Coordinated expansion of memory T follicular helper and B cells mediates spontaneous clearance of HCV reinfection.

Introduction: Follicular helper T cells are essential for helping in the maturation of B cells and the production of neutralizing antibodies (NAbs) during primary viral infections. However, their role during recall responses is unclear. Here, we used hepatitis C virus (HCV) reinfection in humans as a model to study the recall collaborative interaction between circulating CD4 T follicular helper cells (cTfh) and memory B cells (MBCs) leading to the generation of NAbs. Methods: We evaluated this interaction longitudinally in subjects who have spontaneously resolved primary HCV infection during a subsequent reinfection episode that resulted in either another spontaneous resolution (SR/SR, n = 14) or chronic infection (SR/CI, n = 8). Results: Both groups exhibited virus-specific memory T cells that expanded upon reinfection. However, early expansion of activated cTfh (CD4+CXCR5+PD-1+ICOS+FoxP3-) occurred in SR/SR only. The frequency of activated cTfh negatively correlated with time post-infection. Concomitantly, NAbs and HCV-specific MBCs (CD19+CD27+IgM-E2-Tet+) peaked during the early acute phase in SR/SR but not in SR/CI. Finally, the frequency of the activated cTfh1 (CXCR3+CCR6-) subset correlated with the neutralization breadth and potency of NAbs. Conclusion: These results underscore a key role for early activation of cTfh1 cells in helping antigen-specific B cells to produce NAbs that mediate the clearance of HCV reinfection.

Two transcriptionally and functionally distinct waves of neutrophils during mouse acute liver injury.

BACKGROUND: Neutrophils are key mediators of inflammation during acute liver injury (ALI). Emerging evidence suggests that they also contribute to injury resolution and tissue repair. However, the different neutrophil subsets involved in these processes and their kinetics are undefined. Herein, we characterized neutrophil kinetics and heterogeneity during ALI. METHODS: We used the carbon tetrachloride model of ALI and employed flow cytometry, tissue imaging, and quantitative RT-PCR to characterize intrahepatic neutrophils during the necroinflammatory early and late repair phases of the wound healing response to ALI. We FACS sorted intrahepatic neutrophils at key time points and examined their transcriptional profiles using RNA-sequencing. Finally, we evaluated neutrophil protein translation, mitochondrial function and metabolism, reactive oxygen species content, and neutrophil extracellular traps generation. RESULTS: We detected 2 temporarily distinct waves of neutrophils during (1) necroinflammation (at 24 hours after injury) and (2) late repair (at 72 hours). Early neutrophils were proinflammatory, characterized by: (1) upregulation of inflammatory cytokines, (2) activation of the noncanonical NF-κB pathway, (3) reduction of protein translation, (4) decreased oxidative phosphorylation, and (5) higher propensity to generate reactive oxygen species and neutrophil extracellular traps. In contrast, late neutrophils were prorepair and enriched in genes and pathways associated with tissue repair and angiogenesis. Finally, early proinflammatory neutrophils were characterized by the expression of a short isoform of C-X-C chemokine receptor 5, while the late prorepair neutrophils were characterized by the expression of C-X-C chemokine receptor 4. CONCLUSIONS: This study underscores the phenotypic and functional heterogeneity of neutrophils and their dual role in inflammation and tissue repair during ALI.

DAA-mediated HCV cure reduces HIV DNA levels in HCV/HIV coinfected people.

IMPORTANCE: Antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can control virus replication and prolong the life of people living with HIV (PLWH). However, the virus remains dormant within immune cells in what is called the HIV reservoir. Furthermore, 2.3 million PLWH are also coinfected with hepatitis C virus (HCV) and are at risk of developing chronic liver disease and cancer. HCV treatment with direct acting antivirals (DAA) can completely cure the infection in more than 95% of treated individuals and improve their long-term health outcomes. In this study, we investigated how HCV treatment and cure affect the HIV reservoir. We demonstrate the beneficial impact of DAA treatment as it reduces the HIV reservoirs in particular in people infected with HCV before HIV. These results support the need for early ART and DAA treatment in HIV/HCV coinfections.

The multifaceted role of macrophages during acute liver injury.

The liver is situated at the interface of the gut and circulation where it acts as a filter for blood-borne and gut-derived microbes and biological molecules, promoting tolerance of non-invasive antigens while driving immune responses against pathogenic ones. Liver resident immune cells such as Kupffer cells (KCs), a subset of macrophages, maintain homeostasis under physiological conditions. However, upon liver injury, these cells and others recruited from circulation participate in the response to injury and the repair of tissue damage. Such response is thus spatially and temporally regulated and implicates interconnected cells of immune and non-immune nature. This review will describe the hepatic immune environment during acute liver injury and the subsequent wound healing process. In its early stages, the wound healing immune response involves a necroinflammatory process characterized by partial depletion of resident KCs and lymphocytes and a significant infiltration of myeloid cells including monocyte-derived macrophages (MoMFs) complemented by a wave of pro-inflammatory mediators. The subsequent repair stage includes restoring KCs, initiating angiogenesis, renewing extracellular matrix and enhancing proliferation/activation of resident parenchymal and mesenchymal cells. This review will focus on the multifaceted role of hepatic macrophages, including KCs and MoMFs, and their spatial distribution and roles during acute liver injury.

Immunological Monitoring in Hepatitis C Virus Controlled Human Infection Model.

Controlled human infection model trials for hepatitis C virus represent an important opportunity to identify correlates of protective immunity against a well-characterized inoculum of hepatitis C virus and how such responses are modified by vaccination. In this article, we discuss the approach to immunological monitoring during such trials, including a set of recommendations for optimal sampling schedule and preferred immunological assays to examine the different arms of the immune response. We recommend that this approach be adapted to different trial designs. Finally, we discuss how these studies can provide surrogate predictors of the success of candidate vaccines.

Differential immune transcriptomic profiles between vaccinated and resolved HCV reinfected subjects.

Successive episodes of hepatitis C virus (HCV) infection represent a unique natural rechallenge experiment to define correlates of long-term protective immunity and inform vaccine development. We applied a systems immunology approach to characterize longitudinal changes in the peripheral blood transcriptomic signatures in eight subjects who spontaneously resolved two successive HCV infections. Furthermore, we compared these signatures with those induced by an HCV T cell-based vaccine regimen. We identified a plasma cell transcriptomic signature during early acute HCV reinfection. This signature was absent in primary infection and following HCV vaccine boost. Spontaneous resolution of HCV reinfection was associated with rapid expansion of glycoprotein E2-specifc memory B cells in three subjects and transient increase in E2-specific neutralizing antibodies in six subjects. Concurrently, there was an increase in the breadth and magnitude of HCV-specific T cells in 7 out of 8 subjects. These results suggest a cooperative role for both antibodies and T cells in clearance of HCV reinfection and support the development of next generation HCV vaccines targeting these two arms of the immune system.

Distinct spatial distribution and roles of Kupffer cells and monocyte-derived macrophages in mouse acute liver injury.

Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple roles in the liver are not fully understood. We aimed herein to map the intrahepatic macrophage populations and their function(s) during acute liver injury. We used flow cytometry, gene expression analysis, multiplex-immunofluorescence, 3D-reconstruction, and spatial image analysis to characterize the intrahepatic immune landscape in mice post-CCl4-induced acute liver injury during three distinct phases: necroinflammation, and early and late repair. We observed hepatocellular necrosis and a reduction in liver resident lymphocytes during necroinflammation accompanied by the infiltration of circulating myeloid cells and upregulation of inflammatory cytokines. These parameters returned to baseline levels during the repair phase while pro-repair chemokines were upregulated. We identified resident CLEC4F+ Kupffer cells (KCs) and infiltrating IBA1+CLEC4F- monocyte-derived macrophages (MoMFs) as the main hepatic macrophage populations during this response to injury. While occupying most of the necrotic area, KCs and MoMFs exhibited distinctive kinetics, distribution and morphology at the site of injury. The necroinflammation phase was characterized by low levels of KCs and a remarkable invasion of MoMFs suggesting their potential role in phagoctosing necrotic hepatocytes, while opposite kinetics/distribution were observed during repair. During the early repair phase, yolksac - derived KCs were restored, whereas MoMFs diminished gradually then dissipated during late repair. MoMFs interacted with hepatic stellate cells during the necroinflammatory and early repair phases, potentially modulating their activation state and influencing their fibrogenic and pro-repair functions that are critical for wound healing. Altogether, our study reveals novel and distinct spatial and temporal distribution of KCs and MoMFs and provides insights into their complementary roles during acute liver injury.

Sex-Dependent Hepatoprotective Role of IL-22 Receptor Signaling in Non-Alcoholic Fatty Liver Disease-Related Fibrosis.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a major health problem with complex pathogenesis. Although sex differences in NAFLD pathogenesis have been reported, the mechanisms underlying such differences remain understudied. Interleukin (IL)22 is a pleiotropic cytokine with both protective and/or pathogenic effects during liver injury. IL22 was shown to be hepatoprotective in NAFLD-related liver injury. However, these studies relied primarily on exogenous administration of IL22 and did not examine the sex-dependent effect of IL22. Here, we sought to characterize the role of endogenous IL22-receptor signaling during NAFLD-induced liver injury in males and females. METHODS: We used immunofluorescence, flow cytometry, histopathologic assessment, and gene expression analysis to examine IL22 production and characterize the intrahepatic immune landscape in human subjects with NAFLD (n = 20; 11 men and 9 women) and in an in vivo Western high-fat diet-induced NAFLD model in IL22RA knock out mice and their wild-type littermates. RESULTS: Examination of publicly available data sets from 2 cohorts with NAFLD showed increased hepatic IL22 gene expression in females compared with males. Furthermore, our immunofluorescence analysis of liver sections from NAFLD subjects (n = 20) showed increased infiltration of IL22-producing cells in females. Similarly, IL22-producing cells were increased in wild-type female mice with NAFLD and the hepatic IL22/IL22 binding protein messenger RNA ratio correlated with expression of anti-apoptosis genes. The lack of endogenous IL22-receptor signaling (IL22RA knockout) led to exacerbated liver damage, inflammation, apoptosis, and liver fibrosis in female, but not male, mice with NAFLD. CONCLUSIONS: Our data suggest a sex-dependent hepatoprotective antiapoptotic effect of IL22-receptor signaling during NAFLD-related liver injury in females.

Current considerations for clinical management and care of non-alcoholic fatty liver disease: Insights from the 1st International Workshop of the Canadian NASH Network (CanNASH).

Non-alcoholic fatty liver disease (NAFLD) affects approximately 8 million Canadians. NAFLD refers to a disease spectrum ranging from bland steatosis to non-alcoholic steatohepatitis (NASH). Nearly 25% of patients with NAFLD develop NASH, which can progress to liver cirrhosis and related end-stage complications. Type 2 diabetes and obesity represent the main risk factors for the disease. The Canadian NASH Network is a national collaborative organization of health care professionals and researchers with a primary interest in enhancing understanding, care, education, and research around NAFLD, with a vision of best practices for this disease state. At the 1st International Workshop of the CanNASH network in April 2021, a joint event with the single topic conference of the Canadian Association for the Study of the Liver (CASL), clinicians, epidemiologists, basic scientists, and community members came together to share their work under the theme of NASH. This symposium also marked the initiation of collaborations between Canadian and other key opinion leaders in the field representative of international liver associations. The main objective is to develop a policy framework that outlines specific targets, suggested activities, and evidence-based best practices to guide provincial, territorial, and federal organizations in developing multidisciplinary models of care and strategies to address this epidemic.

T cell responses during HBV and HCV infections: similar but not quite the same?

The hepatitis B and C viruses persist by evasion of T cell immunity. Persistence depends upon premature failure of CD4+ T cell help and loss of CD8+ T cell control because of epitope mutational escape and/or functional exhaustion. Powerful new immunological and transcriptomic tools provide insight into the mechanisms of T cell silencing by HBV and HCV. Similarities are apparent, including dysregulated expression of common inhibitory/immune checkpoint receptors and transcription factors. There are also differences. T cell exhaustion is uniform in HCV infection, but varies in HBV infection depending on disease stage and/or protein target. Here, we review recent advances defining similarities and differences in T cell evasion by HBV and HCV, and the potential for reversal following antiviral therapy.

Towards a Systems Immunology Approach to Understanding Correlates of Protective Immunity against HCV.

Over the past decade, tremendous progress has been made in systems biology-based approaches to studying immunity to viral infections and responses to vaccines. These approaches that integrate multiple facets of the immune response, including transcriptomics, serology and immune functions, are now being applied to understand correlates of protective immunity against hepatitis C virus (HCV) infection and to inform vaccine development. This review focuses on recent progress in understanding immunity to HCV using systems biology, specifically transcriptomic and epigenetic studies. It also examines proposed strategies moving forward towards an integrated systems immunology approach for predicting and evaluating the efficacy of the next generation of HCV vaccines.

A Tale of Two Viruses: Immunological Insights Into HCV/HIV Coinfection.

Nearly 2.3 million individuals worldwide are coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Odds of HCV infection are six times higher in people living with HIV (PLWH) compared to their HIV-negative counterparts, with the highest prevalence among people who inject drugs (PWID) and men who have sex with men (MSM). HIV coinfection has a detrimental impact on the natural history of HCV, including higher rates of HCV persistence following acute infection, higher viral loads, and accelerated progression of liver fibrosis and development of end-stage liver disease compared to HCV monoinfection. Similarly, it has been reported that HCV coinfection impacts HIV disease progression in PLWH receiving anti-retroviral therapies (ART) where HCV coinfection negatively affects the homeostasis of CD4+ T cell counts and facilitates HIV replication and viral reservoir persistence. While ART does not cure HIV, direct acting antivirals (DAA) can now achieve HCV cure in nearly 95% of coinfected individuals. However, little is known about how HCV cure and the subsequent resolution of liver inflammation influence systemic immune activation, immune reconstitution and the latent HIV reservoir. In this review, we will summarize the current knowledge regarding the pathogenesis of HIV/HCV coinfection, the effects of HCV coinfection on HIV disease progression in the context of ART, the impact of HIV on HCV-associated liver morbidity, and the consequences of DAA-mediated HCV cure on immune reconstitution and HIV reservoir persistence in coinfected patients.

Expansion of Unique Hepatitis C Virus-Specific Public CD8+ T Cell Clonotypes during Acute Infection and Reinfection.

Hepatitis C virus (HCV) infection resolves spontaneously in ∼25% of acutely infected humans where viral clearance is mediated primarily by virus-specific CD8+ T cells. Previous cross-sectional analysis of the CD8+ TCR repertoire targeting two immunodominant HCV epitopes reported widespread use of public TCRs shared by different subjects, irrespective of infection outcome. However, little is known about the evolution of the public TCR repertoire during acute HCV and whether cross-reactivity to other Ags can influence infectious outcome. In this article, we analyzed the CD8+ TCR repertoire specific to the immunodominant and cross-reactive HLA-A2-restricted nonstructural 3-1073 epitope during acute HCV in humans progressing to either spontaneous resolution or chronic infection and at ∼1 y after viral clearance. TCR repertoire diversity was comparable among all groups with preferential usage of the TCR-ß V04 and V06 gene families. We identified a set of 13 public clonotypes in HCV-infected humans independent of infection outcome. Six public clonotypes used the V04 gene family. Several public clonotypes were long-lived in resolvers and expanded on reinfection. By mining publicly available data, we identified several low-frequency CDR3 sequences in the HCV-specific repertoire matching human TCRs specific for other HLA-A2-restricted epitopes from melanoma, CMV, influenza A, EBV, and yellow fever viruses, but they were of low frequency and limited cross-reactivity. In conclusion, we identified 13 new public human CD8+ TCR clonotypes unique to HCV that expanded during acute infection and reinfection. The low frequency of cross-reactive TCRs suggests that they are not major determinants of infectious outcome.

Cancer immunotherapy: Macs in the middle.

Response to immune checkpoint blockade cancer immunotherapy is variable, but the mechanisms that underlie this variability remain unclear. In a recent issue of Nature Medicine, Yu et al. demonstrate that liver metastases limit immunotherapy efficacy by promoting macrophage-mediated elimination of tumor-specific CD8+ T cells. Liver-directed radiotherapy in preclinical models could partially overcome this effect.

Early T follicular helper cell activity accelerates hepatitis C virus-specific B cell expansion.

Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBCs from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection, using single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (4-6 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV.

Assessment of Treatment Strategies to Achieve Hepatitis C Elimination in Canada Using a Validated Model.

Importance: Achievement of the World Health Organization (WHO) target of eliminating hepatitis C virus (HCV) by 2030 will require an increase in key services, including harm reduction, HCV screening, and HCV treatment initiatives in member countries. These data are not available for Canada but are important for informing a national HCV elimination strategy. Objective: To use a decision analytical model to explore the association of different treatment strategies with HCV epidemiology and HCV-associated mortality in Canada and to assess the levels of service increase needed to meet the WHO elimination targets by 2030. Design, Setting, and Participants: Study participants in this decision analytical model included individuals with hepatitis C virus infection in Canada. Five HCV treatment scenarios (optimistic, very aggressive, aggressive, gradual decrease, and rapid decrease) were applied using a previously validated Markov-type mathematical model. The optimistic and very aggressive treatment scenarios modeled a sustained annual treatment of 10 200 persons and 14 000 persons, respectively, from 2018 to 2030. The aggressive, gradual decrease, and rapid decrease scenarios assessed decreases in treatment uptake from 14 000 persons to 10 000 persons per year, 12 000 persons to 8500 persons per year, and 12 000 persons to 4500 persons per year, respectively, between 2018 and 2030. Main Outcomes and Measures: Hepatitis C virus prevalence and HCV-associated health outcomes were assessed for each of the 5 treatment scenarios with the goal of identifying strategies to achieve HCV elimination by 2030. Results: An estimated mean 180 142 persons (95% CI, 122 786-196 862 persons) in Canada had chronic HCV infection at the end of 2017. The optimistic and gradual decrease scenarios estimated a decrease in HCV prevalence from 180 142 persons to 37 246 persons and 37 721 persons, respectively, by 2030. Relative to 2015, this decrease in HCV prevalence was associated with 74%, 69%, and 69% reductions in the prevalence of decompensated cirrhosis, hepatocellular carcinoma, and liver-associated mortality, respectively, leading to HCV elimination by 2030. More aggressive treatment uptake (very aggressive scenario) could result in goal achievement up to 3 years earlier than 2030, although a rapid decrease in the initiation of treatment (rapid decrease scenario) would preclude Canada from reaching the HCV elimination goal by 2030. Conclusions and Relevance: The study findings suggest that Canada could meet the WHO goals for HCV elimination by 2030 by sustaining the current national HCV treatment rate during the next decade. This target will not be achieved if treatment uptake is allowed to decrease rapidly.

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.