor of poor prognosis and potential targets for future dual-targeting immunotherapy.

BACKGROUND: Gastric cancer (GC) is one of the most prevalent types of cancer worldwide. B7-H3, an immune checkpoint molecule with promising potential, has been found to be overexpressed in various cancers. CD47 is an anti-phagocytic molecule that interacts with the signal regulatory protein alpha (SIRPα) to affect phagocytes. The relationship between the expression of B7-H3 and CD47, two potential therapeutic targets found in tumor cells, remains unknown. In this study, our objective is to investigate the clinical significance of co-expression of B7-H3 and CD47, as well as the potential therapeutic value of combination therapy in GC. METHODS: We utilized immunohistochemistry (IHC) to assess the expression of B7-H3, CD47, CD68, CD86 and CD163 in tissue microarrays obtained from 268 GC patients who underwent surgeries. Western blotting was employed to assess the protein level of B7-H3 and CD47 in GC tissues. The co-localization of B7-H3/CD47 and CD68 in GC tissues was determined using multiplex immunohistochemistry (m-IHC). We further verified the relationship between B7-H3/CD47 and macrophage infiltration via flow cytometry. To estimate the clinical outcomes of patients from different subgroups, we employed the Kaplan-Meier curve and the Cox model. RESULTS: Among the 268 GC cases, a total of 180 cases exhibited positive expression of B7-H3, while 122 cases showed positive expression of CD47. In fresh GC clinical tissues, B7-H3 and CD47 protein level was also higher in tumor tissue than in adjacent normal tissue. Remarkably, 91 cases demonstrated co-expression of B7-H3 and CD47. We observed a significant correlation between B7-H3 expression and tumor stage (P = 0.001), differentiation (P = 0.045), and depth (P = 0.003). Additionally, there was a significant association between B7-H3 and CD47 expression (P = 0.018). The percentage of B7-H3 and CD47 double positive cells in fresh GC tumor tissues were elevated compared with control adjacent tissues regardless of CD45- or CD45+ cells (P = 0.0029, P = 0.0012). Patients with high B7-H3 or CD47 expression had significantly lower overall survival (OS) rates compared to those with low expression levels (P = 0.0176 or P = 0.0042). Surprisingly, patients with combined high expression of B7-H3 and CD47 exhibited a considerably worse prognosis than others (P = 0.0007). Univariate analysis revealed that cases with high expression of B7-H3, CD47, or both had significantly higher hazard ratios (HR) than cases with low expression of these markers. Furthermore, the results of multivariate analysis indicated that B7-H3/CD47 co-expression and CD47 expression alone are independent prognostic factors for overall survival. Moreover, significant correlations were observed between B7-H3 and CD68 expression, CD47 and CD68 expression, as well as B7-H3/CD47 co-expression and CD68 expression in GC patients (P < 0.001, P = 0.003, and P < 0.001). Flow cytometry test showed that the percentage of CD68-positive cells but not CD86-positive cells among B7-H3-positive or CD47-positive immune cells in GC tumor tissue was elevated significantly compared with adjacent tissue. CONCLUSION: Our findings demonstrated a correlation between B7-H3 expression and CD47 expression in GC patient tissues. Co-expression of B7-H3 and CD47 can serve as an indicator of poor prognosis in GC patients. In GC tumor tissue, but not adjacent tissue, B7-H3 and CD47 expression was accompanied with macrophage infiltration.

B7H3-targeting chimeric antigen receptor modification enhances antitumor effect of Vγ9Vδ2 T cells in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. This study investigates the therapeutic potential of human Vγ9Vδ2 T cells in GBM treatment. The sensitivity of different glioma specimens to Vγ9Vδ2 T cell-mediated cytotoxicity is assessed using a patient-derived tumor cell clusters (PTCs) model. METHODS: The study evaluates the anti-tumor effect of Vγ9Vδ2 T cells in 26 glioma cases through the PTCs model. Protein expression of BTN2A1 and BTN3A1, along with gene expression related to lipid metabolism and glioma inflammatory response pathways, is analyzed in matched tumor tissue samples. Additionally, the study explores two strategies to re-sensitize tumors in the weak anti-tumor effect (WAT) group: utilizing a BTN3A1 agonistic antibody or employing bisphosphonates to inhibit farnesyl diphosphate synthase (FPPS). Furthermore, the study investigates the efficacy of genetically engineered Vγ9Vδ2 T cells expressing Car-B7H3 in targeting diverse GBM specimens. RESULTS: The results demonstrate that Vγ9Vδ2 T cells display a stronger anti-tumor effect (SAT) in six glioma cases, while showing a weaker effect (WAT) in twenty cases. The SAT group exhibits elevated protein expression of BTN2A1 and BTN3A1, accompanied by differential gene expression related to lipid metabolism and glioma inflammatory response pathways. Importantly, the study reveals that the WAT group GBM can enhance Vγ9Vδ2 T cell-mediated killing sensitivity by incorporating either a BTN3A1 agonistic antibody or bisphosphonates. Both approaches support TCR-BTN mediated tumor recognition, which is distinct from the conventional MHC-peptide recognition by αß T cells. Furthermore, the study explores an alternative strategy by genetically engineering Vγ9Vδ2 T cells with Car-B7H3, and both non-engineered and Car-B7H3 Vγ9Vδ2 T cells demonstrate promising efficacy in vivo, underscoring the versatile potential of Vγ9Vδ2 T cells for GBM treatment. CONCLUSIONS: Vγ9Vδ2 T cells demonstrate a robust anti-tumor effect in some glioma cases, while weaker in others. Elevated BTN2A1 and BTN3A1 expression correlates with improved response. WAT group tumors can be sensitized using a BTN3A1 agonistic antibody or bisphosphonates. Genetically engineered Vγ9Vδ2 T cells, i.e.,  Car-B7H3, show promising efficacy. These results together highlight the versatility of Vγ9Vδ2 T cells for GBM treatment.

Identification and validation of a novel HOX-related classifier signature for predicting prognosis and immune microenvironment in pediatric gliomas.

Background: Pediatric gliomas (PGs) are highly aggressive and predominantly occur in young children. In pediatric gliomas, abnormal expression of Homeobox (HOX) family genes (HFGs) has been observed and is associated with the development and progression of the disease. Studies have found that overexpression or underexpression of certain HOX genes is linked to the occurrence and prognosis of gliomas. This aberrant expression may contribute to the dysregulation of important pathological processes such as cell proliferation, differentiation, and metastasis. This study aimed to propose a novel HOX-related signature to predict patients' prognosis and immune infiltrate characteristics in PGs. Methods: The data of PGs obtained from publicly available databases were utilized to reveal the relationship among abnormal expression of HOX family genes (HFGs), prognosis, tumor immune infiltration, clinical features, and genomic features in PGs. The HFGs were utilized to identify heterogeneous subtypes using consensus clustering. Then random forest-supervised classification algorithm and nearest shrunken centroid algorithm were performed to develop a prognostic signature in the training set. Finally, the signature was validated in an internal testing set and an external independent cohort. Results: Firstly, we identified HFGs significantly differentially expressed in PGs compared to normal tissues. The individuals with PGs were then divided into two heterogeneous subtypes (HOX-SI and HOX-SII) based on HFGs expression profiles. HOX-SII showed higher total mutation counts, lower immune infiltration, and worse prognosis than HOX-SI. Then, we constructed a HOX-related gene signature (including HOXA6, HOXC4, HOXC5, HOXC6, and HOXA-AS3) based on the cluster for subtype prediction utilizing random forest supervised classification and nearest shrunken centroid algorithm. The signature was revealed to be an independent prognostic factor for patients with PGs by multivariable Cox regression analysis. Conclusion: Our study provides a novel method for the prognosis classification of PGs. The findings also suggest that the HOX-related signature is a new biomarker for the diagnosis and prognosis of patients with PGs, allowing for more accurate survival prediction.

Clinical significance of CD155 expression and correlation with cellular components of tumor microenvironment in gastric adenocarcinoma.

Introduction: CD155 is recently emerging as a promising target in malignancies. However, the relationship between CD155 expression and tumor microenvironment (TME) cell infiltration in gastric adenocarcinoma (GAC) has rarely been clarified. Methods: We measured CD155 expression in specimens of gastric precancerous disease and GAC by immunohistochemistry. The association of CD155 expression with GAC progression and cells infiltration in TME was evaluated through 268 GAC tissues and public dataset analysis. Results: We showed that the expression of CD155 was positively correlated with the pathological development of gastric precancerous disease (r = 0.521, P < 0.0001). GAC patients with high CD155 expression had a poorer overall survival (P = 0.033). Moreover, CD155 expression correlated with aggressive clinicopathological features including tumor volume, tumor stage, lymph node involvement, and cell proliferation (P <0.05). Remarkably, CD155 expression positively related to the infiltration of CD68+ macrophages in TME (P = 0.011). Meanwhile, the positive correlation was observed between CD155 and CD31 (P = 0.026). In addition, patients with high CD155 expression combined with low CD3, CD4, CD8, IL-17, IFN-γ or CD19 expression as well as those with high CD155 and α-SMA expression showed significantly worse overall survival (P < 0.05). Conclusions: CD155 may play a pivotal role in the development of GAC through both immunological and non-immunological mechanisms and be expected to become a novel target of immunotherapy in GAC patients.

Dissecting Sperm Mitochondrial G-Quadruplex Structures Using a Fluorescent Probe Biomarker to Monitor and Regulate Fertilization Capability.

To monitor the levels of mitochondrial DNA G-quadruplexes (mtDNA G4s) in spermatozoa and to explore the possibility using mtDNA G4s as a reliable marker in patients with multiple clinical insemination failures, a novel chemical TPE-mTO probe engineered in our previous work was used on both samples from the mice sperm and from patients with fertilization failure. Expression of valosin-containing protein and the zona-free hamster egg assay were used to evaluate mitophagy and human sperm penetration. RNA-sequencing was used to explore expression changes of key genes affected by mtDNA G4s. Results showed that the probe can track mtDNA G4s in spermatozoa easily and quickly with fewer backgrounds. Significantly increased mtDNA G4s were also found in patients with fertilization failure, using the flow-cytometry-based TPE-mTO probe detection method. A sperm-hamster egg penetration experiment showed that abnormal fertilization caused by increased mtDNA G4s can be effectively restored by a mitophagy inducer. This study provides a novel method for monitoring etiological biomarkers in patients with clinical infertility and treatment for patients with abnormal fertilization caused by mtDNA G4 dysfunction.

Development of a MMAE-based antibody-drug conjugate targeting B7-H3 for glioblastoma.

B7-H3 (immunoregulatory protein B7-homologue 3) is overexpressed in many cancer cells with limited expression in normal tissues, considered to be a promising target for tumor therapeutics. Clinical trials of antibody-drug conjugates (ADCs) against different targets for glioblastoma have been investigated and showed potent efficacies. In this study, we developed a homogeneous ADC 401-4 with a drug-to-antibody ratio (DAR) of 4, which was prepared by conjugation of Monomethyl auristatin E (MMAE) to a humanized anti-B7-H3 mAb 401, through a divinylsulfonamide-mediated disulfide re-bridging approach. In vitro studies, 401-4 displayed specific killing against B7-H3-expressing tumors and was more effective in cells with higher levels of B7-H3 for different glioblastoma cells. 401-4 was furthered labeled with Cy5.5 to yield a fluorescent conjugate 401-4-Cy5.5. The in vivo imaging studies showed that the conjugate accumulated in tumor regions and exhibited the ability to target-specific delivery. In addition, significant antitumor activities for 401-4 was observed against U87-derived tumor xenografts in a dose dependent manner.

CD226 identifies functional CD8+T cells in the tumor microenvironment and predicts a better outcome for human gastric cancer.

It is well-known that CD226 serves as a critical activating receptor on various immune cells, such as lymphocytes and monocytes, and it is suggested to promote anti-tumor immunity in the tumor microenvironment (TME). Herein, we showed a crucial regulatory role of CD226 in CD8+T cell-mediated anti-tumor response in TME of human gastric cancer (GC). Specifically, the increased CD226 expression in cancer tissues was significantly associated with better clinical outcomes in GC patients. Moreover, the increased infiltrating CD226+CD8+T cells and the increased ratio of infiltrating CD226+CD8+T cells in CD8+T subpopulation within cancer tissues could also be valuable prognostic predictors for GC patients. Mechanically, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis revealed that the chromatin accessibility of CD226 in CD4+ and CD8+TILs was significantly higher than that in CD8+T cells in normal tissues. Further analysis showed that CD8+TILs highly expressed immune checkpoint molecules, such as TIGIT, LAG3, and HAVCR2, which means CD8+TILs are more exhausted. In addition, our multi-color immunohistochemical staining (mIHC) revealed that GC patients with higher frequency of IFN-γ+CD226+CD8+TILs showed poorer prognosis. Combined with the single-cell transcriptome sequencing (scRNA-seq) data analysis, we found that the expressions of IFN-γ and TIGIT in CD8+TILs were significantly and positively correlated. The expression of TIGIT in IFN-γ+CD226+CD8+TILs was higher, while that in IFN-γ-CD226+CD8+TILs was significantly lower. The correlation analysis showed that the expression of CD226 was positively correlated with the score of effector T cells but negatively correlated with that of immunosuppressive factors, such as Tregs and tumor-associated macrophages (TAMs). Collectively, we showed that the frequency of CD226+CD8+TILs was an excellent prognostic predictor for GC patients. Our findings provided insights into the interaction pattern between co-stimulatory receptor CD226 and tumor cells as well as the infiltrating immune cells in the TME in GC.

Vascular Normalization Was Associated with Colorectal Tumor Regression upon Anti-PD-L1 Combinational Therapy.

Anti-PD-L1 therapy exhibits durable efficacy, but only in a small fraction of cancer patients. The immunosuppressive tumor microenvironment (TME) is a crucial obstacle that impedes cancer immunotherapy. Here, we found that anti-PD-L1 therapy coupled with CD4+ T cell depletion induced colorectal tumor regression and vascular normalization, while monotherapy only retarded tumor growth without affecting the tumor vasculature. Moreover, simultaneous PD-L1 blockade and CD4+ T cell depletion eradicated intratumoral PD-L1+ lymphoid and myeloid cell populations, while additively elevating the proportions of CD44+CD69+CD8+, central memory CD44+CD62L+CD8+, and effector memory CD44+CD62L-CD8+ T cells, suggesting a reduction in immunosuppressive cell populations and the activation of CD8+ T cells in the TME. Moreover, anti-PD-L1 therapy reduced the proportions of intratumoral PD-L1+ immune cells and suppressed tumor growth in a CD8+ T cell dependent manner. Together, these results suggest that anti-PD-L1 therapy induces tumor vascular normalization and colorectal tumor regression via CD8+ T cells, which is antagonized by CD4+ T cells. Our findings unveil the positive correlation of tumor regression and vascular normalization in colorectal tumor models upon anti-PD-L1 therapy, providing a potential new strategy to improve its efficacy.