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T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8+ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory. Lauer and colleagues examine CD8+ T cells following cure of human hepatitis C virus (HCV) infection. CD8+ T cells exposed to chronic HCV-specific activation show durable functional, phenotypic and transcriptional exhaustion that is maintained even after antigen stimulus is removed.

ObjectiveChronic HBV infection affects more than 250 million people worldwide and remains a global healthcare problem in part because we lack curative treatment. Sustained viral control requires HBV-specific T cells, but these become functionally impaired in chronic infection. Clinical evidence indicates that functional cure of HBV infection by the host immune response is feasible. Developing T cell-based therapies able to achieve functional cure will require identification of the requirements for a successful T cell response against HBV and the relative contribution of individual T cell specificities to HBV control.DesignThe phenotype and function of HBV-specific T cells were studied directly ex vivo using fluorochrome-labelled multimers. We studied multiple HBV-specific T cell specificities targeting different HBV proteins in individuals with either an acute self-limiting or chronic HBV infection.ResultsWe detected strong T cell responses targeting multiple HBV viral proteins in acute self-limiting and low-frequency core and polymerase-specific T cells in chronic infection. Expression of the T cell inhibitory receptor PD-1, as well as T cell differentiation, T cell function and T cell regulation differed by stages and outcomes of infection. In addition, these features differed significantly between T cells targeting different HBV specificities.ConclusionHBV-specific T cells with different target specificities are characterised by distinct phenotypical and functional profiles. These results have direct implications for the design of immunological studies in HBV infection, and are potentially relevant for informing immunotherapeutic approaches to induce functional cure.

CD4(+) T cell failure is a hallmark of chronic hepatitis C virus (HCV) infection. However, the mechanisms underlying the impairment and loss of virus-specific CD4(+) T cells in persisting HCV infection remain unclear. Here we examined HCV-specific CD4(+) T cells longitudinally during acute infection with different infection outcomes. We found that HCV-specific CD4(+) T cells are characterized by expression of a narrower range of T cell inhibitory receptors compared with CD8(+) T cells, with initially high expression levels of PD-1 and CTLA-4 that were associated with negative regulation of proliferation in all patients, irrespective of outcome. In addition, HCV-specific CD4(+) T cells were phenotypically similar during early resolving and persistent infection and secreted similar levels of cytokines. However, upon viral control, CD4(+) T cells quickly downregulated inhibitory receptors and differentiated into long-lived memory cells. In contrast, persisting viremia continued to drive T cell activation and PD-1 and CTLA-4 expression, and blocked T cell differentiation, until the cells quickly disappeared from the circulation. Our data support an important and physiological role for inhibitory receptor–mediated regulation of CD4(+) T cells in early HCV infection, irrespective of outcome, with persistent HCV viremia leading to sustained upregulation of PD-1 and CTLA-4.

Approximately three quarters of acute HCV infections evolve to a chronic state, while one quarter are spontaneously cleared. Genetic predispositions strongly contribute to the development of chronicity. We have conducted a genome-wide association study to identify genomic variants underlying HCV spontaneous clearance using Immunochip in European and African ancestries. We confirmed two previously reported significant associations, in the IL28B/IFNL41,2 and MHC regions, with spontaneous clearance in the European population. We further fine-mapped the MHC association to a region of about 50 kilo base pairs, down from 1 mega base pairs in the previous study. Additional analyses suggested that the association in the MHC locus might be significantly stronger for virus subtype 1a than 1b, suggesting that viral subtype may have influenced the genetic mechanism underlying the clearance of HCV.

Background & Aims There is an unclear relationship between inhibitory receptor expression on T cells and their ability to control viral infections. Studies of human immune cells have been mostly limited to T cells from blood, which is often not the site of infection. We investigated the relationship between T-cell location, expression of inhibitory receptors, maturation, and viral control using blood and liver T cells from patients with hepatitis C virus (HCV) and other viral infections. Methods We analyzed 36 liver samples from HCV antibody–positive patients (30 from patients with chronic HCV infection, 5 from patients with sustained virological responses to treatment, and 1 from a patient with spontaneous clearance) with 19 paired blood samples and 51 liver samples from HCV-negative patients with 17 paired blood samples. Intrahepatic and circulating lymphocytes were extracted; T-cell markers and inhibitory receptors were quantified for total and virus-specific T cells by flow cytometry. Results Levels of the markers PD-1 and 2B4 (but not CD160, TIM-3, or LAG-3) were increased on intrahepatic T cells from healthy and diseased liver tissues compared with T cells from blood. HCV-specific intrahepatic CD8 + T cells from patients with chronic HCV infection were distinct in that they expressed TIM-3 along with PD-1 and 2B4. In comparison, HCV-specific CD8 + T cells from patients with sustained virological responses and T cells that recognized cytomegalovirus lacked TIM-3 but expressed higher levels of LAG-3; these cells also had different memory phenotypes and proliferative capacity. Conclusions T cells from liver express different inhibitory receptors than T cells from blood, independent of liver disease. HCV-specific and cytomegalovirus-specific CD8 + T cells can be differentiated based on their expression of inhibitory receptors; these correlate with their memory phenotype and levels of proliferation and viral control.

Early after symptom onset, HCV-specific CD4+ T cell responses are primed and detectable in patients regardless of clinical outcome, but without early antiviral therapy these T cells become exhausted or deleted in chronically infected patients., Vigorous proliferative CD4+ T cell responses are the hallmark of spontaneous clearance of acute hepatitis C virus (HCV) infection, whereas comparable responses are absent in chronically evolving infection. Here, we comprehensively characterized the breadth, specificity, and quality of the HCV-specific CD4+ T cell response in 31 patients with acute HCV infection and varying clinical outcomes. We analyzed in vitro T cell expansion in the presence of interleukin-2, and ex vivo staining with HCV peptide-loaded MHC class II tetramers. Surprisingly, broadly directed HCV-specific CD4+ T cell responses were universally detectable at early stages of infection, regardless of the clinical outcome. However, persistent viremia was associated with early proliferative defects of the HCV-specific CD4+ T cells, followed by rapid deletion of the HCV-specific response. Only early initiation of antiviral therapy was able to preserve CD4+ T cell responses in acute, chronically evolving infection. Our results challenge the paradigm that HCV persistence is the result of a failure to prime HCV-specific CD4+ T cells. Instead, broadly directed HCV-specific CD4+ T cell responses are usually generated, but rapid exhaustion and deletion of these cells occurs in the majority of patients. The data further suggest a short window of opportunity to prevent the loss of CD4+ T cell responses through antiviral therapy.

Although epidemiological and functional studies have implicated NK cells in protection and early clearance of HCV, the mechanism by which they may contribute to viral control is poorly understood, particularly at the site of infection, the liver. We hypothesized that a unique immunophenotypic/functional NK cell signature exists in the liver that may provide insights into the contribution of NK cells to viral control. Intrahepatic and blood NK cells were profiled from chronically infected HCV-positive and HCV-negative individuals. Baseline expression of activating and inhibitory receptors was assessed, as well as functional responses following stimulation through classic NK cell pathways. Independent of HCV infection, the liver was enriched for the immunoregulatory CD56[superscript bright] NK cell population, which produced less IFNγ and CD107a but comparable levels of MIP1β, and was immunophenotypically distinct from their blood counterparts. This profile was mostly unaltered in chronic HCV infection, though different expression levels of NKp46 and NKG2D were associated with different grades of fibrosis. In contrast to the liver, chronic HCV infection associated with an enrichment of CD161[superscript low]perforin[superscript high] NK cells in the blood correlated with increased AST and 2B4 expression. However, the association of relatively discrete changes in the NK cell phenotype in the liver with the fibrosis stage nevertheless suggests an important role for the NK response. Overall these data suggest that tissue localization has a more pervasive effect on NK cells than the presence of chronic viral infection, during which these cells might be mostly attuned to limiting immunopathology. It will be important to characterize NK cells during early HCV infection, when they should have a critical role in limiting infection., National Institutes of Health (U.S.) (Grant U19 #AI08230), National Institutes of Health (U.S.) (Grant U19 #AI066345)