Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8+ T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8+ T cells and Tregs and represses the CD8+ T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers.

Exosome-based delivery of super-repressor IκBα ameliorates kidney ischemia-reperfusion injury.

Ischemia-reperfusion injury is a major cause of acute kidney injury. Recent studies on the pathophysiology of ischemia-reperfusion-induced acute kidney injury showed that immunologic responses significantly affect kidney ischemia-reperfusion injury and repair. Nuclear factor (NF)-ĸB signaling, which controls cytokine production and cell survival, is significantly involved in ischemia-reperfusion-induced acute kidney injury, and its inhibition can ameliorate ischemic acute kidney injury. Using EXPLOR, a novel, optogenetically engineered exosome technology, we successfully delivered the exosomal super-repressor inhibitor of NF-ĸB (Exo-srIĸB) into B6 wild type mice before/after kidney ischemia-reperfusion surgery, and compared outcomes with those of a control exosome (Exo-Naïve)-injected group. Exo-srIĸB treatment resulted in lower levels of serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin in post-ischemic mice than in the Exo-Naïve treatment group. Systemic delivery of Exo-srIĸB decreased NF-ĸB activity in post-ischemic kidneys and reduced apoptosis. Post-ischemic kidneys showed decreased gene expression of pro-inflammatory cytokines and adhesion molecules with Exo-srIĸB treatment as compared with the control. Intravital imaging confirmed the uptake of exosomes in neutrophils and macrophages. Exo-srIĸB treatment also significantly affected post-ischemic kidney immune cell populations, lowering neutrophil, monocyte/macrophage, and T cell frequencies than those in the control. Thus, modulation of NF-ĸB signaling through exosomal delivery can be used as a novel therapeutic method for ischemia-reperfusion-induced acute kidney injury.

Viral infection of the lung: host response and sequelae.

Because of its essential role in gas exchange and oxygen delivery, the lung has evolved a variety of strategies to control inflammation and maintain homeostasis. Invasion of the lung by pathogens (and in some instances exposure to certain noninfectious particulates) disrupts this equilibrium and triggers a cascade of events aimed at preventing or limiting colonization (and more importantly infection) by pathogenic microorganisms. In this review we focus on viral infection of the lung and summarize recent advances in our understanding of the triggering of innate and adaptive immune responses to viral respiratory tract infection, mechanisms of viral clearance, and the well-recognized consequences of acute viral infection complicating underlying lung diseases, such as asthma.

Effects of NF-κB Inhibitor on Sepsis Depend on the Severity and Phase of the Animal Sepsis Model.

Hyperinflammation occurs in sepsis, especially in the early phase, and it could have both positive and negative effects on sepsis. Previously, we showed that a new concept of NF-κB inhibitor, exosome-based super-repressor IκBα (Exo-srIκB) delivery, has a beneficial effect on sepsis. Here, we further investigate the therapeutic effects of Exo-srIκB at different severities and phases of sepsis using an animal polymicrobial intra-abdominal infection model. We used a rat model of fecal slurry polymicrobial sepsis. First, we determined the survival effects of Exo-srIκB on sepsis according to the severity. We used two different severities of the animal sepsis model. The severe model had a mortality rate of over 50%. The mild/moderate model had a less than 30% mortality rate. Second, we administered the Exo-srIκB at various time points (1 h, 6 h, and 24 h after fecal slurry administration) to determine the therapeutic effect of Exo-srIκB at different phases of sepsis. Lastly, we determined the effects of the Exo-srIκB on cytokine production, arterial blood gas, electrolyte, and lactate. The survival gain was statistically significant in the severe sepsis model when Exo-srIκB was administered 6 h after sepsis. Interleukin 6 and interleukin-10 were significantly decreased in the kidney when administered with Exo-srIκB. The laboratory data showed that lactate, glucose, and potassium levels were significantly lowered in the NF-κB inhibitor group. In conclusion, Exo-srIκB exhibited a beneficial therapeutic effect when administered 6 h post fecal slurry administration in a severe sepsis model.

Exosome-mediated delivery of super-repressor IκBα alleviates inflammation and joint damages in rheumatoid arthritis.

BACKGROUND: This study aims to investigate the potential anti-inflammatory effects of exosomes engineered to carry super-repressor IκB (Exo-srIκB), an exosome-based NF-κB inhibitor, in the context of RA. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from patients diagnosed with RA and treated with Exo-srIκB to test the therapeutic potential. Flow cytometry analysis was performed to assess the production of inflammatory cytokines (IL-17A and GM-CSF) by the cells. ELISA was utilized to measure the levels of TNF-α, IL-17A, IL-6, and GM-CSF. Arthritis was induced in SKG mice by intraperitoneal injection of curdlan. DBA/1 J mice were used in collagen-induced arthritis (CIA) experiments. After the development of arthritis, mice were injected with either Exo-Naïve (control exosome) or Exo-srIκB. Arthritis scores were recorded biweekly, and histological observations of the ankle joint were conducted using H&E and safranin-O staining. Additionally, bone erosion was evaluated using micro-CT imaging. RESULTS: In the ex vivo study involving human PBMCs and SFMCs, treatment with Exo-srIκB demonstrated a notable reduction in inflammatory cytokines. Furthermore, in both the SKG and CIA models, Exo-srIκB treatment exhibited significant reductions in inflammation, cartilage destruction, and bone erosion within the joint tissues when compared to the Exo-Naive control group. Additionally, the radiographic score assessed through microCT showed a significant decrease compared to the Exo-Naive control group. CONCLUSION: Overall, these findings suggest that Exo-srIκB possesses anti-inflammatory properties in human RA cells and animal models, making it a promising therapeutic candidate for the treatment of RA.

Exosome-Based Delivery of Super-Repressor IκBα Alleviates Alcohol-Associated Liver Injury in Mice.

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) instigates nuclear factor-κB (NF-κB)-mediated inflammatory responses in alcohol-associated liver diseases (ALD). Here, we utilized a novel optogenetically engineered exosome technology called 'exosomes for protein loading via optically reversible protein-protein interactions (EXPLOR)' to efficiently deliver the super-repressor IκB-loaded exosomes (Exo-srIκB) to the liver and examined its therapeutic potential in acute-on-chronic alcohol-associated liver injury. We detected enhanced uptake of DiI-labeled Exo-srIκB by LPS-treated inflammatory KCs, which suppressed LPS-induced inflammatory gene expression levels. In animal experiments, a single intravenous injection of Exo-srIκB prior to alcohol binge drinking significantly attenuated alcohol-associated hepatic steatosis and infiltration of neutrophils and macrophages but not a liver injury. Notably, three consecutive days of Exo-srIκB injection remarkably reduced alcohol-associated liver injury, steatosis, apoptosis of hepatocytes, fibrosis-related gene expression levels in hepatic stellate cells, infiltration of neutrophils and macrophages, and inflammatory gene expression levels in hepatocytes and KCs. In particular, the above effects occurred with inhibition of nuclear translocation of NF-κB in liver tissues, and these beneficial effects of Exo-srIκB on ALD were shown regardless of doses. Our results suggest an exosome-based modulation of NF-κB activity in KCs by Exo-srIκB as a novel and efficient therapeutic approach in ALD.

Biodistribution of Exosomes and Engineering Strategies for Targeted Delivery of Therapeutic Exosomes.

Exosomes are cell-secreted nano-sized vesicles which deliver diverse biological molecules for intercellular communication. Due to their therapeutic potential, exosomes have been engineered in numerous ways for efficient delivery of active pharmaceutical ingredients to various target organs, tissues, and cells. In vivo administered exosomes are normally delivered to the liver, spleen, kidney, lung, and gastrointestinal tract and show rapid clearance from the blood circulation after systemic injection. The biodistribution and pharmacokinetics (PK) of exosomes can be modulated by engineering various factors such as cellular origin and membrane protein composition of exosomes. Recent advances accentuate the potential of targeted delivery of engineered exosomes even to the most challenging organs including the central nervous system. Major breakthroughs have been made related to various imaging techniques for monitoring in vivo biodistribution and PK of exosomes, as well as exosomal surface engineering technologies for inducing targetability. For inducing targeted delivery, therapeutic exosomes can be engineered to express various targeting moieties via direct modification methods such as chemically modifying exosomal surfaces with covalent/non-covalent bonds, or via indirect modification methods by genetically engineering exosome-producing cells. In this review, we describe the current knowledge of biodistribution and PK of exosomes, factors determining the targetability and organotropism of exosomes, and imaging technologies to monitor in vivo administered exosomes. In addition, we highlight recent advances in strategies for inducing targeted delivery of exosomes to specific organs and cells.

Exosomal delivery of NF-κB inhibitor delays LPS-induced preterm birth and modulates fetal immune cell profile in mouse models.

Accumulation of immune cells and activation of the pro-inflammatory transcription factor NF-κB in feto-maternal uterine tissues is a key feature of preterm birth (PTB) pathophysiology. Reduction of the fetal inflammatory response and NF-κB activation are key strategies to minimize infection-associated PTB. Therefore, we engineered extracellular vesicles (exosomes) to contain an NF-κB inhibitor, termed super-repressor (SR) IκBα. Treatment with SR exosomes (1 × 1010 per intraperitoneal injection) after lipopolysaccharide (LPS) challenge on gestation day 15 (E15) prolonged gestation by over 24 hours (PTB ≤ E18.5) and reduced maternal inflammation (n ≥ 4). Furthermore, using a transgenic model in which fetal tissues express the red fluorescent protein tdTomato while maternal tissues do not, we report that LPS-induced PTB in mice is associated with influx of fetal innate immune cells, not maternal, into feto-maternal uterine tissues. SR packaged in exosomes provides a stable and specific intervention for reducing the inflammatory response associated with PTB.

p53 deficiency and defective mitotic checkpoint in proliferating T lymphocytes increase chromosomal instability through aberrant exit from mitotic arrest.

During the proliferation of T cells for successful immune responses against pathogens, the fine regulation of cell cycle is important to the maintenance of T cell homeostasis and the prevention of lymphoproliferative disorders. However, it remains to be elucidated how the cell cycle is controlled at the mitotic phase in proliferating T cells. Here, we show that during the proliferation of primary T cells, the disruption of the mitotic spindle leads to cell-cycle arrest at mitosis and that prolonged mitotic arrest results in not only apoptosis but also the form of chromosomal instability observed in human cancers. It is interesting that in response to spindle damage, the phosphorylation of BubR1, a mitotic checkpoint kinase, was significantly induced in proliferating T cells, and the expression of the dominant-negative mutant of BubR1 compromised mitotic arrest and subsequent apoptosis and thus led to the augmentation of polyploidy formation. We also show that in response to prolonged spindle damage, the expression of p53 but not of p73 was significantly induced. In addition, following sustained mitotic arrest, p53-deficient T cells were found to be more susceptible to polyploidy formation than the wild type. These results suggest that during flourishing immune response, mitotic checkpoint and p53 play important roles in the prevention of chromosomal instability and in the maintenance of the genomic integrity of proliferating T cells.

IL-21 promotes late activator APC-mediated T follicular helper cell differentiation in experimental pulmonary virus infection.

IL-21 is a type-I cytokine that has pleiotropic immuno-modulatory effects. Primarily produced by activated T cells including NKT and TFH cells, IL-21 plays a pivotal role in promoting TFH differentiation through poorly understood cellular and molecular mechanisms. Here, employing a mouse model of influenza A virus (IAV) infection, we demonstrate that IL-21, initially produced by NKT cells, promotes TFH differentiation by promoting the migration of late activator antigen presenting cell (LAPC), a recently identified TFH inducer, from the infected lungs into the draining lymph nodes (dLN). LAPC migration from IAV-infected lung into the dLN is CXCR3-CXCL9 dependent. IL-21-induced TNF-α production by conventional T cells is critical to stimulate CXCL9 expression by DCs in the dLN, which supports LAPC migration into the dLN and ultimately facilitates TFH differentiation. Our results reveal a previously unappreciated mechanism for IL-21 modulation of TFH responses during respiratory virus infection.

LAPCs promote follicular helper T cell differentiation of Ag-primed CD4+ T cells during respiratory virus infection.

The humoral immune response to most respiratory virus infections plays a prominent role in virus clearance and is essential for resistance to reinfection. T follicular helper (Tfh) cells are believed to support the development both of a potent primary antibody response and of the germinal center response critical for memory B cell development. Using a model of primary murine influenza A virus (IAV) infection, we demonstrate that a novel late activator antigen-presenting cell (LAPC) promotes the Tfh response in the draining lymph nodes (dLNs) of the IAV-infected lungs. LAPCs migrate from the infected lungs to the dLN "late," i.e., 6 d after infection, which is concomitant with Tfh differentiation. LAPC migration is CXCR3-dependent, and LAPC triggering of Tfh cell development requires ICOS-ICOSL-dependent signaling. LAPCs appear to play a pivotal role in driving Tfh differentiation of Ag-primed CD4(+) T cells and antiviral antibody responses.

Interferon-ß modulates type 1 immunity during influenza virus infection.

Influenza viruses are important human pathogens, associated throughout history with worldwide outbreaks and pandemics. The antiviral effects of interferon (IFN)-αs/ß against influenza virus infections are well recognized, yet the mechanisms whereby IFNs exert their immunomodulatory effects on an anti-influenza response remain ill-defined. Here, we describe the effects of IFN-ß treatment on the immune response during a respiratory influenza (A/WSN/33) A virus infection of mice. A single dose of IFN-ß (1×10(5)U) enhanced DC migration into the draining lymph node (DLN) on day 3 post-intranasal infection, and subsequently inhibited the migration from the lungs into the DLN of a newly identified late activator antigen-presenting cell population associated with type 2 immunity, LAPC. IFN-ß treatment polarized the immune response towards a type 1 immune response, eliciting enhanced T(H)1 effector and cytolytic T cell responses, but diminished T(H)2 effector T cell responses in both the DLN and lung tissues of influenza virus-infected mice. Associated with the polarization towards a type 1 immune response, IFN-ß treatment of mice resulted in accelerated viral clearance and diminished pulmonary eosinophilia in infected lung tissues.

Identification of a novel antigen-presenting cell population modulating antiinfluenza type 2 immunity.

Antiinfluenza type 2 (T2) immunity contributes to both immunopathology and immunoprotection, yet the underlying mechanisms modulating T2 immunity remain ill defined. We describe a novel mouse antigen (Ag)-presenting cell (APC), designated late-activator APC (LAPC). After pulmonary influenza A (H1N1) virus infection, LAPCs enter the lungs, capture viral Ag, and subsequently migrate to the draining lymph node (DLN) and spleen, with delayed kinetics relative to dendritic cells (DCs). In the DLN, influenza virus-activated LAPCs present Ag and selectively induce T helper type 2 (Th2) effector cell polarization by cell-cell contact-mediated modulation of GATA-3 expression. In adoptive transfer experiments, influenza virus-activated LAPCs augmented Th2 effector T cell responses in the DLN, increased production of circulating antiinfluenza immunoglobulin, and increased levels of T2 cytokines in bronchoalveolar lavage fluid in recipient influenza virus-infected mice. LAPC-recipient mice exhibited exacerbated pulmonary pathology, with delayed viral clearance and enhanced pulmonary eosinophilia. Collectively, our results identify and highlight the importance of LAPCs as immunomodulators of T2 immunity during influenza A virus infection.

IL-18 induces monocyte chemotactic protein-1 production in macrophages through the phosphatidylinositol 3-kinase/Akt and MEK/ERK1/2 pathways.

Macrophages are activated during an inflammatory response and produce multiple inflammatory cytokines. IL-18 is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. Monocyte chemoattractant protein (MCP-1) is expressed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and its expression is correlated with the severity of the disease. Both IL-18 and MCP-1 have been shown to be involved in inflammatory immune responses. However, it has been unclear whether IL-18 is involved in the induction of MCP-1. This investigation was initiated to determine whether IL-18 can induce MCP-1 production, and if so, by which signal transduction pathways. We found that IL-18 induced the production of MCP-1 in macrophages, which was IL-12-independent and was not mediated by autocrine cytokines such as IFN-gamma or TNF-alpha. We then examined signal transduction pathways involved in IL-18-induced MCP-1 production. We found that IL-18 did not activate the IkappaB kinase/NF-kappaB pathway, evidenced by no degradation of IkappaBalpha and no translocation of NF-kappaB p65 to the nucleus in IL-18-stimulated macrophages. Instead, IL-18 activated the PI3K/Akt and MEK/ERK1/2 pathways. Inhibition of either of these pathways attenuated MCP-1 production in macrophages, and inhibition of both signaling pathways resulted in the complete inhibition of MCP-1 production. On the basis of these observations, we conclude that IL-18 induces MCP-1 production through the PI3K/Akt and MEK/ERK1/2 pathways in macrophages.

Inhibition of TCR-induced CD8 T cell death by IL-12: regulation of Fas ligand and cellular FLIP expression and caspase activation by IL-12.

In this study we demonstrate the anti-apoptotic effect of IL-12 and its underlying mechanism in CD8 T cells. The prolonged stimulation of CD8 T cells with anti-CD3 alone caused apoptosis mediated by Fas and the caspase signaling pathway. However, costimulation with IL-12 significantly prevented anti-CD3-induced apoptosis of CD8 T cells. IL-12 decreased the number of Fas ligand-positive CD8 T cells and inhibited the activation of caspase-8 and caspase-3. In addition, IL-12 up-regulated cellular FLIPs but not Bcl-2 family proteins or cellular inhibitor of apoptosis proteins. These data suggest that IL-12 provides survival signals to CD8 T cells by down-regulating Fas ligand and up-regulating cellular FLIPs, followed by inhibiting caspase activation, which implies a role for IL-12 in peripheral responses of CD8 T cells in vivo.

IL-12 provides proliferation and survival signals to murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway.

IL-12 is a pleiotropic cytokine that plays an important role in innate and adaptive immunity. IL-12 induces T cell proliferation and IFN-gamma secretion from activated T cells. It was also reported that IL-12 prevents apoptosis of CD4(+) T cells. However, the signaling mechanism that regulates these IL-12-induced responses is poorly understood yet. In this study, we demonstrated that IL-12 activates phosphatidylinositol 3-kinase (PI3K)/Akt pathway in murine CD4(+) T cells, and that this signaling pathway is required for IL-12-induced T cell proliferation and antiapoptotic function, but not for IFN-gamma induction. Through PI3K/Akt pathway, IL-12 up-regulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins-2, followed by down-regulation of active caspase-3. These results suggest that PI3K/Akt pathway is critical for mediating IL-12-induced CD4(+) T cell responses such as T cell proliferation and survival.