Antigen-driven CD8 + T cell clonal expansion is a prominent feature of MASH in humans and mice.

Background and Aims: Chronic liver disease due to metabolic dysfunction-associated steatohepatitis (MASH) is a rapidly increasing global epidemic. MASH progression is a consequence of the complex interplay between inflammatory insults and dysregulated hepatic immune responses. T lymphocytes have been shown to accumulate in the liver during MASH, but the cause and consequence of T cell accumulation in the liver remain unclear. Our study aimed to define the phenotype and T cell receptor diversity of T cells from human cirrhotic livers and an animal model of MASH to begin resolving their function in disease. Approach and Results: In these studies, we evaluated differences in T cell phenotype in the context of liver disease we isolated liver resident T cell populations from individuals with cirrhosis and a murine model of MASH. Using both 5' single cell sequencing and flow cytometry we defined the phenotype and T cell receptor repertoire of liver resident T cells during health and disease. Conclusions: MASH-induced cirrhosis and diet-induced MASH in mice resulted in the accumulation of activated and clonally expanded T cells in the liver. The clonally expanded T cells in the liver expressed markers of chronic antigenic stimulation, including PD1 , TIGIT and TOX . Overall, this study establishes for the first time that T cells undergo antigen-dependent clonal expansion and functional differentiation during the progression of MASH. These studies could lead to the identification of potential antigenic targets that drive T cell activation, clonal expansion, and recruitment to the liver during MASH.

NF-κB/NLRP3 Translational Inhibition by Nanoligomer Therapy Mitigates Ethanol and Advanced Age-Related Neuroinflammation.

Binge alcohol use is increasing among aged adults (>65 years). Alcohol-related toxicity in aged adults is associated with neurodegeneration, yet the molecular underpinnings of age-related sensitivity to alcohol are not well described. Studies utilizing rodent models of neurodegenerative disease reveal heightened activation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Nod like receptor 3 (NLRP3) mediate microglia activation and associated neuronal injury. Our group, and others, have implicated hippocampal-resident microglia as key producers of inflammatory mediators, yet the link between inflammation and neurodegeneration has not been established in models of binge ethanol exposure and advanced age. Here, we report binge ethanol increased the proportion of NLRP3+ microglia in the hippocampus of aged (18-20 months) female C57BL/6N mice compared to young (3-4 months). In primary microglia, ethanol-induced expression of reactivity markers and NLRP3 inflammasome activation were more pronounced in microglia from aged mice compared to young. Making use of an NLRP3-specific inhibitor (OLT1177) and a novel brain-penetrant Nanoligomer that inhibits NF-κB and NLRP3 translation (SB_NI_112), we find ethanol-induced microglial reactivity can be attenuated by OLT1177 and SB_NI_112 in microglia from aged mice. In a model of intermittent binge ethanol exposure, SB_NI_112 prevented ethanol-mediated microglia reactivity, IL-1ß production, and tau hyperphosphorylation in the hippocampus of aged mice. These data suggest early indicators of neurodegeneration occurring with advanced age and binge ethanol exposure are NF-κB- and NLRP3-dependent. Further investigation is warranted to explore the use of targeted immunosuppression via Nanoligomers to attenuate neuroinflammation after alcohol consumption in the aged.

Oxidized low density lipoprotein in the liver causes decreased permeability of liver lymphatic- but not liver sinusoidal-endothelial cells via VEGFR-3 regulation of VE-Cadherin.

The lymphatic vasculature of the liver is vital for liver function as it maintains fluid and protein homeostasis and is important for immune cell transport to the lymph node. Chronic liver disease is associated with increased expression of inflammatory mediators including oxidized low-density lipoprotein (oxLDL). Intrahepatic levels of oxLDL are elevated in nonalcoholic fatty liver disease (NAFLD), chronic hepatitis C infection (HCV), alcohol-associated liver disease (ALD), and cholestatic liver diseases. To determine if liver lymphatic function is impaired in chronic liver diseases, in which increased oxLDL has been documented, we measured liver lymphatic function in murine models of NAFLD, ALD and primary sclerosing cholangitis (PSC). We found that Mdr2-/- (PSC), Lieber-DeCarli ethanol fed (ALD) and high fat and high cholesterol diet fed (NAFLD) mice all had a significant impairment in the ability to traffic FITC labeled dextran from the liver parenchyma to the liver draining lymph nodes. Utilizing an in vitro permeability assay, we found that oxLDL decreased the permeability of lymphatic endothelial cells (LEC)s, but not liver sinusoidal endothelial cells (LSEC)s. Here we demonstrate that LECs and LSECs differentially regulate SRC-family kinases, MAPK kinase and VE-Cadherin in response to oxLDL. Furthermore, Vascular Endothelial Growth Factor (VEGF)C or D (VEGFR-3 ligands) appear to regulate VE-Cadherin expression as well as decrease cellular permeability of LECs in vitro and in vivo after oxLDL treatment. These findings suggest that oxLDL acts to impede protein transport through the lymphatics through tightening of the cell-cell junctions. Importantly, engagement of VEGFR-3 by its ligands prevents VE-Cadherin upregulation and improves lymphatic permeability. These studies provide a potential therapeutic target to restore liver lymphatic function and improve liver function.

Proteomic analysis of alcohol-associated hepatitis reveals glycoprotein NMB (GPNMB) as a novel hepatic and serum biomarker.

Alcohol consumption remains a leading cause of liver disease worldwide, resulting in a complex array of hepatic pathologies, including steatosis, steatohepatitis, and cirrhosis. Individuals who progress to a rarer form of alcohol-associated liver disease (ALD), alcohol-associated hepatitis (AH), require immediate life-saving intervention in the form of liver transplantation. Rapid onset of AH is poorly understood and the metabolic mechanisms contributing to the progression to liver failure remain undetermined. While multiple mechanisms have been identified that contribute to ALD, no cures exist and mortality from AH remains high. To identify novel pathways associated with AH, our group utilized proteomics to investigate AH-specific biomarkers in liver explant tissues. The goal of the present study was to determine changes in the proteome as well as epigenetic changes occurring in AH. Protein abundance and acetylomic analyses were performed utilizing nHPLC-MS/MS, revealing significant changes to proteins associated with metabolic and inflammatory fibrosis pathways. Here, we describe a novel hepatic and serum biomarker of AH, glycoprotein NMB (GPNMB). The anti-inflammatory protein GPNMB was significantly increased in AH explant liver and serum compared to healthy donors by 50-fold and 6.5-fold, respectively. Further, bioinformatics analyses identified an AH-dependent decrease in protein abundance across fatty acid degradation, biosynthesis of amino acids, and carbon metabolism. The greatest increases in protein abundance were observed in pathways for focal adhesion, lysosome, phagosome, and actin cytoskeleton. In contrast with the hyperacetylation observed in murine models of ALD, protein acetylation was decreased in AH compared to normal liver across fatty acid degradation, biosynthesis of amino acids, and carbon metabolism. Interestingly, immunoblot analysis found epigenetic marks were significantly increased in AH explants, including Histone H3K9 and H2BK5 acetylation. The increased acetylation of histones likely plays a role in the altered proteomic profile observed, including increases in GPNMB. Indeed, our results reveal that the AH proteome is dramatically impacted through unanticipated and unknown mechanisms. Understanding the origin and consequences of these changes will yield new mechanistic insight for ALD as well as identify novel hepatic and serum biomarkers, such as GPNMB.

Tumor NLRP3-Derived IL-1ß Drives the IL-6/STAT3 Axis Resulting in Sustained MDSC-Mediated Immunosuppression.

Tumors evade the immune system by inducing inflammation. In melanoma, tumor-derived IL-1ß drives inflammation and the expansion of highly immunosuppressive myeloid-derived suppressor cells (MDSCs). Similar in many tumors, melanoma is also linked to the downstream IL-6/STAT3 axis. In this study, we observed that both recombinant and tumor-derived IL-1ß specifically induce pSTAT3(Y705), creating a tumor-autoinflammatory loop, which amplifies IL-6 signaling in the human melanoma cell line 1205Lu. To disrupt IL-1ß/IL-6/STAT3 axis, we suppressed IL-1ß-mediated inflammation by inhibiting the NOD-like receptor protein 3 (NLRP3) using OLT1177, a safe-in-humans specific NLRP3 oral inhibitor. In vivo, using B16F10 melanoma, OLT1177 effectively reduced tumor progression (p< 0.01); in primary tumors, OLT1177 decreased pSTAT3(Y705) by 82% (p<0.01) and II6 expression by 53% (p<0.05). Disruption of tumor-derived NLRP3, either pharmacologically or genetically, reduced STAT3 signaling in bone marrow cells. In PMN-MDSCs isolated from tumor-bearing mice treated with OLT1177, we observed significant reductions in immunosuppressive genes such as Pdcd1l1, Arg1, Il10 and Tgfb1. In conclusion, the data presented here show that the inhibition of NLRP3 reduces IL-1ß induction of pSTAT3(Y705) preventing expression of immunosuppressive genes as well as activity in PMN-MDSCs.

Single-cell transcriptomic analyses of T cells in chronic HCV-infected patients dominated by DAA-induced interferon signaling changes.

Chronic infection with HCV is manifested by dysregulation of innate immune responses and impaired T cell function at multiple levels. These changes may impact susceptibility to other infections, responsiveness to antiviral therapies, vaccine responsiveness, and development of complications such as hepatocellular carcinoma. Highly effective direct-acting antiviral (DAA) therapy has revolutionized the management of chronic HCV, with expected cure rates exceeding 95%. DAA treatment represents a unique opportunity to investigate to what extent elimination of viral replication and chronic antigen stimulation can restore immunologic phenotype. In this study we interrogated the global transcriptional profile of isolated peripheral blood T cells before, during and after IFN-free DAA therapy using single-cell mRNA sequencing. Our results demonstrate that T cells mapped at single-cell resolution have dramatic transcriptomic changes early after initiation of DAA and many of these changes are sustained after completion of DAA therapy. Specifically, we see a significant reduction in transcripts associated with innate immune activation and interferon signaling such as ISG15, ISG20, IFIT3, OAS and MX1 in many different T cell subsets. Furthermore, we find an early upregulation of a gene involved in suppression of immune activation, DUSP1, in circulating T cells. Conclusion: This study provides the first in-depth transcriptomic analysis at the single-cell level of patients undergoing DAA therapy, demonstrating that IFN-free antiviral therapy in chronic HCV infection induces hitherto unrecognized shifts in innate immune and interferon signaling within T cell populations early, during, and long-term after treatment. The present study provides a rich data source to explore the effects of DAA treatment on bulk T cells.

Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

The lymphatic system is a network of ducts that transports fluid, proteins, and immune cells from different organs around the body. Lymph nodes provide pit stops at hundreds of points along this network where immune cells reside, and lymph fluid can be filtered and cleaned. When pathogens, such as viruses or bacteria, enter the body during an infection, fragments of their proteins can get swept into the lymph nodes. These pathogenic proteins or protein fragments activate resident immune cells and kickstart the immune response. Vaccines are designed to mimic this process by introducing isolated pathogenic proteins in a controlled way to stimulate similar immune reactions in lymph nodes. Once an infection has been cleared by the immune system, or a vaccination has triggered the immune system, most pathogenic proteins get cleared away. However, a small number of pathogenic proteins remain in the lymph nodes to enable immune cells to respond more strongly and quickly the next time they see the same pathogen. Yet it is largely unclear how much protein remains for training and how or where it is all stored. Current techniques are not sensitive or long-lived enough to accurately detect and track these small protein deposits over time. Walsh, Sheridan, Lucas, et al. have addressed this problem by developing biological tags that can be attached to the pathogenic proteins so they can be traced. These tags were designed so the body cannot easily break them down, helping them last as long as the proteins they are attached to. Walsh, Sheridan, Lucas et al. tested whether vaccinating mice with the tagged proteins allowed the proteins to be tracked. The method they used was designed to identify individual cell types based on their genetic information along with the tag. This allowed them to accurately map the complex network of cells involved in storing and retrieving archived protein fragments, as well as those involved in training new immune cells to recognize them. These results provide important insights into the protein archiving system that is involved in enhancing immune memory. This may help guide the development of new vaccination strategies that can manipulate how proteins are archived to establish more durable immune protection. The biological tags developed could also be used to track therapeutic proteins, allowing scientists to determine how long cancer drugs, antibody therapies or COVID19 anti-viral agents remain in the body. This information could then be used by doctors to plan specific and personalized treatment timetables for patients.

Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis.

BACKGROUND: Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT. METHODS: We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments. RESULTS: In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence. CONCLUSIONS: Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches.

Oxidized Low-Density Lipoprotein Drives Dysfunction of the Liver Lymphatic System.

BACKGROUND AND AIMS: As the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease. METHODS: To answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo. RESULTS: Confirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries. CONCLUSIONS: We identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease.

JAK1 Inhibition Blocks Lethal Immune Hypersensitivity in a Mouse Model of Down Syndrome.

Individuals with Down syndrome (DS; trisomy 21) display hyperactivation of interferon (IFN) signaling and chronic inflammation, which could potentially be explained by the extra copy of four IFN receptor (IFNR) genes encoded on chromosome 21. However, the clinical effects of IFN hyperactivity in DS remain undefined. Here, we report that a commonly used mouse model of DS overexpresses IFNR genes and shows hypersensitivity to IFN ligands in diverse immune cell types. When treated repeatedly with a TLR3 agonist to induce chronic inflammation, these animals overexpress key IFN-stimulated genes, induce cytokine production, exhibit liver pathology, and undergo rapid weight loss. Importantly, the lethal immune hypersensitivity and cytokine production and the ensuing pathology are ameliorated by JAK1 inhibition. These results indicate that individuals with DS may experience harmful hyperinflammation upon IFN-inducing immune stimuli, as observed during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, pointing to JAK1 inhibition as a strategy to restore immune homeostasis in DS.

PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.

Although the function of the extracellular region of programmed death ligand 1 (PD-L1) through its interactions with PD-1 on T cells is well studied, little is understood regarding the intracellular domain of PD-L1. Here, we outline a major role for PD-L1 intracellular signaling in the control of dendritic cell (DC) migration from the skin to the draining lymph node (dLN). Using a mutant mouse model, we identify a TSS signaling motif within the intracellular domain of PD-L1. The TSS motif proves critical for chemokine-mediated DC migration to the dLN during inflammation. This loss of DC migration, in the PD-L1 TSS mutant, leads to a significant decline in T cell priming when DC trafficking is required for antigen delivery to the dLN. Finally, the TSS motif is required for chemokine receptor signaling downstream of the Gα subunit of the heterotrimeric G protein complex, ERK phosphorylation, and actin polymerization in DCs.

Chronic Liver Disease in Humans Causes Expansion and Differentiation of Liver Lymphatic Endothelial Cells.

Liver lymphatic vessels support liver function by draining interstitial fluid, cholesterol, fat, and immune cells for surveillance in the liver draining lymph node. Chronic liver disease is associated with increased inflammation and immune cell infiltrate. However, it is currently unknown if or how lymphatic vessels respond to increased inflammation and immune cell infiltrate in the liver during chronic disease. Here we demonstrate that lymphatic vessel abundance increases in patients with chronic liver disease and is associated with areas of fibrosis and immune cell infiltration. Using single-cell mRNA sequencing and multi-spectral immunofluorescence analysis we identified liver lymphatic endothelial cells and found that chronic liver disease results in lymphatic endothelial cells (LECs) that are in active cell cycle with increased expression of CCL21. Additionally, we found that LECs from patients with NASH adopt a transcriptional program associated with increased IL13 signaling. Moreover, we found that oxidized low density lipoprotein, associated with NASH pathogenesis, induced the transcription and protein production of IL13 in LECs both in vitro and in a mouse model. Finally, we show that oxidized low density lipoprotein reduced the transcription of PROX1 and decreased lymphatic stability. Together these data indicate that LECs are active participants in the liver, expanding in an attempt to maintain tissue homeostasis. However, when inflammatory signals, such as oxidized low density lipoprotein are increased, as in NASH, lymphatic function declines and liver homeostasis is impeded.

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

Emerging Roles for Lymphatics in Chronic Liver Disease.

Chronic liver disease (CLD) is a global health epidemic causing ∼2 million deaths annually worldwide. As the incidence of CLD is expected to rise over the next decade, understanding the cellular and molecular mediators of CLD is critical for developing novel therapeutics. Common characteristics of CLD include steatosis, inflammation, and cholesterol accumulation in the liver. While the lymphatic system in the liver has largely been overlooked, the liver lymphatics, as in other organs, are thought to play a critical role in maintaining normal hepatic function by assisting in the removal of protein, cholesterol, and immune infiltrate. Lymphatic growth, permeability, and/or hyperplasia in non-liver organs has been demonstrated to be caused by obesity or hypercholesterolemia in humans and animal models. While it is still unclear if changes in permeability occur in liver lymphatics, the lymphatics do expand in number and size in all disease etiologies tested. This is consistent with the lymphatic endothelial cells (LEC) upregulating proliferation specific genes, however, other transcriptional changes occur in liver LECs that are dependent on the inflammatory mediators that are specific to the disease etiology. Whether these changes induce lymphatic dysfunction or if they impact liver function has yet to be directly addressed. Here, we will review what is known about liver lymphatics in health and disease, what can be learned from recent work on the influence of obesity and hypercholesterolemia on the lymphatics in other organs, changes that occur in LECs in the liver during disease and outstanding questions in the field.

Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.

Lymph node (LN) expansion during an immune response is a complex process that involves the relaxation of the fibroblastic network, germinal center formation, and lymphatic vessel growth. These processes require the stromal cell network of the LN to act deliberately to accommodate the influx of immune cells to the LN. The molecular drivers of these processes are not well understood. Therefore, we asked whether the immediate cytokines type 1 IFN produced during viral infection influence the lymphatic network of the LN in mice. We found that following an IFN-inducing stimulus such as viral infection or polyI:C, programmed cell death ligand 1 (PD-L1) expression is dynamically upregulated on lymphatic endothelial cells (LECs). We found that reception of type 1 IFN by LECs is important for the upregulation of PD-L1 of mouse and human LECs and the inhibition of LEC expansion in the LN. Expression of PD-L1 by LECs is also important for the regulation of LN expansion and contraction after an IFN-inducing stimulus. We demonstrate a direct role for both type 1 IFN and PD-L1 in inhibiting LEC division and in promoting LEC survival. Together, these data reveal a novel mechanism for the coordination of type 1 IFN and PD-L1 in manipulating LEC expansion and survival during an inflammatory immune response.

Rapid reversal of innate immune dysregulation in blood of patients and livers of humanized mice with HCV following DAA therapy.

RESULTS: First, in patients receiving two different combinations of DAAs, we found that DAAs induced not only rapid viral clearance, but also a re-setting of antiviral immune responses in the peripheral blood. Specifically, we see a rapid decline in the expression of genes associated with chronic IFN stimulation (IFIT3, USP18, IFIT1) as well as a rapid decline in genes associated with inflammation (IL1ß, CXCL10, CXCL11) in the peripheral blood that precedes the complete removal of virus from the blood. Interestingly, this rapid reversal of innate immune activation was not seen in patients who successfully clear chronic HCV infection using IFN-based therapy. Next, using a novel humanized mouse model (Fah-/-RAG2-/-IL2rgnull-FRG), we assessed the changes that occur in the hepatic tissue following DAA treatment. DAA-mediated rapid HCV clearance resulted in blunting of the expression of proinflammatory responses while functionally restoring the RIG-I/MAVS axis in the liver of humanized mice. CONCLUSIONS: Collectively, our data demonstrate that the rapid viral clearance following treatment with DAAs results in the rebalancing of innate antiviral response in both the peripheral blood and the liver as well as enhanced antiviral signaling within previously infected hepatocytes.

Virtual memory T cells develop and mediate bystander protective immunity in an IL-15-dependent manner.

Virtual memory cells (VM) are an antigen-specific, memory phenotype CD8 T-cell subset found in lymphoreplete, unchallenged mice. Previous studies indicated that VM cells were the result of homeostatic proliferation (HP) resembling the proliferation observed in a lymphopenic environment. Here we demonstrate that HP is ongoing in lymphoreplete mice, the degree of which is dictated by the number of naive CD8 T cells with a sufficiently high affinity for self-antigen interacting with peripheral IL-15. VM cell transcriptional profiles suggest a capacity to mediate protective immunity via antigen non-specific bystander killing, a function we show is dependent on IL-15. Finally, we show a VM-like population of human cells that accumulate with age and traffic to the liver, displaying phenotypic and functional attributes consistent with the bystander protective functions of VM cells identified in the mouse. These data identify developmental and functional attributes of VM cells, including their likely role in protective immunity.

T cells compete by cleaving cell surface CD27 and blocking access to CD70-bearing APCs.

T cells compete against each other for access to molecules on APCs in addition to peptide/MHC complexes. However, the identity of cell surface molecules that influence T-cell competition, other than peptide/MHC, have yet to be defined. Here, we identify CD70, a TNF ligand expressed on activated APCs, as an important mediator of T-cell competition for APCs. Upon engagement of CD27 by CD70, CD27 is proteolytically cleaved from the surface of the interacting CD8(+) T cell and captured by CD70 expressing dendritic cells. The capture of CD27 effectively masks CD70 on APCs, disallowing the interaction with CD27 on other competing T cells. Collectively, our data indicate that T cells compete against each other for access to the TNF-ligand CD70, an interaction that affects the duration and potency of T cell/DC interactions, thus influencing the repertoire of responding CD8(+) T cells to self or foreign antigens.

Antigen capture and archiving by lymphatic endothelial cells following vaccination or viral infection.

Antigen derived from viral infections with influenza and vesicular stomatitis virus can persist after resolution of infection. Here we show that antigen can similarly persist for weeks following viral challenge and vaccination. Antigen is captured by lymphatic endothelial cells (LECs) under conditions that induce LEC proliferation. Consistent with published data showing that viral antigen persistence impacts the function of circulating memory T cells, we find that vaccine-elicited antigen persistence, found on LECs, positively influences the degree of protective immunity provided by circulating memory CD8(+) T cells. The coupling of LEC proliferation and antigen capture identifies a mechanism by which the LECs store, or 'archive', antigens for extended periods of time after antigen challenge, thereby increasing IFNγ/IL-2 production and enhancing protection against infection. These findings therefore have the potential to have an impact on future vaccination strategies and our understanding of the role for persisting antigen in both vaccine and infectious settings.