Microbiome dynamics in immune checkpoint blockade.

Immune checkpoint blockade (ICB) is one of the leading immunotherapies, although a variable extent of resistance has been observed among patients and across cancer types. Among the efforts underway to overcome this challenge, the microbiome has emerged as a factor affecting the responsiveness and efficacy of ICB. Active research, facilitated by advances in sequencing techniques, is assessing the predominant influence of the intestinal microbiome, as well as the effects of the presence of an intratumoral microbiome. In this review, we describe recent findings from clinical trials, observational studies of human patients, and animal studies on the impact of the microbiome on the efficacy of ICB, highlighting the role of the intestinal and tumor microbiomes and the contribution of methodological advances in their study.

γδ T cells as a potential therapeutic agent for glioblastoma.

Although γδ T cells comprise a small population of T cells, they perform important roles in protecting against infection and suppressing tumors. With their distinct tissue-localizing properties, combined with their various target recognition mechanisms, γδ T cells have the potential to become an effective solution for tumors that do not respond to current therapeutic procedures. One such tumor, glioblastoma (GBM), is a malignant brain tumor with the highest World Health Organization grade and therefore the worst prognosis. The immune-suppressive tumor microenvironment (TME) and immune-evasive glioma stem cells are major factors in GBM immunotherapy failure. Currently, encouraged by the strong anti-tumoral function of γδ T cells revealed at the preclinical and clinical levels, several research groups have shown progression of γδ T cell-based GBM treatment. However, several limitations still exist that block effective GBM treatment using γδ T cells. Therefore, understanding the distinct roles of γδ T cells in anti-tumor immune responses and the suppression mechanism of the GBM TME are critical for successful γδ T cell-mediated GBM therapy. In this review, we summarize the effector functions of γδ T cells in tumor immunity and discuss current advances and limitations of γδ T cell-based GBM immunotherapy. Additionally, we suggest future directions to overcome the limitations of γδ T cell-based GBM immunotherapy to achieve successful treatment of GBM.

Supplementation with a high-glucose drink stimulates anti-tumor immune responses to glioblastoma via gut microbiota modulation.

A high-sugar diet induces lifestyle-associated metabolic diseases, such as obesity and diabetes, which may underlie the pro-tumor effects of a high-sugar diet. We supply GL261 syngeneic glioblastoma (GBM) mice with a short-term high-glucose drink (HGD) and find an increased survival rate with no evidence of metabolic disease. Modulation of the gut microbiota through HGD supplementation is critical for enhancing the anti-tumor immune response. Single-cell RNA sequencing shows that gut microbiota modulation by HGD supplementation increases the T cell-mediated anti-tumor immune response in GBM mice. We find that the cytotoxic CD4+ T cell population in GBM is increased due to synergy with anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint inhibitors, but this effect depends upon HGD supplementation. Thus, we determine that HGD supplementation enhances anti-tumor immune responses in GBM mice through gut microbiota modulation and suggest that the role of HGD supplementation in GBM should be re-examined.

Double-edged sword: γδ T cells in mucosal homeostasis and disease.

The mucosa is a tissue that covers numerous body surfaces, including the respiratory tract, digestive tract, eye, and urogenital tract. Mucosa is in direct contact with pathogens, and γδ T cells perform various roles in the tissue. γδ T cells efficiently defend the mucosa from various pathogens, such as viruses, bacteria, and fungi. In addition, γδ T cells are necessary for the maintenance of homeostasis because they select specific organisms in the microbiota and perform immunoregulatory functions. Furthermore, γδ T cells directly facilitate pregnancy by producing growth factors. However, γδ T cells can also play detrimental roles in mucosal health by amplifying inflammation, thereby worsening allergic responses. Moreover, these cells can act as major players in autoimmune diseases. Despite their robust roles in the mucosa, the application of γδ T cells in clinical practice is lacking because of factors such as gaps between mice and human cells, insufficient knowledge of the target of γδ T cells, and the small population of γδ T cells. However, γδ T cells may be attractive targets for clinical use due to their effector functions and low risk of inducing graft-versus-host disease. Therefore, robust research on γδ T cells is required to understand the crucial features of these cells and apply these knowledges to clinical practices.

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.

Harnessing type I interferon-mediated immunity to target malignant brain tumors.

Type I interferons have long been appreciated as a cytokine family that regulates antiviral immunity. Recently, their role in eliciting antitumor immune responses has gained increasing attention. Within the immunosuppressive tumor microenvironment (TME), interferons stimulate tumor-infiltrating lymphocytes to promote immune clearance and essentially reshape a "cold" TME into an immune-activating "hot" TME. In this review, we focus on gliomas, with an emphasis on malignant glioblastoma, as these brain tumors possess a highly invasive and heterogenous brain TME. We address how type I interferons regulate antitumor immune responses against malignant gliomas and reshape the overall immune landscape of the brain TME. Furthermore, we discuss how these findings can translate into future immunotherapies targeting brain tumors in general.

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.

Pellino-1 promotes intrinsic activation of skin-resident IL-17A-producing T cells in psoriasis.

BACKGROUND: Psoriasis is a chronically relapsing inflammatory skin disease primarily perpetuated by skin-resident IL-17-producing T (T17) cells. Pellino-1 (Peli1) belongs to a member of E3 ubiquitin ligase mediating immune receptor signaling cascades, including nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) pathway. OBJECTIVE: We explored the potential role of Peli1 in psoriatic inflammation in the context of skin-resident T17 cells. METHODS: We performed single-cell RNA sequencing of relapsing and resolved psoriatic lesions with analysis for validation data set of psoriasis. Mice with systemic and conditional depletion of Peli1 were generated to evaluate the role of Peli1 in imiquimod-induced psoriasiform dermatitis. Pharmacologic inhibition of Peli1 in human CD4+ T cells and ex vivo human skin cultures was also examined to evaluate its potential therapeutic implications. RESULTS: Single-cell RNA sequencing analysis revealed distinct T-cell subsets in relapsing psoriasis exhibiting highly enriched gene signatures for (1) tissue-resident T cells, (2) T17 cells, and (3) NF-κB signaling pathway including PELI1. Peli1-deficient mice were profoundly protected from psoriasiform dermatitis, with reduced IL-17A production and NF-κB activation in γδ T17 cells. Mice with conditional depletion of Peli1 treated with FTY720 revealed that Peli1 was intrinsically required for the skin-resident T17 cell immune responses. Notably, pharmacologic inhibition of Peli1 significantly ameliorated murine psoriasiform dermatitis and IL-17A production from the stimulated human CD4+ T cells and ex vivo skin explants modeling psoriasis. CONCLUSION: Targeting Peli1 would be a promising therapeutic strategy for psoriasis by limiting skin-resident T17 cell immune responses.

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.

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.

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.

Frame-rate up-conversion detection based on convolutional neural network for learning spatiotemporal features.

With the advance in user-friendly and powerful video editing tools, anyone can easily manipulate videos without leaving prominent visual traces. Frame-rate up-conversion (FRUC), a representative temporal-domain operation, increases the motion continuity of videos with a lower frame-rate and is used by malicious counterfeiters in video tampering such as generating fake frame-rate video without improving the quality or mixing temporally spliced videos. FRUC is based on frame interpolation schemes and subtle artifacts that remain in interpolated frames are often difficult to distinguish. Hence, detecting such forgery traces is a critical issue in video forensics. This paper proposes a frame-rate conversion detection network (FCDNet) that learns forensic features caused by FRUC in an end-to-end fashion. The proposed network uses a stack of consecutive frames as the input and effectively learns interpolation artifacts using network blocks to learn spatiotemporal features. Moreover, it can cover the following three types of frame interpolation schemes: nearest neighbor interpolation, bilinear interpolation, and motion-compensated interpolation. In contrast to existing methods that exploit all frames to verify integrity, the proposed approach achieves a high detection speed because it observes only six frames to test its authenticity. Extensive experiments were conducted with conventional forensic methods and neural networks for video forensics to validate our research. The proposed work achieved an outstanding performance in terms of detecting the interpolated artifacts of FRUC. The experimental results also demonstrate that our model is robust against an unseen dataset, unlearned frame-rate, and unlearned quality factor. Furthermore, FCDNet can precisely localize the tampered region applied to manipulation along the time-domain through temporal localization.

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).

Dendritic Cell-Based Immunotherapy in Hot and Cold Tumors.

Dendritic cells mediate innate and adaptive immune responses and are directly involved in the activation of cytotoxic T lymphocytes that kill tumor cells. Dendritic cell-based cancer immunotherapy has clinical benefits. Dendritic cell subsets are diverse, and tumors can be hot or cold, depending on their immunogenicity; this heterogeneity affects the success of dendritic cell-based immunotherapy. Here, we review the ontogeny of dendritic cells and dendritic cell subsets. We also review the characteristics of hot and cold tumors and briefly introduce therapeutic trials related to hot and cold tumors. Lastly, we discuss dendritic cell-based cancer immunotherapy in hot and cold tumors.

Host and Microbiome Interplay Shapes the Vaginal Microenvironment.

The female reproductive tract harbors a unique microbiome, especially the vagina. The human vaginal microbiome exhibits a low diversity and is dominated by Lactobacillus species, compared to the microbiome of other organs. The host and vaginal microbiome mutually coexist in the vaginal microenvironment. Host cells provide Lactobacillus glycogen as an energy source, and Lactobacillus produce lactic acid, which lowers vaginal pH thereby preventing growth of other bacteria. Bacterial vaginosis can modulate host immune systems, and is frequently associated with various aspects of disease, including sexually transmitted infection, gynecologic cancer, and poor pregnancy outcomes. Because of this, numerous studies focused on the impact of the vaginal microbiome on women`s health and disease. Furthermore, numerous epidemiologic studies also have demonstrated various host factors regulate the vaginal microbiome. The female reproductive tract undergoes constant fluctuations due to hormonal cycle, pregnancy, and other extrinsic factors. Depending on these fluctuations, the vaginal microbiome composition can shift temporally and dynamically. In this review, we highlight the current knowledge of how host factors modulate vaginal microbiome composition and how the vaginal microbiome contributes to maintaining homeostasis or inducing pathogenesis. A better understanding of relationship between host and vaginal microbiome could identify novel targets for diagnosis, prognosis, or treatment of microbiome-related diseases.

A Microbiota-Dependent Subset of Skin Macrophages Protects Against Cutaneous Bacterial Infection.

Microbiota is essential to the development and functional maturation of the immune system. The effects of the gut microbiota on myeloid cells remote from the gut, especially the skin remain unclear. Transcriptomic analysis revealed that type I interferon (IFN) signaling was down-regulated in the skin of germ-free mice compared to that in specific pathogen-free mice. The decrease in type I IFN signaling was closely related to the presence of microbiota and macrophage-specific marker CD169. The absence of CD169+ macrophages resulted in increased bacterial burden and impaired immune responses against Staphylococcus aureus skin infection. CD169+ macrophages mediated the recruitment of γδ T cells as well as the activation of γδ T cells via interleukin (IL)-23. Our findings demonstrate the role of the microbiota in establishment of a specific myeloid cell subset expressing CD169 in the skin and provide evidence of a specific mechanism by which this subset protects against bacterial skin infection.

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.

The Role of Autophagy in the Function of CD4+ T Cells and the Development of Chronic Inflammatory Diseases.

Uncontrolled acute inflammation progresses to persistent inflammation that leads to various chronic inflammatory diseases, including asthma, Crohn's disease, rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus. CD4+ T cells are key immune cells that determine the development of these chronic inflammatory diseases. CD4+ T cells orchestrate adaptive immune responses by producing cytokines and effector molecules. These functional roles of T cells vary depending on the surrounding inflammatory or anatomical environment. Autophagy is an important process that can regulate the function of CD4+ T cells. By lysosomal degradation of cytoplasmic materials, autophagy mediates CD4+ T cell-mediated immune responses, including cytokine production, proliferation, and differentiation. Furthermore, through canonical processes involving autophagy machinery, autophagy also contributes to the development of chronic inflammatory diseases. Therefore, a targeted intervention of autophagy processes could be used to treat chronic inflammatory diseases. This review focuses on the role of autophagy via CD4+ T cells in the pathogenesis and treatment of such diseases. In particular, we explore the underlying mechanisms of autophagy in the regulation of CD4+ T cell metabolism, survival, development, proliferation, differentiation, and aging. Furthermore, we suggest that autophagy-mediated modulation of CD4+ T cells is a promising therapeutic target for treating chronic inflammatory diseases.