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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.

A crucial component of nonalcoholic fatty liver disease (NAFLD) pathogenesis is lipid stress, which may contribute to hepatic inflammation and activation of innate immunity in the liver. However, little is known regarding how dietary lipids, including fat and cholesterol, may facilitate innate immune activation in vivo. We hypothesized that dietary fat and cholesterol drive NAFLD progression to steatohepatitis and hepatic fibrosis by altering the transcription and phenotype of hepatic macrophages. This hypothesis was tested by using RNA-sequencing methods to characterize and analyze sort-purified hepatic macrophage populations that were isolated from mice fed diets with varying amounts of fat and cholesterol. The addition of cholesterol to a high-fat diet triggered hepatic pathology reminiscent of advanced nonalcoholic steatohepatitis (NASH) in humans characterized by signs of cholesterol dysregulation, generation of oxidized low-density lipoprotein, increased recruitment of hepatic macrophages, and significant fibrosis. RNA-sequencing analyses of hepatic macrophages in this model revealed that dietary cholesterol induced a tissue repair and regeneration phenotype in Kupffer cells (KCs) and recruited infiltrating macrophages to a greater degree than fat. Furthermore, comparison of diseased KCs and infiltrating macrophages revealed that these two macrophage subsets are transcriptionally diverse. Finally, direct stimulation of murine and human macrophages with oxidized low-density lipoprotein recapitulated some of the transcriptional changes observed in the RNA-sequencing study. These findings indicate that fat and cholesterol synergize to alter macrophage phenotype, and they also challenge the dogma that KCs are purely proinflammatory in NASH. CONCLUSION: This comprehensive view of macrophage populations in NASH indicates mechanisms by which cholesterol contributes to NASH progression and identifies potential therapeutic targets for this common disease.

Summary: 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.

Within in 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 were 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 (LEC) from the liver or specific markers that could be used to evaluated 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.

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 lymph node to act deliberately in order to accommodate the influx of immune cells to the lymph node. The molecular drivers of these processes are not well understood. Therefore, we asked whether the immediate cytokines, type 1 interferon, produced during viral infection influences the lymphatic network of the lymph node in mice. We found that following an interferon inducing stimulus, such as viral infection or polyI:C, programmed death ligand 1 (PD-L1) expression is dynamically upregulated on lymphatic endothelial cells (LEC). We find that reception of type 1 interferon 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 lymph node expansion and contraction after an interferon inducting stimulus. We demonstrate a direct role for both type 1 interferon 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.

SUMMARY 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., Graphical Abstract, In Brief Lucas et al. define three residues within the cytoplasmic tail of PD-L1 that are required for proper dendritic cell migration from the skin to the lymph node. These three-amino-acid residues promote chemokine signaling in dendritic cells and productive T cell responses to skin infections.

The TNF superfamily members CD70 and OX40 ligand (OX40L) were reported to be important for CD4+ T cell expansion and differentiation. However, the relative contribution of these costimulatory signals in driving CD4+ T cell responses has not been addressed. In this study, we found that OX40L is a more important determinant than CD70 of the primary CD4+ T cell response to multiple immunization regimens. Despite the ability of a combined TLR and CD40 agonist (TLR/CD40) stimulus to provoke appreciable expression of CD70 and OX40L on CD8+ dendritic cells, resulting CD4+ T cell responses were substantially reduced by Ab blockade of OX40L and, to a lesser degree, CD70. In contrast, the CD8+ T cell responses to combined TLR/CD40 immunization were exclusively dependent on CD70. These requirements for CD4+ and CD8+ T cell activation were not limited to the use of combined TLR/CD40 immunization, because vaccinia virus challenge elicited primarily OX40L-dependent CD4 responses and exclusively CD70-dependent CD8+ T cell responses. Attenuation of CD4+ T cell priming induced by OX40L blockade was independent of signaling through the IL-12R, but it was reduced further by coblockade of CD70. Thus, costimulation by CD70 or OX40L seems to be necessary for primary CD4+ T cell responses to multiple forms of immunization, and each may make independent contributions to CD4+ T cell priming.

SummaryAppropriate development of regulatory T (Treg) cells is necessary to prevent autoimmunity. Neonatal mice, unlike adults, lack factors required for Treg cell development. It is unclear what these missing factors are. However, signals emanating from the T cell receptor (TCR), the costimulatory receptor CD28, and the family of γc-dependent cytokine receptors are required for Treg cell development. Herein we demonstrate that expression of a constitutively active Stat5b transgene (Stat5b-CA) allowed for Treg cell development in neonatal mice and restored Treg cell numbers in Cd28−/− mice. Sequence analysis of TCR genes in Stat5b-CA Treg cells indicated that ectopic STAT5 activation resulted in a TCR repertoire that more closely resembled that of naive T cells. Using MHCII tetramers to identify antigen-specific T cells, we showed that STAT5 signals diverted thymocytes normally destined to become naive T cells into the Treg cell lineage. Our data support a two-step model of Treg cell differentiation in which TCR and CD28 signals induce cytokine responsiveness and STAT5-inducing cytokines then complete the program of Treg cell differentiation.

Interleukin-2 (IL2) was initially identified from supernatants of activated lymphocytes over 30 years ago [1]. In the ensuing 15 years, the cDNAs for both IL2 and the three chains of the interleukin-2 receptor (IL2R) were cloned [2–7]. Subsequently, many of the downstream biochemical pathways activated by the IL2 receptor complex were identified [8] and the structure of IL2 bound to this tripartite receptor complex was solved [9]. Thus, we now have a very good understanding of how each chain contributes to high affinity IL2 binding and signal transduction [10,11]. In contrast, over the past 30 years the role that IL2 plays in regulating lymphocyte function has involved many surprising twists and turns. For example, IL2 has been shown, paradoxically, to regulate both lymphocyte proliferation and lymphocyte death. In this review, we briefly outline the original findings suggesting a role for IL2 as a T cell growth factor, as well as subsequent studies pointing to its function as an initiator of Activation-induced Cell Death, but then focus on the newly appreciated role for IL2 and IL2R signaling in the development and homeostasis of regulatory T cells.

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., Virtual memory T cells are CD8 T cells with memory phenotype present in unimmunized mice. Here the authors show that these cells have higher affinity for self-antigen, depend on IL-15 for proliferation and antigen-non-specific cytotoxicity in mice, and that a similar population exists in humans.

T cells compete against each other for access to molecules on antigen presenting cells (APC) 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 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.

The objective of modern vaccine development is the safe generation of protective long-term immune memory, both prophylactic and therapeutic. Live attenuated vaccines generate potent cellular and humoral immunity [1–3], but numerous problems exist with these vaccines, ranging from production and storage issues to adverse reactions and reversion to virulence. Subunit vaccines are safer, more stable, and more amenable to mass production. However the protection they produce is frequently inferior to live attenuated vaccines and is typically confined to humoral, and not cellular immunity. Unfortunately, there are presently no subunit vaccines available clinically that are effective at eliciting cellular responses let alone cellular memory [4]. This article will provide and overview of areas of investigation that we see as important for the development of vaccines with the capacity to induce robust and enduring cellular immune responses.

Despite expression of the high-affinity IL-2R, CD4+CD25+ regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4+CD25+ T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4+CD25+ Tregs to IL-2. IL-2R stimulation results in a G1 cell cycle arrest, cellular enlargement and increased cellular survival of CD4+CD25+ T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4+CD25+ T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.