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Blood samples are frequently collected in human studies of the immune system but poorly represent tissue-resident immunity. Understanding the immunopathogenesis of tissue-restricted diseases, such as chronic hepatitis B, necessitates direct investigation of local immune responses. We developed a workflow that enables frequent, minimally invasive collection of liver fine-needle aspirates in multi-site international studies and centralized single-cell RNA sequencing data generation using the Seq-Well S3 picowell-based technology. All immunological cell types were captured, including liver macrophages, and showed distinct compartmentalization and transcriptional profiles, providing a systematic assessment of the capabilities and limitations of peripheral blood samples when investigating tissue-restricted diseases. The ability to electively sample the liver of chronic viral hepatitis patients and generate high-resolution data will enable multi-site clinical studies to power fundamental and therapeutic discovery.

HBV is a non-cytopathic virus and the progression of liver fibrosis is attributed to the host immune response. Complete suppression of viral replication using nucleotide or nucleoside analogues (NUCs) can prevent most complications related to chronic HBV infection. Unfortunately, antiviral treatment has to be administered lifelong to the majority of patients as HBV persists in the hepatocytes. However, although NUCs are very frequently administered in clinical practice, their effects on vital parts of the host immune response to HBV are not well established. In this review we summarize the currently available data gathered from longitudinal studies that investigated treatment-associated alterations of HBV-specific CD4+and CD8+T-cells, regulatory T-cells and natural killer (NK) cells. These observations are important, as they can guide the design of studies that investigate the efficacy of new immune therapeutic agents. Novel experimental compounds will likely be added to ongoing NUC treatment, which leads to a functional cure in only a small minority of patients.

In this study, we describe a method to reliably characterize intrahepatic leukocyte populations using flow cytometry and next‐generation RNA sequencing on fresh human liver biopsies. Over the last decades, immune responses of viral hepatitis patients, and of other liver diseases, have been incompletely characterized. Most studies include peripheral blood samples only, foregoing the possibility to investigate the site of inflammation directly. Here, we show that with an optimized protocol that combines cell sorting and RNA sequencing, we can perform a side by side comparison of both intrahepatic and peripheral immune cells. Using core liver biopsies from chronic hepatitis B virus patients, we show that the expression levels of IFN‐stimulated genes and leukocyte‐specific genes are markedly different in the liver compartment as compared to the peripheral blood. These observations emphasize the need to sample the liver directly. The variation of gene expression profiles in these chronic hepatitis B patients was considerable, despite the uniform treatment with nucleotide analogs and absence of liver inflammation in these patients. Finally, we show that this method can provide a detailed characterization of previously undetected liver‐specific effects of novel candidate therapeutic compounds., The use of fluorescence‐activated cell sorting and next generation RNA sequencing to maximize the information yield of studies investigating tissue‐resident leukocytes in human liver.

Background Mucosal-associated invariant T (MAIT) cells might play a role in control of viral replication during chronic hepatitis B (cHBV) infection, but little is known of their number, phenotype, or function in cHBV patients. Methods We performed flow cytometry on CD3+Vɑ7.2+CD161+ MAIT cells in blood of 55 cHBV patients. Nine patients were sampled before and on entecavir treatment. Six patients on therapy underwent a liver biopsy for flow cytometric analysis. Measurements included MAIT cell frequency, phenotype, and cytokine-producing capacity. Results The MAIT cells were not deleted in blood or liver of cHBV patients compared with healthy controls, but they had higher percentages of CD38+ MAIT cells in blood, which declined on entecavir treatment. Peripheral MAIT cells of patients in the HBeAg-negative phase were least activated. Cytokine-producing MAIT cells were as frequent, but granzyme B-producing MAIT cells were more frequent upon stimulation with Escherichia coli compared with healthy controls. Conclusions We demonstrate that, in sharp contrast to hepatitis C virus and human immunodeficiency virus patients, MAIT cells isolated from HBV patients are not deleted but are more activated, which can be normalized by nucleoside analog therapy. These observations may aid in deciphering the role of MAIT cells in immune responses to HBV.

Natural killer (NK) cells during chronic viral infection have been well studied in the past. We performed an unbiased next-generation RNA-sequencing approach to identify commonalities or differences of the effect of HIV, HCV, and HBV viremia on NK cell transcriptomes. Using cell sorting, we obtained CD3(−) CD56(+) NK cells from blood of 6 HIV-, 8 HCV-, and 32 HBV-infected patients without treatment. After library preparation and sequencing, we used an in-house analytic pipeline to compare expression levels with matched healthy controls. In NK cells from HIV-, HCV-, and HBV-infected patients, transcriptome analysis identified 272, 53, and 56 differentially expressed genes, respectively (fold change, >1.5; q-value, 0.2). Interferon-stimulated genes were induced in NK cells from HIV/HCV patients, but not during HBV infection. HIV viremia downregulated ribosome assembly genes in NK cells. In HBV-infected patients, viral load and alanine aminotransferase (ALT) variation had little effect on genes related to NK effector function. In conclusion, we compare, for the first time, NK cell transcripts of viremic HIV, HCV, and HBV patients. We clearly demonstrate distinctive NK cell gene signatures in three different populations, suggestive for a different degree of functional alterations of the NK cell compartment compared to healthy individuals. IMPORTANCE Three viruses exist that can result in persistently high viral loads in immunocompetent humans: human immunodeficiency virus (HIV), hepatitis C virus, and hepatitis B virus. In the last decades, by using flow cytometry and in vitro assays on NK cells from patients with these types of infections, several impairments have been established, particularly during and possibly contributing to HIV viremia. However, the background of NK cell impairments in viremic patients is not well understood. In this study, we describe the NK cell transcriptomes of patients with high viral loads of different etiologies. We clearly demonstrate distinctive NK cell gene signatures with regard to interferon-stimulated gene induction and the expression of genes coding for activation markers or proteins involved in cytotoxic action, as well immunological genes. This study provides important details necessary to uncover the origin of functional and phenotypical differences between viremic patients and healthy subjects and provides many leads that can be confirmed using future in vitro manipulation experiments.

Clinical events and the host immune response during hepatitis B virus (HBV) infection are intricately linked. Despite decades of research, important questions concerning the immunopathogenesis of chronic HBV infection remain unanswered. For example, it is unclear which immune parameters facilitate persistence, and if HBV can be completely cleared from the human liver. Recent technological breakthroughs now allow researchers to address these seemingly basic, but essential questions surrounding HBV immunity. It will be important to better define the molecular underpinnings of immune cell function and dysfunction during chronic disease and in controlled infection, with particular focus on the liver, as little information is available on the intrahepatic compartment. In the near future, it may be possible to solve some of the controversy surrounding the immune responses to HBV, and establish the features of both the innate and adaptive arms of the immune system required to achieve sustained control of HBV infection.

Evidence over the past decades have shown that HIV/HCV coinfected patients did not respond as well to HCV therapy as HCV mono-infected patients. However, these paradigms are being recently reassessed with the improvements of care for HIV and HCV patients. This article reviews these original paradigms and how the new data is impacting upon them. Treatment efficacy now appears comparable for HIV/HCV coinfected and HCV mono-infected patients, while liver fibrosis progression is increasingly similar in optimally managed patients. Additional importance of therapy is directed to drug-drug interactions and the impact of HCV reinfection, as well as the possibility of transmitted drug resistance.