Identification of Two Distinct Immune Subtypes in Hepatitis B Virus (HBV)-Associated Hepatocellular Carcinoma (HCC).

HBV is the most common risk factor for HCC development, accounting for almost 50% of cases worldwide. Despite significant advances in immunotherapy, there is limited information on the HBV-HCC tumor microenvironment (TME), which may influence the response to checkpoint inhibitors. Here, we characterize the TME in a unique series of liver specimens from HBV-HCC patients to identify who might benefit from immunotherapy. By combining an extensive immunohistochemistry analysis with the transcriptomic profile of paired liver samples (tumor vs. nontumorous tissue) from 12 well-characterized Caucasian patients with HBV-HCC, we identified two distinct tumor subtypes that we defined immune-high and immune-low. The immune-high subtype, seen in half of the patients, is characterized by a high number of infiltrating B and T cells in association with stromal activation and a transcriptomic profile featuring inhibition of antigen presentation and CTL activation. All the immune-high tumors expressed high levels of CTLA-4 and low levels of PD-1, while PD-L1 was present only in four of six cases. In contrast, the immune-low subtype shows significantly lower lymphocyte infiltration and stromal activation. By whole exome sequencing, we documented that four out of six individuals with the immune-low subtype had missense mutations in the CTNNB1 gene, while only one patient had mutations in this gene in the immune-high subtype. Outside the tumor, there were no differences between the two subtypes. This study identifies two distinctive immune subtypes in HBV-associated HCC, regardless of the microenvironment observed in the surrounding nontumorous tissue, providing new insights into pathogenesis. These findings may be instrumental in the identification of patients who might benefit from immunotherapy.

Ongoing production of tissue-resident macrophages from hematopoietic stem cells in healthy adult macaques.

ABSTRACT: Macrophages orchestrate tissue immunity from the initiation and resolution of antimicrobial immune responses to the repair of damaged tissue. Murine studies demonstrate that tissue-resident macrophages are a heterogenous mixture of yolk sac-derived cells that populate the tissue before birth, and bone marrow-derived replacements recruited in adult tissues at steady-state and in increased numbers in response to tissue damage or infection. How this translates to species that are constantly under immunologic challenge, such as humans, is unknown. To understand the ontogeny and longevity of tissue-resident macrophages in nonhuman primates (NHPs), we use a model of autologous hematopoietic stem progenitor cell (HSPC) transplantation with HSPCs genetically modified to be marked with clonal barcodes, allowing for subsequent analysis of clonal ontogeny. We study the contribution of HSPCs to tissue macrophages, their clonotypic profiles relative to leukocyte subsets in the peripheral blood, and their transcriptomic and epigenetic landscapes. We find that HSPCs contribute to tissue-resident macrophage populations in all anatomic sites studied. Macrophage clonotypic profiles are dynamic and overlap significantly with the clonal hierarchy of contemporaneous peripheral blood monocytes. Epigenetic and transcriptomic landscapes of HSPC-derived macrophages are similar to tissue macrophages isolated from NHPs that did not undergo transplantation. We also use in vivo bromodeoxyuridine infusions to monitor tissue macrophage turnover in NHPs that did not undergo transplantation and find evidence for macrophage turnover at steady state. These data demonstrate that the life span of most tissue-resident macrophages is limited and can be replenished continuously from HSPCs.

Cell-free chromatin immunoprecipitation to detect molecular pathways in heart transplantation.

Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy, an invasive and blind procedure that lacks the precision to reliably obtain biopsy samples from diseased sites. This study examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) as a noninvasive proxy to define molecular gene sets and sources of tissue injury in heart transplant patients. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of relevant immune and nonimmune molecular pathways that were predominantly down-regulated in immunosuppressed heart transplant patients compared with healthy controls. cfChIP-seq also identified cell-free DNA tissue sources. Compared with healthy controls, heart transplant patients demonstrated greater cell-free DNA from tissue types associated with heart transplant complications, including the heart, hematopoietic cells, lungs, liver, and vascular endothelium. cfChIP-seq may therefore be a reliable approach to profile dynamic assessments of molecular pathways and sources of tissue injury in heart transplant patients.

Cell-free Chromatin Immunoprecipitation to detect molecular pathways in Physiological and Disease States.

Patient monitoring is a cornerstone in clinical practice to define disease phenotypes and guide clinical management. Unfortunately, this is often reliant on invasive and/or less sensitive methods that do not provide deep phenotype assessments of disease state to guide treatment. This paper examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) to define molecular gene sets in physiological and heart transplant patients taking immunosuppression medications. We show cfChIP-seq reliably detect gene signals that correlate with gene expression. In healthy controls and in heart transplant patients, cfChIP-seq reliably detected housekeeping genes. cfChIP-seq identified differential gene signals of the relevant immune and non-immune molecular pathways that were predominantly downregulated in immunosuppressed heart transplant patients compared to healthy controls. cfChIP-seq also identified tissue sources of cfDNA, detecting greater cell-free DNA from cardiac, hematopoietic, and other non-hematopoietic tissues such as the pulmonary, digestive, and neurological tissues in transplant patients than healthy controls. cfChIP-seq gene signals were reproducible between patient populations and blood collection methods. cfChIP-seq may therefore be a reliable approach to provide dynamic assessments of molecular pathways and tissue injury associated to disease.

Small-molecule screening identifies Syk kinase inhibition and rutaecarpine as modulators of macrophage training and SARS-CoV-2 infection.

Biologically active small molecules can impart modulatory effects, in some cases providing extended long-term memory. In a screen of biologically active small molecules for regulators of tumor necrosis factor (TNF) induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing lipopolysaccharide (LPS)-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages. Rutaecarpine inhibits ß-glucan-induced H3K4Me3 marks at the promoters of several pro-inflammatory cytokines, highlighting the potential of this molecule to modulate chromosomal topology. Syk kinase inhibitor (SYKi IV), another screen hit, promotes an enhanced response to LPS similar to that previously reported for ß-glucan-induced training. Macrophages trained with SYKi IV show a high degree of resistance to influenza A, multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and OC43 coronavirus infection, highlighting a potential application of this molecule and other SYKis as prophylactic treatments for viral susceptibility.

Early B cell factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells.

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease, including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLLs), otherwise known as "COVID toes," remains unclear. Studying multinational cohorts, we found that, in CLLs, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs after disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased NET levels when compared with other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including MIS-C and chilblain-like lesions (CLL), otherwise known as "COVID toes", remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients. Summary: NET formation and degradation are dysregulated in pediatric and symptomatic adult patients with various complications of COVID-19, in association with disease severity. NET degradation impairments are multifactorial and associated with natural inhibitors of DNase 1, G-actin and anti-DNase1L3 and anti-NET antibodies. Infection with the Omicron variant is associated with decreased levels of NETs when compared to other SARS-CoV-2 strains.

Molecular Signature and Immune Landscape of HCV-Associated Hepatocellular Carcinoma (HCC): Differences and Similarities with HBV-HCC.

INTRODUCTION: HCC is the third leading cause of cancer-related death worldwide, with chronic viral hepatitis accounting for more than 70% of the cases. Therapeutic options are limited and ineffective. The increasing use of immune-based therapies in solid tumors highlights the need to expand our knowledge on the immunologic microenvironment of HCC. METHODS: Access to liver samples from 20 well-characterized patients with HCC associated with HCV (n = 9) or HBV (n = 11) gave us the opportunity to study the immunologic landscape in these tumors. For each patient, RNA-sequencing was performed on the tumor and surrounding nontumorous tissue. RESULTS: We found that both HCV- and HBV-HCC are associated with a predominance of downregulated genes (74% and 67%, respectively). Analysis of the immune landscape using a curated gene list showed 216 of 2481 (9%) immune genes in HCV-HCC and 164 of 2560 (6%) in HBV-HCC. However, only 8 immune genes (4%) were upregulated in HCV-HCC and 27 (16.5%) in HBV-HCC. HCV-HCC was characterized by an enrichment of downregulated genes related to T-cell activation and oxidative stress. The dramatic downregulation of immune genes related to T-cell activation in HCV-HCC prompted us to perform an extensive immunohistochemistry analysis on paraffin-embedded liver specimen. Interestingly, we found a significant reduction of immune-cell infiltration (CD3, CD8 and CD20 positive cells) within the tumor. Moreover, we observed that HCV-HCC is characterized by an enrichment of M2-like CD68-positive cells. These data are consistent with the dramatic downregulation of immune-cell infiltration seen in HCV-HCC. Conversely, HBV-HCC was characterized by upregulation of genes related to monocyte/macrophage activation and cell cycle control, and downregulation of genes involved in various cell metabolisms. CONCLUSION: This study demonstrates a distinctive molecular signature and immune landscape in HCC of different viral etiology, which could provide new insights into pathogenesis and lead to the development of novel immune-based therapies.

Recurrent integration of human papillomavirus genomes at transcriptional regulatory hubs.

Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers, and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites but often only one "driver" site that expressed viral RNA. As common fragile sites and active enhancer elements are cell-type-specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration "hotspots" that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters and accidental integration could promote viral oncogene expression and carcinogenesis.

Spatial and Functional Organization of Human Papillomavirus Replication Foci in the Productive Stage of Infection.

The life cycle of human papillomavirus (HPV) depends on keratinocyte differentiation as the virus modulates and takes advantage of cellular pathways to replicate its genome and assemble viral particles in differentiated cells. Viral genomes are amplified in nuclear replication foci in differentiated keratinocytes, and DNA repair factors from the DNA damage response signaling pathway are recruited to replicate viral DNA. The HPV genome is associated with cellular histones at all stages of the infectious cycle, and here, we show that the histone variant macroH2A1 is bound to the HPV genome and enriched in viral replication foci in differentiated cells. macroH2A1 isoforms play important roles in cellular transcriptional repression, double-strand break repair, and replication stress. The viral E8^E2 protein also binds to the HPV genome and inhibits viral replication and gene expression by recruiting NCoR/SMRT complexes. We show here that E8^E2 and SMRT also localize within replication foci, though independently from macroH2A1. Conversely, transcription complexes containing RNA polymerase II and Brd4 are located on the surface of the foci. Foci generated with an HPV16 E8^E2 mutant genome are not enriched for SMRT or macroH2A1 but contain transcriptional complexes throughout the foci. We propose that both the cellular macroH2A1 protein and viral E8^E2 protein help to spatially separate replication and transcription activities within viral replication foci. IMPORTANCE Human papillomaviruses are small DNA viruses that cause chronic infection of cutaneous and mucosal epithelium. In some cases, persistent infection with HPV can result in cancer, and 5% of human cancers are the result of HPV infection. In differentiated cells, HPV amplifies viral DNA in nuclear replication factories and transcribes late mRNAs to produce capsid proteins. However, very little is known about the spatial organization of these activities in the nucleus. Here, we show that repressive viral and cellular factors localize within the foci to suppress viral transcription, while active transcription takes place on the surface. The cellular histone variant macroH2A1 is important for this spatial organization.

Genome-wide Screens Identify Lineage- and Tumor-Specific Genes Modulating MHC-I- and MHC-II-Restricted Immunosurveillance of Human Lymphomas.

Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.