Experimental Study of the Moisture Resistance of Cement Mortar Using Pozzolan Materials and Calcium Stearate.

Nanosilica and diatomite are pozzolanic resources rich in SiO2. In this study, the purpose of this study was to improve the moisture resistance of the specimen by producing a mixed material using pozzolanic materials and calcium stearate and adding it to cement mortar while stirring. The results showed that the hydration reaction was not activated when calcium stearate adhered to the fine particles of nanosilica; it existed simply in the form of a filler inside the specimen. Diatomite, due to its atypical particles and porosity, may have greater water tightness than nanosilica because of the pozzolanic reaction in particles to which calcium stearate is not attached.

Protocol for analyzing γδ T cells from the vagina of diet-induced obese mice using single-cell RNA sequencing and flow cytometry.

Obesity affects susceptibility to sexually transmitted diseases like genital herpes, caused by herpes simplex virus (HSV) 2. The γδ T cells in the vagina play a major role in HSV-2 suppression. Here, we present a protocol for inducing HSV-2 infection intravaginally in high-fat diet-induced obese mice. We describe steps for isolating single cells from vaginal tissue and analyzing cells using single-cell RNA sequencing and flow cytometry. We then detail confirmation of the γδ T cell phenotype in vitro. For complete details on the use and execution of this protocol, please refer to Park et al.1.

Intranasal delivery of an adenovirus-vector vaccine co-expressing a modified spike protein and a genetic adjuvant confers lasting mucosal immunity against SARS-CoV-2.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 infection has threatened global health. Since the first case of infection was reported in December 2019, SARS-CoV-2 has rapidly spread worldwide and caused millions of deaths. As vaccination is the best way to protect the host from invading pathogens, several vaccines have been developed to prevent the infection of SARS-CoV-2, saving numerous lives thus far. However, SARS-CoV-2 constantly changes its antigens, resulting in escape from vaccine-induced protection, and the longevity of immunity induced by vaccines remains an issue. Additionally, traditional intramuscular COVID-19 vaccines are insufficient at evoking mucosal-specific immune responses. Because the respiratory tract is the primary route of SARS-CoV-2 entry, the need for mucosal vaccines is strong. Using an adenoviral (Ad) vector platform, we generated Ad5-S.Mod, a recombinant COVID-19 vaccine that encodes modified-spike (S) antigen and the genetic adjuvant human CXCL9. Intranasal delivery of Ad5-S.Mod elicited superior airway humoral and T-cell responses over traditional intramuscular vaccines and protected mice from lethal SARS-CoV-2 infection. cDC1 cells were required for the generation of antigen-specific CD8+ T-cell responses and CD8+ tissue-resident memory T-cell development in intranasal Ad5-S.Mod vaccinated mice. Furthermore, we confirmed the efficacy of the intranasal Ad5-S.Mod vaccine in terms of transcriptional changes and identified lung macrophages as a key supporter of maintenance of lung-resident memory T and B cells. Our study demonstrates Ad5-S.Mod has the potential to confer protective immunity against SARS-CoV-2 and that lung macrophages support the maintenance of vaccine-induced tissue-resident memory lymphocytes.

Regulation of c-SMAC formation and AKT-mTOR signaling by the TSG101-IFT20 axis in CD4+ T cells.

CD4+ T cells play major roles in the adaptive immune system, which requires antigen recognition, costimulation, and cytokines for its elaborate orchestration. Recent studies have provided new insight into the importance of the supramolecular activation cluster (SMAC), which comprises concentric circles and is involved in the amplification of CD4+ T cell activation. However, the underlying mechanism of SMAC formation remains poorly understood. Here, we performed single-cell RNA sequencing of CD4+ T cells left unstimulated and stimulated with anti-CD3 and anti-CD28 antibodies to identify novel proteins involved in their regulation. We found that intraflagellar transport 20 (IFT20), previously known as cilia-forming protein, was upregulated in antibody-stimulated CD4+ T cells compared to unstimulated CD4+ T cells. We also found that IFT20 interacted with tumor susceptibility gene 101 (TSG101), a protein that endocytoses ubiquitinated T-cell receptors. The interaction between IFT20 and TSG101 promoted SMAC formation, which led to amplification of AKT-mTOR signaling. However, IFT20-deficient CD4+ T cells showed SMAC malformation, resulting in reduced CD4+ T cell proliferation, aerobic glycolysis, and cellular respiration. Finally, mice with T-cell-specific IFT20 deficiency exhibited reduced allergen-induced airway inflammation. Thus, our data suggest that the IFT20-TSG101 axis regulates AKT-mTOR signaling via SMAC formation.

The Role of Hypoxia in Brain Tumor Immune Responses.

Oxygen is a vital component of living cells. Low levels of oxygen in body tissues, known as hypoxia, can affect multiple cellular functions across a variety of cell types and are a hallmark of brain tumors. In the tumor microenvironment, abnormal vasculature and enhanced oxygen consumption by tumor cells induce broad hypoxia that affects not only tumor cell characteristics but also the antitumor immune system. Although some immune reactions require hypoxia, hypoxia generally negatively affects immunity. Hypoxia induces tumor cell invasion, cellular adaptations to hypoxia, and tumor cell radioresistance. In addition, hypoxia limits the efficacy of immunotherapy and hinders antitumor responses. Therefore, understanding the role of hypoxia in the brain tumor, which usually does not respond to immunotherapy alone is important for the development of effective anti-tumor therapies. In this review, we discuss recent evidence supporting the role of hypoxia in the context of brain tumors.

Human allogenic γδ T cells kill patient-derived glioblastoma cells expressing high levels of DNAM-1 ligands.

Adoptive transfer of γδ T cells is a novel immunotherapeutic approach to glioblastoma. Few recent studies have shown the efficacy of γδ T cells against glioblastoma, but no previous studies have identified the ligand-receptor interactions between γδ T cells and glioblastoma cells. Here, we identify those ligand-receptor interactions and provide a basis for using γδ T cells to treat glioblastoma. Vγ9Vδ2 T cells were generated from peripheral blood mononuclear cells of healthy donors using artificial antigen presenting cells. MICA, ULBP, PVR and Nectin-2 expression in 10 patient-derived glioblastoma (PDG) cells were analyzed. The in vitro cytokine secretion from the γδ T cells and their cytotoxicity toward the PDG cells were also analyzed. The in vivo anti-tumor effects were evaluated using a U87 orthotopic xenograft glioblastoma model. Expression of ligands and cytotoxicity of the γδ T cells varied among the PDG cells. IFN-γ and Granzyme B secretion levels were significantly higher when γδ Tcells were co-cultured with high-susceptible PDG cells than when they were co-cultured with low-susceptible PDG cells. Cytotoxicity correlated significantly with the expression levels of DNAM-1 ligands of the PDG cells. Blocking DNAM-1 resulted in a decrease in γδ T cell-mediated cytotoxicity and cytokine secretion. Intratumoral injection of γδ T cells showed anti-tumor effects in an orthotopic mouse model. Allogenic γδ T cells showed potent anti-tumor effects on glioblastoma in a DNAM-1 axis dependent manner. Our findings will facilitate the development of clinical strategies using γδ T cells for glioblastoma treatment.

Obesity enhances antiviral immunity in the genital mucosa through a microbiota-mediated effect on γδ T cells.

Obesity is detrimental to the immune system. It impairs lymphatics, T cell development, and lymphopoiesis; induces dysfunction of antitumor immunity; and also promotes tumor progression. However, direct evidence of the impact of obesity on viral infection is lacking. We report a protective role of obesity against herpes simplex virus 2 infection of the genital mucosa in mice. Although conventional antiviral immunity is comparable between obese mice and lean mice, obesity enhances the cytotoxic subset of γδ T cells. This effect is mediated by L-arginine produced by commensal microbiota in the genital mucosa, which induces "pseudonormoxia" of γδ T cells, resulting in increased natural killer (NK) group 2 D (NKG2D) expression of γδ T cells through the downregulation of hypoxia-inducible factor 1-alpha (HIF1A) by inducing nitric oxide (NO) production, thereby protecting mice from lethal genital herpes. Thus, our work illuminates one mechanism by which obesity-induced compositional changes in the vaginal microbiota can affect mucosal immune responses against viral infection.

Blood monocyte-derived CD169+ macrophages contribute to antitumor immunity against glioblastoma.

Infiltrating tumor-associated macrophages (TAM) are known to impede immunotherapy against glioblastoma (GBM), however, TAMs are heterogeneous, and there are no clear markers to distinguish immunosuppressive and potentially immune-activating populations. Here we identify a subset of CD169+ macrophages promoting an anti-tumoral microenvironment in GBM. Using single-cell transcriptome analysis, we find that CD169+ macrophages in human and mouse gliomas produce pro-inflammatory chemokines, leading to the accumulation of T cells and NK cells. CD169 expression on macrophages facilitates phagocytosis of apoptotic glioma cells and hence tumor-specific T cell responses. Depletion of CD169+ macrophages leads to functionally impaired antitumor lymphocytes and poorer survival of glioma-bearing mice. We show that NK-cell-derived IFN-γ is critical for the accumulation of blood monocyte-derived CD169+ macrophages in gliomas. Our work thus identifies a well-distinguished TAM subset promoting antitumor immunity against GBM, and identifies key factors that might shift the balance from immunosuppressive to anti-tumor TAM.

Properties and Durability of Cement Mortar Using Calcium Stearate and Natural Pozzolan for Concrete Surface Treatment.

Applying a concrete surface treatment method (epoxy or primer) can prevent water from penetrating concrete through surface pores. However, if the concrete surface is damaged, the subsequent reconstruction can be expensive and time-consuming. Concrete that is resistant to internal and external water has been extensively developed and used to supplement the surface treatment method. Herein, we prepared specimens by mixing cement mortar with fatty-acid-salt-based calcium stearate attached to two natural pozzolanic materials­diatomite and yellow clay. The physical tests measured (1) the air content, (2) flow test, (3) compressive strength, and (4) activity Factor. Durability experiments were performed on (1) the contact angle, (2) chloride ion diffusion coefficient, and (3) water absorption test. The results revealed that the compressive strength of concrete decreased as the calcium stearate content increased. Furthermore, it was confirmed that the contact angle of the test piece using the pozzolanic substance and calcium stearate was twice as high. It was confirmed that the sand test specimen had the highest water absorption rate, and the DT3% had the lowest. (Sand%: 11.8 > OPC: 6.5 > DT3%: 2.4), the chloride diffusion coefficient also showed similar results. (Sand%: 12.5 > OPC: 8.4 > DT1%: 8.8)Due to its unique insolubility, calcium stearate retards hydrate formation when mixed alone and negates compressive strength loss when combined with pozzolanic mixtures rich in SiO2 and Al2O3. Furthermore, the ideal method for producing water-resistant cement mortar is to evenly disperse calcium stearate in the porous powder of cement mortar.

Protocol to analyze antitumor immunity of orthotopic injection and spontaneous murine high-grade glioma models using flow cytometry and single-cell RNA sequencing.

Despite the recognized importance of antitumor immunity, our understanding of brain tumor immunity is poor. Orthotopic injection models have been widely used for immunological analyses. However, these models have limitations in analysis of antitumor immunity because the approach involves drilling skulls and injecting tumor cells, which can induce adverse effects. We describe a protocol for the induction of spontaneous brain tumor model, isolation of single cells from brain tumor microenvironment, and analysis of the immune responses using scRNA-seq and flow cytometry. For complete details on the use and execution of this protocol, please refer to Park et al. (2021).

γδ T Cells in Brain Homeostasis and Diseases.

γδ T cells are a distinct subset of T cells expressing γδ T cell receptor (TCR) rather than αßTCR. Since their discovery, the critical roles of γδ T cells in multiple physiological systems and diseases have been investigated. γδ T cells are preferentially located at mucosal surfaces, such as the gut, although a small subset of γδ T cells can circulate the blood. Additionally, a subset of γδ T cells reside in the meninges in the central nervous system. Recent findings suggest γδ T cells in the meninges have critical roles in brain function and homeostasis. In addition, several lines of evidence have shown γδ T cells can infiltrate the brain parenchyma and regulate inflammatory responses in multiple diseases, including neurodegenerative diseases. Although the importance of γδ T cells in the brain is well established, their roles are still incompletely understood due to the complexity of their biology. Because γδ T cells rapidly respond to changes in brain status and regulate disease progression, understanding the role of γδ T cells in the brain will provide critical information that is essential for interpreting neuroimmune modulation. In this review, we summarize the complex role of γδ T cells in the brain and discuss future directions for research.

Survey of Airborne Microorganisms in an Arcade-Type Traditional Market in Anseong, South Korea.

We aimed to analyze airborne microorganisms and assess air quality, temperature, and relative humidity at "J" Market, an arcade-type traditional market in Anseong (South Korea). Measurements were taken 16 times, twice per quarter (January, April, July, and October), at both the entrance and intersection of the market in 2020. The concentrations of airborne bacteria and fungi at the entrance and intersection were highest in October and lowest in April; however, they were below the recommended indoor levels (airborne bacteria: <800 CFU/m3, airborne fungi: <500 CFU/m3) in January (second measurement) and April (first and second measurements). The concentrations of microbes during the first measurement in January and both measurements in July and October exceeded the allowed limits. The concentration of microorganisms exceeded the acceptable levels at relative humidity ≥60%. At all time points, except during the eighth survey, when the microorganisms were too numerous to count, microbial concentrations were higher at the intersection than at the entrance. It was confirmed that the microorganisms detected in this experiment were 26 species of bacteria and 21 species of fungi. Three of the four species of bacteria and fungi detected in more than 50% of the 16 experimental results were pathogenic. Our findings suggest that air purification systems must be installed in the market to improve sanitary conditions.

Current Understanding of Hypoxia in Glioblastoma Multiforme and Its Response to Immunotherapy.

Hypoxia is a hallmark of glioblastoma multiforme (GBM), the most aggressive cancer of the central nervous system, and is associated with multiple aspects of tumor pathogenesis. For example, hypoxia induces resistance to conventional cancer therapies and inhibits antitumor immune responses. Thus, targeting hypoxia is an attractive strategy for GBM therapy. However, traditional studies on hypoxia have largely excluded the immune system. Recently, the critical role of the immune system in the defense against multiple tumors has become apparent, leading to the development of effective immunotherapies targeting numerous cancer types. Critically, however, GBM is classified as a "cold tumor" due to poor immune responses. Thus, to improve GBM responsiveness against immunotherapies, an improved understanding of both immune function in GBM and the role of hypoxia in mediating immune responses within the GBM microenvironment is needed. In this review, we discuss the role of hypoxia in GBM from a clinical, pathological, and immunological perspective.

The immune landscape of high-grade brain tumor after treatment with immune checkpoint blockade.

Despite the therapeutic success of immune checkpoint blockade (ICB) therapy against multiple tumors, many patients still do not benefit from ICB. In particular, high-grade brain tumors, such as glioblastoma multiforme (GBM), have a very low response rate to ICB, resulting in several failed clinical trials. This low response rate might be caused by a lack of understanding of the unique characteristics of brain immunity. To overcome this knowledge gap, macroscopic studies of brain immunity are needed. We use single cell RNA sequencing to analyze the immune landscape of the tumor microenvironment (TME) under anti-PD-1 antibody treatment in a murine GBM model. We observe that CD8 T cells show a mixed phenotype overall that includes reinvigoration and re-exhaustion states. Furthermore, we find that CCL5 induced by anti-PD-1 treatment might be related to an increase in the number of anti-inflammatory macrophages in the TME. Therefore, we hypothesize that CCL5-mediated recruitment of anti-inflammatory macrophages may be associated with re-exhaustion of CD8 T cells in the TME. We compare our observations in the murine GBM models with publicly available data from human patients with recurrent GBM. Our study provides critical information for the development of novel immunotherapies to overcome the limitations of anti-PD-1 therapy.

Delivery Routes for COVID-19 Vaccines.

The novel coronavirus, SARS-CoV-2, which causes COVID-19, has resulted in a pandemic with millions of deaths. To eradicate SARS-CoV-2 and prevent further infections, many vaccine candidates have been developed. These vaccines include not only traditional subunit vaccines and attenuated or inactivated viral vaccines but also nucleic acid and viral vector vaccines. In contrast to the diversity in the platform technology, the delivery of vaccines is limited to intramuscular vaccination. Although intramuscular vaccination is safe and effective, mucosal vaccination could improve the local immune responses that block the spread of pathogens. However, a lack of understanding of mucosal immunity combined with the urgent need for a COVID-19 vaccine has resulted in only intramuscular vaccinations. In this review, we summarize the history of vaccines, current progress in COVID-19 vaccine technology, and the status of intranasal COVID-19 vaccines. Future research should determine the most effective route for vaccine delivery based on the platform and determine the mechanisms that underlie the efficacy of different delivery routes.

Function of γδ T cells in tumor immunology and their application to cancer therapy.

T cells of the γδ lineage are unconventional T cells with functions not restricted to MHC-mediated antigen presentation. Because of their broad antigen specificity and NK-like cytotoxicity, γδ T-cell importance in tumor immunology has been emphasized. However, some γδ T-cell subsets, especially those expressing IL-17, are immunosuppressive or tumor-promoting cells. Their cytokine profile and cytotoxicity are seemingly determined by cross-talk with microenvironment components, not by the γδTCR chain. Furthermore, much about the TCR antigen of γδ T cells remains unknown compared with the extreme diversity of their TCR chain pairs. Thus, the investigation and application of γδ T cells have been relatively difficult. Nevertheless, γδ T cells remain attractive targets for antitumor therapy because of their independence from MHC molecules. Because tumor cells have the ability to evade the immune system through MHC shedding, heterogeneous antigens, and low antigen spreading, MHC-independent γδ T cells represent good alternative targets for immunotherapy. Therefore, many approaches to using γδ T cells for antitumor therapy have been attempted, including induction of endogenous γδ T cell activation, adoptive transfer of expanded cells ex vivo, and utilization of chimeric antigen receptor (CAR)-T cells. Here, we discuss the function of γδ T cells in tumor immunology and their application to cancer therapy.

Tumor hypoxia represses γδ T cell-mediated antitumor immunity against brain tumors.

The anatomic location and immunologic characteristics of brain tumors result in strong lymphocyte suppression. Consequently, conventional immunotherapies targeting CD8 T cells are ineffective against brain tumors. Tumor cells escape immunosurveillance by various mechanisms and tumor cell metabolism can affect the metabolic states and functions of tumor-infiltrating lymphocytes. Here, we discovered that brain tumor cells had a particularly high demand for oxygen, which affected γδ T cell-mediated antitumor immune responses but not those of conventional T cells. Specifically, tumor hypoxia activated the γδ T cell protein kinase A pathway at a transcriptional level, resulting in repression of the activatory receptor NKG2D. Alleviating tumor hypoxia reinvigorated NKG2D expression and the antitumor function of γδ T cells. These results reveal a hypoxia-mediated mechanism through which brain tumors and γδ T cells interact and emphasize the importance of γδ T cells for antitumor immunity against brain tumors.

Autophagic protein ATG5 controls antiviral immunity via glycolytic reprogramming of dendritic cells against respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infections in infants. Macroautophagy/autophagy is a catalytic metabolic process required for cellular homeostasis. Although intracellular metabolism is important for immune responses in dendritic cells, the link between autophagy and immunometabolism remains unknown. Here, we show that the autophagy-related protein ATG5 regulates immunometabolism. Atg5-deficient mouse dendritic cells showed increased CD8A+ T-cell response and increased secretion of proinflammatory cytokines upon RSV infection. Transcriptome analysis showed that Atg5 deficiency alters the expression of metabolism-related genes. Atg5-deficient dendritic cells also showed increased activation of glycolysis and the AKT-MTOR-RPS6KB1 pathway and decreased mitochondrial activity, all of which are cellular signatures for metabolic activation. These cells also showed elevated CD8A+ T-cell priming and surface major histocompatibility complex (MHC) class I expression. Our results suggested that ATG5 regulated host immune responses by modulating dendritic cell metabolism. These findings may help develop potential antiviral therapies that alter host immunity by regulating autophagy and immunometabolism.Abbreviations : 2-DG: 2-deoxyglucose; AAK1: AP2 associated kinase 1; AKT: AKT serine/threonine kinase; AM: alveolar macrophage; ATG: autophagy; ATP: adenosine triphosphate; BAL: bronchoalveolar lavage; BMDC: bone marrow dendritic cell; CSF2/GM-CSF: colony-stimulating factor 2 (granulocyte-macrophage); CTL: cytotoxic T lymphocyte; ELISA: enzyme-linked immunosorbent assay; GFP: green fluorescent protein; GSEA: gene-set enrichment analysis; H-2Db: H-2 class I histocompatibility antigen, D-B alpha chain; H-2Kb: MHC class I H2-K-b; HIF1A: hypoxia-inducible factor 1 alpha; IFNG: interferon-gamma; IL: interleukin; ITGAX: integrin alpha X; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MHC: major histocompatibility complex; MTORC1: mammalian target of rapamycin kinase complex 1; PBS: phosphate-buffered saline; PFU: plaque-forming unit; RLR: retinoic acid-inducible-I-like receptor; ROS: reactive oxygen species; RPMI: Roswell Park Memorial Institute; RPS6KB1/S6K: ribosomal protein S6 kinase, polypeptide 1; RSV: respiratory syncytial virus; Th: T helper; TLR: toll-like receptor; Treg: regulatory T cells; UMAP: uniform manifold approximation and projection.

Re-analysis of Single Cell Transcriptome Reveals That the NR3C1-CXCL8-Neutrophil Axis Determines the Severity of COVID-19.

SARS-CoV-2 infection has recently been declared a pandemic. Some patients showing severe symptoms exhibit drastic inflammation and airway damage. In this study, we re-analyzed published scRNA-seq data of COVID-19 patient bronchoalveolar lavage fluid to further classify and compare immunological features according to the patient's disease severity. Patients with severe symptoms showed DNA damage and apoptotic features of epithelial cells. Our results suggested that epithelial damage was associated with neutrophil infiltration. Myeloid cells of severe patients showed higher expression of proinflammatory cytokines and chemokines such as CXCL8. As a result, neutrophils were abundant in lungs of patients from the severe group. Furthermore, recruited neutrophils highly expressed genes related to neutrophil extracellular traps. Neutrophil-mediated inflammation was regulated by glucocorticoid receptor expression and activity. Based on these results, we suggest that severe COVID-19 symptoms may be determined by differential expression of glucocorticoid receptors and neutrophils.