Harsimran D. Singh

Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell–mediated deletion

Hepatic NK cells eliminate HBV-specific T cells dependent on TRAIL and TRAIL-R2 interactions to limit antiviral immunity in chronic infection.

Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.

The Third Signal Cytokine IL-12 Rescues the Anti-Viral Function of Exhausted HBV-Specific CD8 T Cells

Optimal immune activation of naïve CD8 T cells requires signal 1 mediated by the T cell receptor, signal 2 mediated by co-stimulation and signal 3 provided by pro-inflammatory cytokines. However, the potential for signal 3 cytokines to rescue anti-viral responses in functionally exhausted T cells has not been defined. We investigated the effect of using third signal cytokines IL-12 or IFN-α to rescue the exhausted CD8 T cell response characteristic of patients persistently infected with hepatitis B virus (HBV). We found that IL-12, but not IFN-α, potently augmented the capacity of HBV-specific CD8 T cells to produce effector cytokines upon stimulation by cognate antigen. Functional recovery mediated by IL-12 was accompanied by down-modulation of the hallmark inhibitory receptor PD-1 and an increase in the transcription factor T-bet. PD-1 down-regulation was observed in HBV but not CMV-specific T cells, in line with our finding that the highly functional CMV response was not further enhanced by IL-12. IL-12 enhanced a number of characteristics of HBV-specific T cells important for viral control: cytotoxicity, polyfunctionality and multispecificity. Furthermore, IL-12 significantly decreased the pro-apoptotic molecule Bim, which is capable of mediating premature attrition of HBV-specific CD8 T cells. Combining IL-12 with blockade of the PD-1 pathway further increased CD8 functionality in the majority of patients. These data provide new insights into the distinct signalling requirements of exhausted T cells and the potential to recover responses optimised to control persistent viral infections.

Interferon Alpha Induces Sustained Changes in NK Cell Responsiveness to Hepatitis B Viral Load Suppression In Vivo

NK cells are important antiviral effectors, highly enriched in the liver, with the potential to regulate immunopathogenesis in persistent viral infections. Here we examined whether changes in the NK pool are induced when patients with eAg-positive CHB are ‘primed’ with PegIFNα and importantly, whether these changes are sustained or further modulated long-term after switching to nucleos(t)ides (sequential NUC therapy), an approach currently tested in the clinic. Longitudinal sampling of a prospectively recruited cohort of patients with eAg+CHB showed that the cumulative expansion of CD56bright NK cells driven by 48-weeks of PegIFNα was maintained at higher than baseline levels throughout the subsequent 9 months of sequential NUCs. Unexpectedly, PegIFNα-expanded NK cells showed further augmentation in their expression of the activating NK cell receptors NKp30 and NKp46 during sequential NUCs. The expansion in proliferating, functional NK cells was more pronounced following sequential NUCs than in comparison cohorts of patients treated with de novo NUCs or PegIFNα only. Reduction in circulating HBsAg concentrations, a key goal in the path towards functional cure of CHB, was only achieved in those patients with enhancement of NK cell IFNγ and cytotoxicity but decrease in their expression of the death ligand TRAIL. In summary, we conclude that PegIFNα priming can expand a population of functional NK cells with an altered responsiveness to subsequent antiviral suppression by NUCs. Patients on sequential NUCs with a distinct NK cell profile show a decline in HBsAg, providing mechanistic insights for the further optimisation of treatment strategies to achieve sustained responses in CHB.

T Cells Infiltrating Diseased Liver Express Ligands for the NKG2D Stress Surveillance System

NK cells, which are highly enriched in the liver, are potent regulators of antiviral T cells and immunopathology in persistent viral infection. We investigated the role of the NKG2D axis in T cell/NK cell interactions in hepatitis B. Activated and hepatitis B virus (HBV)–specific T cells, particularly the CD4 fraction, expressed NKG2D ligands (NKG2DL), which were not found on T cells from healthy controls (p < 0.001). NKG2DL-expressing T cells were strikingly enriched within HBV-infected livers compared with the periphery or to healthy livers (p < 0.001). NKG2D+NK cells were also increased and preferentially activated in the HBV-infected liver (p < 0.001), in direct proportion to the percentage of MICA/B-expressing CD4 T cells colocated within freshly isolated liver tissue (p < 0.001). This suggests that NKG2DL induced on T cells within a diseased organ can calibrate NKG2D-dependent activation of local NK cells; furthermore, NKG2D blockade could rescue HBV-specific and MICA/B-expressing T cells from HBV-infected livers. To our knowledge, this is the first ex vivo demonstration that non-virally infected human T cells can express NKG2DL, with implications for stress surveillance by the large number of NKG2D-expressing NK cells sequestered in the liver.

Harnessing alveolar macrophages for sustained mucosal T-cell recall confers long-term protection to mice against lethal influenza challenge without clinical disease.

Vaccines that induce T cells, which recognize conserved viral proteins, could confer universal protection against seasonal and pandemic influenza strains. An effective vaccine should generate sufficient mucosal T cells to ensure rapid viral control before clinical disease. However, T cells may also cause lung injury in influenza, so this approach carries inherent risks. Here we describe intranasal immunization of mice with a lentiviral vector expressing influenza nucleoprotein (NP), together with an NFκB activator, which transduces over 75% of alveolar macrophages (AM). This strategy recalls and expands NP-specific CD8+ T cells in the lung and airway of mice that have been immunized subcutaneously, or previously exposed to influenza. Granzyme B-high, lung-resident T-cell populations persist for at least 4 months and can control a lethal influenza challenge without harmful cytokine responses, weight loss, or lung injury. These data demonstrate that AM can be harnessed as effective antigen-presenting cells for influenza vaccination.

TRAIL regulatory receptors constrain human hepatic stellate cell apoptosis

The TRAIL pathway can mediate apoptosis of hepatic stellate cells to promote the resolution of liver fibrosis. However, TRAIL has the capacity to bind to regulatory receptors in addition to death-inducing receptors; their differential roles in liver fibrosis have not been investigated. Here we have dissected the contribution of regulatory TRAIL receptors to apoptosis resistance in primary human hepatic stellate cells (hHSC). hHSC isolated from healthy margins of liver resections from different donors expressed variable levels of TRAIL-R2/3/4 (but negligible TRAIL-R1) ex vivo and after activation. The apoptotic potential of TRAIL-R2 on hHSC was confirmed by lentiviral-mediated knockdown. A functional inhibitory role for TRAIL-R3/4 was revealed by shRNA knockdown and mAb blockade, showing that these regulatory receptors limit apoptosis of hHSC in response to both oligomerised TRAIL and NK cells. A close inverse ex vivo correlation between hHSC TRAIL-R4 expression and susceptibility to apoptosis underscored its central regulatory role. Our data provide the first demonstration of non-redundant functional roles for the regulatory TRAIL receptors (TRAIL-R3/4) in a physiological setting. The potential for these inhibitory TRAIL receptors to protect hHSC from apoptosis opens new avenues for prognostic and therapeutic approaches to the management of liver fibrosis.

Molecular Recalibration of PD-1+ Antigen-Specific T Cells from Blood and Liver

Checkpoint inhibitors and adoptive cell therapy provide promising options for treating solid cancers such as HBV-related HCC, but they have limitations. We tested the potential to combine advantages of each approach, genetically reprogramming T cells specific for viral tumor antigens to overcome exhaustion by down-modulating the co-inhibitory receptor PD-1. We developed a novel lentiviral transduction protocol to achieve preferential targeting of endogenous or TCR-redirected, antigen-specific CD8 T cells for shRNA knockdown of PD-1 and tested functional consequences for antitumor immunity. Antigen-specific and intrahepatic CD8 T cells transduced with lentiviral (LV)-shPD-1 consistently had a marked reduction in PD-1 compared to those transduced with a control lentiviral vector. PD-1 knockdown of human T cells rescued antitumor effector function and promoted killing of hepatoma cells in a 3D microdevice recapitulating the pro-inflammatory PD-L1hi liver microenvironment. However, upon repetitive stimulation, PD-1 knockdown drove T cell senescence and induction of other co-inhibitory pathways. We provide the proof of principle that T cells with endogenous or genetically engineered specificity for HBV-associated HCC viral antigens can be targeted for functional genetic editing. We show that PD-1 knockdown enhances immediate tumor killing but is limited by compensatory engagement of alternative co-inhibitory and senescence program upon repetitive stimulation.

Effect of in-vivo hepatitis B viral load suppression after interferon exposure on natural killer and T-cell responsiveness

Abstract Background There is a pressing need for new treatment strategies in chronic hepatitis B. Natural killer (NK) cells are important antiviral effectors, and are potently expanded with peginterferon alfa in chronic hepatitis B. Robust antiviral T-cell responses are crucial for resolution of this disease and can be expanded with nucleos(t)ide analogues (NAs). We assessed whether changes in the NK and T-cell pool would be altered when patients are primed with peginterferon alfa and whether these changes are modulated upon switching to sequential NAs. Methods Peripheral blood mononuclear cells from 52 patients were analysed. 33 underwent a course of peginterferon alfa and were sampled longitudinally; 24 of them progressed to sequential NAs. 19 patients receiving de-novo NA therapy were analysed for comparison. NK cell subsets and global T cells were analysed by multicolour flow cytometry, and hepatitis B virus (HBV)-specific T cells were analysed by enzyme-linked immunospot assay and correlated with treatment outcomes. Findings There was cumulative expansion of CD56 bright NK cells driven by peginterferon alfa, which was maintained at higher than baseline concentrations throughout subsequent sequential NAs (p=0·0039). The expansion in proliferating, functional NK cells was more pronounced after sequential NAs compared with de-novo NAs. Patients treated with peginterferon alfa progressing to sequential NAs expressed higher levels of CD69, CD62L, and CXCR3 (implicated in antifibrogenesis) than did patients on de-novo therapy. Reduction in circulating HBsAg concentrations was only achieved in patients with enhancement of NK cell interferon γ and cytotoxicity. Partial recovery of HBV-specific T cells was seen during sequential NAs after peginterferon alfa cessation. Interestingly, no difference in the magnitude of HBV-specific T-cell responses was seen in patients on sequential NAs compared with de novo NAs. Interpretation Peginterferon alfa priming expands a population of functional NK cells, with altered responsiveness to subsequent viral suppression by NAs. We have previously reported that NK cells can delete HBV-specific T cells, but our findings here suggest that the peginterferon alfa expanded NK cell pool does not exhibit this negative effect. We are investigating whether peginterferon alfa is able to protect T cells from NK cell attack, as demonstrated in a mouse model by other investigators. Funding Wellcome Trust, Barts and The London Charity, National Medical Research Council Singapore.

P0485 : Functional immune restoration correlates with HBsAg decline and may predict treatment response on sequential NUC therapy in chronic hepatitis B

Results: Frequencies of CD3(+)CD56(+) NK-like T cells did not differ significantly between the study groups. Phenotypic analysis revealed that acutely HCV infected patients displayed a significantly lower frequency of CD161(+)CD3(+)CD56(+) NKlike T cells compared to controls. Of note, frequency of CD161expressing cells was positively correlated with IFN-g production. We found, IL-12/IL-15 stimulated CD3(+)CD56(+) NK-like T cells from healthy donors effectively block HCV replication in vitro. Supernatants of IL-12/IL-15 stimulated CD3(+)CD56(+) NK-like T cells also significantly inhibited HCV replication in vitro, suggesting that non-cytolytic mechanisms may play a major role. In addition, we only observed minimal killing of HuH7A2HCVreplicon cells by CD3(+)CD56(+) NK-like T cells and could confirm that blocking of IFN-g with a specific antibody significantly reduced the antiviral activity of these cells. However, when CD3(+)CD56(+) NKlike T cells from HIV(+) patients were studied we found HIV infection to be associated with a significantly impaired IFN-g production, irrespective of HCV co-infection. In line with this observation, CD3(+)CD56(+) NK-like T cells from HIV(+) patients were significantly less effective in blocking HCV replication in vitro than cells from healthy individuals. Conclusions: Taken together, our data indicate that CD3(+)CD56(+) NK-like T cells have the potential to block HCV replication but are functionally impaired in HIV(+) patients. This might represent a novel mechanism of dysregulated immune response in HIV/HCV co-infected patients.