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In Gut, two compelling papers1 2 provide novel insights into the complexity of HBV-specific T-cell responses through a detailed analysis of whether T-cell immunity targeting different HBV proteins in HBV-infected patients might exhibit distinct features.
CD8 T cells are a major component of antiviral immunity. Through their ability to recognise and target the cells where viral replication occurs, they can block and even terminate the spread of viral infection in the host. Their highly selective recognition is mediated by the T-cell receptor (TCR) which detects HLA-class I/viral peptide complexes on the cell surface. These protein complexes are made by short viral peptides generated by the processing of viral proteins synthesised within the infected cells (called epitopes) non-covalently associated with HLA-class I molecules. The recognition of viral peptide/HLA complexes by the specific TCR activates the CD8 T cells, which can directly lyse the virus-producing cell or secrete cytokines that inhibit virus replication occurring in the targeted cells.3 Since viruses produce different viral proteins, CD8 T cells are armed with distinct TCRs that recognise epitopes derived from different proteins, and this multispecificity of CD8 T cells should provide the host with a greater chance to effectively control the virus infection.
This is a paradigm in HBV infection, where CD8 T cells have been shown to have a crucial role in the sustained viral control4 and where the ability to mount a multispecific CD8 T-cell response against epitopes derived from different HBV proteins is present in patients with self-limited hepatitis B.5 6 Conversely, patients with chronic HBV are characterised by a defect in the number, specificity and function of HBV-specific CD8 T cells (reviewed in Bertoletti and Ferrari7).
Immunological characterisation in HBV-infected patients to date has supported this neat dichotomy of HBV-specific CD8 T-cell response in different stages of infection, but has discounted the possibility that CD8 T cells specific for epitopes derived from different HBV proteins could possess distinct features. This knowledge gap is noteworthy since work in animal models has shown that CD8 T cells specific for different epitopes can have different antiviral efficacies.8 In addition, the virological characteristics of HBV justify the possibility that CD8 T cells targeting distinct HBV proteins might exert different antiviral effects. Unlike HCV, which produces its proteins from a single translated polyprotein, HBV encodes its proteins from distinct open reading frames, which are differentially regulated. As a consequence, the different HBV proteins are produced in varying quantities in the infected cells.9 10
The two manuscripts, Schuch et al 1 and Hoogeveen et al,2 in Gut tackle this knowledge gap and undertake a direct and detailed ex vivo analysis of the phenotypic and functional features of CD8 T cells targeting epitopes located in different HBV proteins in patients with acute self-limited hepatitis B and in patients with eAg-negative chronic hepatitis B (CHB).
Schuch et al focused their analysis on CD8 T cells present in patients with anti-HBe-positive CHB with low levels of HBV DNA (<3000 IU/mL). The authors, using a sophisticated method of T-cell enrichment, were able to directly visualise HBV-specific T cells in most of the patients (~80%) and convincingly demonstrate that CD8 T cells specific for core and polymerase epitopes present different phenotypic, transcriptional and functional features. Even though all HBV-specific CD8 T cells exhibit common features of exhaustion (they all expressed the exhaustion/inhibitory receptors PD-1, KLRG1, 2B4, TIGIT), it was also apparent that polymerase epitope–specific CD8 T cells were characterised by a higher expression of CD38, KLRG and of the transcriptional factor Eomes than the core-specific CD8 T cells. These features are indicative of a greater degree of T-cell exhaustion and functional in vitro experiments showed an enhanced ability of core-specific CD8 T cells to proliferate and survive after antigenic stimulation.
In the other study, Hoogeveen and colleagues have undertaken a detailed longitudinal analysis of the phenotype and function of HBV-specific T cells present first in patients with acute self-limited hepatitis B and then in patients with eAg-negative CHB. They confirmed that multispecific CD8 T cells targeting all HBV proteins are detected at a higher frequency in patients with acute HBV and, notably, that the CD8 T-cell phenotype changes after the control of the infection. However, they also demonstrate that CD8 T cells targeting epitopes located in different HBV proteins exhibit differences in both phenotype and function, which are maintained even after viral control in both acute and chronic patients. The most striking difference was observed in the expression of the inhibitory receptor PD-1 and in functionality (degranulation and IFN-gamma production).
Thus, both manuscripts show that CD8 T cells specific for epitopes located in distinct proteins differ in frequency (envelope responses would appear to be particularly compromised in patients with CHB), but also in phenotype and function. Moreover, they demonstrate that the exhausted HBV-specific CD8+ T-cell population in patients with CHB is not homogeneous, but conversely consists of heterogeneous subsets of less differentiated progenitor/memory-like and further differentiated/terminally exhausted T cells.
Both manuscripts also propose that these differences in epitope-specific CD8 T cells are related to the protein of origin, thus implying that all core and polymerase epitopes would behave like those reported. The authors have to be commended for undertaking a study without exclusive focus on CD8 T cells specific for the ‘classical’ HLA-A0201 core and polymerase epitopes. Both studies have extended their characterisation on some CD8 T cells specific for different HBV epitopes restricted by other HLA-class I molecules. However, as a note of caution, we would point out that the bulk of the data presented still describe the features of the HLA-A0201 core 18–27 and HLA-A0201 pol 455–63 specific CD8 T cells. A more comprehensive study, demonstrating that all the possible polymerase-specific CD8 T cells restricted by distinct HLA-class I molecules present in different patients with CHB are identical, is still needed. Nevertheless, these new and interesting findings convincingly show that HBV-specific CD8 T cells are not equal and in fact are differentially characterised. This begs the obvious question: what are the implications of these findings?
First, as Lauer et al argue, studies ‘monitoring HBV-specific CD8 T cells in CHB patients needs to be interpreted in the context of these intrinsic T cell differences’. The other practical point proposed by both groups is that the reported differences indicate that CD8 T cells targeting distinct epitopes might have different antiviral efficacy or varying potential to be rescued by immunotherapeutic strategies, such as check point inhibitor therapy or therapeutic vaccination. Both manuscripts also make the case that their ability to detect some HBV-specific CD8 T cells in the majority of patients with CHB analysed indicates that complete deletion of HBV-specific CD8 T cells is not common in patients with CHB. This would imply that therapy designed to rescue HBV-specific CD8 T-cell function might be applicable in the majority of patients with CHB.
Although the authors put forward a cogent argument, the conclusions should be also taken with some degree of caution. The CHB patient populations analysed in both studies is characterised by ‘spontaneous’ low replicative levels of HBV-DNA and is composed only of patients with eAg-negative CHB. Previous analyses of direct ex vivo frequencies of HBV-specific CD8 T cells in different clinical categories of patients with CHB did not detect any peripheral HBV-specific T cells in patients with HBeAg-positive CHB.11 A larger and more detailed analysis of the various clinical categories of patients with CHB with similar enrichment methods should be performed to confirm these latest findings.
Other interesting findings are also reported. It is remarkable in both studies that the level of expression of PD-1 on HBV-specific CD8 T cells of patients with CHB is not directly proportional to their level of exhaustion. For example, in both studies, polymerase-specific CD8 T cells express lower levels of PD-1, but these virus-specific T cells were found to be more functionally exhausted. These data are consistent with the concept that PD-1 expression might also rescue T cells from rapid apoptosis12 and are in line with the recent demonstration that PD-1-positive HBV–specific CD8 T cells are important for viral control after withdrawal of nucleos(t)ide analogue therapy.13
What drives the different features of the CD8 T cells targeting distinct HBV epitopes is still unclear (figure 1). The authors acknowledge that understanding these drivers of differences in phenotype and function of the HBV-specific CD8 T cells will require additional studies. One possible explanation was that the different phenotype of polymerase-specific and core-specific CD8 T cells was primarily caused by the different quantities of viral antigen in the infected hepatocytes. However, this interpretation is not supported by the observation that core and polymerase CD8 T cells maintain their phenotypic differences even after complete HBV control and that the presence of viral mutations in the core epitopes, targeted by CD8 T cells, did not alter their function or phenotype.
If the persistence of HBV antigens in hepatocytes is not the primary cause of the differences detected on CD8 T cells, an alternative hypothesis could be that the initial CD8 T-cell priming might have a determining role. HBV proteins are produced in different quantities and, thus, a possibility could be that higher quantities of viral antigen (like envelope and core) might be presented by hepatocytes as well as professional antigen-presenting cells (monocytes/macrophages, dendritic cells) with the ability to cross-present the secreted antigens.14 It is accepted that, under inflammatory conditions, HBV envelope antigens can be cross-presented during natural HBV infection,15 but what remains less clear is the eventual functional consequences of priming performed by hepatocytes as opposed to professional antigen-presenting cells.
Finally, even though both studies show that CD8 T cells specific for distinct epitopes are endowed with different antiviral potential, we still cannot conclude which one might have a better antiviral efficacy in vivo. On first impressions, one might surmise that core-specific CD8 T cells, which show a less exhausted phenotype ex vivo, might possess greater antiviral efficacy. However, the ability of HBV to mutate in epitopes targeted by core-specific CD8 T cells without any apparent loss of viral fitness16 17 would suggest that rescuing the more ‘exhausted’ polymerase-specific CD8 T cells might be a more efficient strategy.
In summary, the reality is that these two compelling studies open a new chapter in the study of HBV adaptive immunity, offering a novel perspective on the complexity of the T-cell response present in patients with CHB, which to date has been overlooked and only recently investigated also by other groups.18 Combining these latest findings with the recent evidence of marked heterogeneity of HBV antigen-expressing hepatocytes present in CHB-infected livers,19–21 we can conclude that the task of controlling chronic HBV infection with immunological therapies will be both challenging and scientifically stimulating.
Contributors Both authors contributed to the writing of this commentary.
Funding This work was supported by a Singapore Translational Research (STaR) investigator award (NMRC/STaR/Nov 004/2018) of National Medical Research Council of Singapore to AB and a Barts and The London Charity Large Project grant (723/1795) to PTFK.
Competing interests AB receives research support from Gilead Sciences to test the effect of HBV antigens on immune cell function. He acted as a consultant and served on the advisory boards of Gilead Sciences, Janssen-Cilag, Vir, HUMABS BioMed, SpringBank and Jiangsu Simcere Pharmaceutical. He is also a cofounder of Lion TCR, a biotech company developing T-cell receptors for treatment of virus-related cancers. PTFK has collaborative grant funding from Gilead Sciences, participates in advisory board/provides consultancy to Gilead Sciences and Janssen, and is an investigator for industry-led trials with Gilead Sciences, Janssen, Alere and Assembly Biosciences.
Provenance and peer review Commissioned; internally peer reviewed.
Patient consent for publication Not required.
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