A. Schuch

The Role of Natural Killer Cells and CD8+ T Cells in Hepatitis B Virus Infection

Hepatitis B virus (HBV) infection is one of the main causes of chronic liver diseases that may progress to liver cirrhosis and hepatocellular carcinoma. Host immune responses are important factors that determine whether HBV infection is cleared or persists. Natural killer (NK) cells represent the main effector population of the innate immune system and are abundant in the human liver. Recently, it has been demonstrated that NK cells not only exhibit antiviral functions but may also regulate adaptive immune responses by deletion of HBV-specific CD8+ T cells. It is well-established that HBV-specific CD8+ T cells contribute to virus elimination. However, the mechanisms contributing to CD8+ T cell failure in chronic HBV infection are not well-understood. In this review, we will summarize the current knowledge about NK cells and CD8+ T cells and illustrate their contribution to viral clearance and persistence in HBV infection. Moreover, novel immunological in vitro model systems and techniques to analyze HBV-specific CD8+ T cells, which are barely detectable using current multimer staining methods, will be discussed.

TCF1 + hepatitis C virus-specific CD8 + T cells are maintained after cessation of chronic antigen stimulation

Differentiation and fate of virus-specific CD8+ T cells after cessation of chronic antigen stimulation is unclear. Here we show that a TCF1+CD127+PD1+ hepatitis C virus (HCV)-specific CD8+ T-cell subset exists in chronically infected patients with phenotypic features of T-cell exhaustion and memory, both before and after treatment with direct acting antiviral (DAA) agents. This subset is maintained during, and for a long duration after, HCV elimination. After antigen re-challenge the less differentiated TCF1+CD127+PD1+ population expands, which is accompanied by emergence of terminally exhausted TCF1-CD127-PD1hi HCV-specific CD8+ T cells. These results suggest the TCF1+CD127+PD1+ HCV-specific CD8+ T-cell subset has memory-like characteristics, including antigen-independent survival and recall proliferation. We thus provide evidence for the establishment of memory-like virus-specific CD8+ T cells in a clinically relevant setting of chronic viral infection and we uncover their fate after cessation of chronic antigen stimulation, implicating a potential strategy for antiviral immunotherapy.

Hepatitis B Virus-Infected HepG2hNTCP Cells Serve as a Novel Immunological Tool To Analyze the Antiviral Efficacy of CD8+ T Cells In Vitro

ABSTRACT CD8+ T cells are the main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems, such as low infection rates, restrict mechanistic studies of HBV-specific CD8+ T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2hNTCP cells that endogenously processed viral antigens and presented them to HBV-specific CD8+ T cells. This induced cytolytic and noncytolytic CD8+ T-cell effector functions and reduction of viral loads.

NK-cell responses are biased towards CD16-mediated effector functions in chronic Hepatitis B virus infection

BACKGROUND & AIMS Phenotypic and functional natural killer (NK)-cell alterations are well described in chronic hepatitis B virus (cHBV) infection. However, it is largely unknown whether these alterations result from general effects on the overall NK-cell population or the emergence of distinct NK-cell subsets. Human cytomegalovirus (HCMV) is common in cHBV and is associated with the emergence of memory-like NK cells. We aimed to assess the impact of these cells on cHBV infection. METHODS To assess the impact of memory-like NK cells on phenotypic and functional alterations in cHBV infection, we performed in-depth analyses of circulating NK cells in 52 patients with cHBV, 45 with chronic hepatitis C virus infection and 50 healthy donors, with respect to their HCMV serostatus. RESULTS In patients with cHBV/HCMV+, FcεRIγ- memory-like NK cells were present in higher frequencies and with higher prevalence than in healthy donors with HCMV+. This pronounced HCMV-associated memory-like NK-cell expansion could be identified as key determinant of the NK-cell response in cHBV infection. Furthermore, we observed that memory-like NK cells consist of epigenetically distinct subsets and exhibit key metabolic characteristics of long-living cells. Despite ongoing chronic infection, the phenotype of memory-like NK cells was conserved in patients with cHBV/HCMV+. Functional characteristics of memory-like NK cells also remained largely unaffected by cHBV infection with the exception of an increased degranulation capacity in response to CD16 stimulation that was, however, detectable in both memory-like and conventional NK cells. CONCLUSIONS The emergence of HCMV-associated memory-like NK cells shapes the overall NK-cell response in cHBV infection and contributes to a general shift towards CD16-mediated effector functions. Therefore, HCMV coinfection needs to be considered in the design of immunotherapeutic approaches that target NK cells in cHBV. LAY SUMMARY In chronic hepatitis B virus infection, natural killer (NK)-cell phenotype and function is altered. In this study, we demonstrate that these changes are linked to the emergence of a distinct NK-cell subset, namely memory-like NK cells. The emergence of these memory-like NK cells is associated with coinfection of human cytomegalovirus that affects the majority of patients with chronic hepatitis B.

Unique and Common Features of Innate-Like Human Vδ2+ γδT Cells and Mucosal-Associated Invariant T Cells

Mucosal-associated invariant T (MAIT) cells are innate-like T cells abundant in humans that can be activated in a TCR-independent manner by inflammatory and antiviral cytokines. In humans, the capacity for TCR-independent activation is functionally linked to a transcriptional program that can be identified by the expression of the C-type lectin receptor, CD161. In addition to MAIT cells, it has been demonstrated that a subset of γδT cells expresses CD161 and can be activated by TCR-independent cytokine stimulation. In this study, we sought to clarify the nature of cytokine-responsive human γδT cells. We could link CD161 expression on Vδ2+ versus Vδ1+ γδT cells to the observation that Vδ2+ γδT cells, but not Vδ1+ γδT cells, robustly produced IFN-γ upon stimulation with a variety of cytokine combinations. Interestingly, both CD161+ and CD161− Vδ2+ γδT cells responded to these stimuli, with increased functionality within the CD161+ subset. This innate-like responsiveness corresponded to high expression of PLZF and IL-18Rα, analogous to MAIT cells. Vδ2+ γδT cells in human duodenum and liver maintained a CD161+ IL-18Rα+ phenotype and produced IFN-γ in response to IL-12 and IL-18 stimulation. In contrast to MAIT cells, we could not detect IL-17A production but observed higher steady-state expression of Granzyme B by Vδ2+ γδT cells. Finally, we investigated the frequency and functionality of γδT cells in the context of chronic hepatitis C virus infection, as MAIT cells are reduced in frequency in this disease. By contrast, Vδ2+ γδT cells were maintained in frequency and displayed unimpaired IFN-γ production in response to cytokine stimulation. In sum, human Vδ2+ γδT cells are a functionally distinct population of cytokine-responsive innate-like T cells that is abundant in blood and tissues with similarities to human MAIT cells.

Priming persistence of HCV

Acute infection with hepatitis C virus (HCV) is cleared in about 30% of cases with CD8+ T-cells being the main effector cells in viral elimination. Yet, about 70% of patients progress to chronic HCV infection that can result in chronic immunopathology. This immune-mediated liver diseases can progress to liver cirrhosis and ultimately to hepatocellular carcinoma. Chronic HCV infection is characterized by an impaired virus-specific CD8+ T-cell response caused by several viral and host factors. However, the relative contribution of those and the exact mechanisms of CD8+ T-cell failure are still not fully illuminated. Viral escape and CD8+ T-cell exhaustion due to prolonged antigen exposure, for example, cause impaired virus-specific CD8+ T-cell responses and therefore contribute to HCV persistence. Exhausted HCV-specific CD8+ T-cells are characterized by impaired effector functions and the co-expression of inhibitory receptors such as PD-1, 2B4 and KLRG1 (Figure ​(Figure1).1). In contrast, HCV-specific CD8+ T-cells that target epitopes that underwent viral escape mutations express fewer inhibitory receptors and are marked by CD127 expression [1] (Figure ​(Figure1).1). Importantly, the identification of these distinct CD8+ T-cell phenotypes as markers for different mechanisms of CD8+ T-cell failure (viral escape versus CD8+ T-cell exhaustion) was based on analyses performed in patients with HCV-specific CD8+ T-cell populations that were detectable directly ex vivo by conventional peptide/MHC multimer staining. However, HCV-specific CD8+ T-cells are not detectable in most chronically HCV-infected patients by this method. The lack of ex vivo detectable HCV-specific CD8+ T-cells in the majority of chronically HCV-infected patients has raised the question whether this is due to technical constraints or to the absence of HCV-specific CD8+ T-cells in these patients. Figure 1 Mechanisms underlying HCV persistence reflected by CD8+ T-cell differentiation Recently, a protocol adopted from mouse studies [2] elegantly combined peptide/MHC tetramer staining and magnetic bead enrichment to detect and analyze rare epitope-specific CD8+ T-cells ex vivo not only in infected individuals, but also in unexposed donors [3, 4]. Using this approach Nitschke et al. [5] were able to enrich HLA-A*02:01-restricted HCV-specific CD8+ T-cells specific for two different viral epitopes in seventeen analyzed patients as well as in twelve uninfected individuals. Hence, HCV-specific CD8+ T-cells were present in all chronically HCV-infected patients. These results indicate that the absence of virus-specific CD8+ T-cells does not contribute to HCV persistence in this cohort. As expected, chronically HCV-infected patients with virus-specific CD8+ T-cell responses that have been detectable directly ex vivo showed significantly higher frequencies of enriched HCV-specific CD8+ T-cells compared to patients without virus-specific CD8+ T-cell responses detectable by conventional peptide/MHC multimer staining methods (enriched detectable). Yet, enriched detectable HCV-specific CD8+ T-cell populations were more frequent in chronically HCV-infected patients than HCV-specific CD8+ T-cell precursor populations in healthy donors. A detailed phenotypic characterization of enriched HCV-specific CD8+ T-cells revealed that HCV-specific CD8+ precursor T-cells obtained from healthy donors predominantly displayed a naive phenotype (CD45RA+CCR7+CD27+). In contrast, directly detectable virus-specific CD8+ T-cell populations in chronically HCV-infected patients had an antigen-experienced phenotype (CD45RA−CCR7− CD27+). Interestingly, enriched detectable HCV-specific CD8+ T-cell populations had a highly heterogeneous differentiation profile with an antigen-experienced phenotype in two third and a predominantly naive-like phenotype in one third of chronically HCV-infected patients. In addition, enriched HCV-specific CD8+ T-cells with an effector memory phenotype also expressed PD-1 according to previous data obtained with directly detectable HCV-specific CD8+ T-cells that have been defined as exhausted. Notably, the presence of both antigen-experienced and naive-like phenotypes in different HCV-specific CD8+ T-cell populations within the same patients argues against a role of host genetic factors. In a next series of experiments, the authors analyzed the functionality of enriched detectable HCV-specific CD8+ T-cells after co-culture with peptide-pulsed autologous monocyte-derived DC. As expected, enriched detectable HCV-specific CD8+ T-cells with an antigen-experienced phenotype accompanied by high PD-1 expression did not expand after co-culture, indicating CD8+ T-cell exhaustion. Of note, bona fide virus-specific CD8+ T-cell memory populations should expand upon antigen-specific stimulation. Surprisingly, only two of five naive-like enriched detectable HCV-specific CD8+ T-cell populations expanded and produced effector cytokines such as IFN-γ and TNF and mobilized CD107a, a surrogate marker for degranulation. Those two patients harbored viral escape variants carrying mutations in the corresponding HCV epitope. Consequently, stimulation with the autologous variant peptide sequence did not lead to recognition and thus to successful priming of the naive-like HCV-specific CD8+ T-cells in these two patients. This indicates an initial infection with a variant HCV strain, since viral escape occurring at later time points during infection would engender HCV-specific CD8+ T-cells with a non-naive phenotype (Figure ​(Figure1).1). In contrast, the three enriched detectable naive-like HCV-specific CD8+ T-cell populations that did not expand upon in vitro priming indicate a functional impairment in priming of naive-like HCV-specific CD8+ T-cells. In sum, these results indicate that in addition to viral escape and CD8+ T-cell exhaustion lack of priming may also represent a mechanism for HCV-specific CD8+ T-cell failure in chronically infected patients promoting viral persistence. However, it remains to be elucidated whether intrinsic CD8+ T-cell defects account for this phenomenon or whether impaired activation by DC, lack of CD4+ T-cell help or the presence of immunomodulatory cytokines such as IL-10 or TGF-β may play a role. It will also be important to analyze whether similar observations can be made in other chronic infections, such as chronic HBV infection, to gain further insights into the contribution of priming defects to CD8+ T-cell impairment and viral persistence in general.